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Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of2009influenza A (H1N1)isolates

Xiaofan Ding•Lifang Jiang•Changwen Ke•Zhan Yang•Chunliang Lei•

Kaiyuan Cao•Jun Xu•Lin Xu•Xingfen Yang•Yonghui Zhang•

Ping Huang•Weijun Huang•Xun Zhu•Zhenjian He•Liping Liu•

Jun Li•Jie Yuan•Jueheng Wu•Xiaoping Tang•Mengfeng Li

Received:4March2010/Accepted:17August2010/Published online:31August2010

ÓSpringer Science+Business Media,LLC2010

Abstract The2009flu pandemic is caused by a new strain of influenza A(H1N1)virus,A/H1N1/09.With its high transmissibility,this novel virus has caused a pandemic and infected over600,000people globally.By comparing the hemaglutinin(HA)gene and protein sequences among over 700A/H1N1/09isolates,mutations in the receptor-binding sites and antigenic epitope regions were identified.Among these mutations,T220and E/G239were found to be strongly positively selected over the course of spreading of the A/H1N1/09virus worldwide.Interestingly,both sites are located in the highly variable epitope regions of HA1,and residue239also plays an important role in the receptor-binding process.Further analyses demonstrated that the percentage of T220mutants among all isolates increased rapidly during the evolution,and that an E/G239mutation could decrease the binding affinity of the virus with its cellular receptor.Thus,due to a potential functional importance of residues220and239,mutations at these sites, as well as the significant of positive selection on these sites deserves more attention,while new vaccines and therapeutic drugs are developed against this novel virus.

Keywords H1N1influenza virusÁHemaglutininÁMutationÁPositive selection

Abbreviations

IAV Influenza A virus

WHO World Health Organization

Xiaofan Ding,Lifang Jiang,and Changwen Ke contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11262-010-0526-z)contains supplementary material,which is available to authorized users.

X.DingÁL.JiangÁK.CaoÁL.XuÁX.ZhuÁZ.HeÁ

L.LiuÁJ.LiÁJ.YuanÁJ.WuÁM.Li

Key Laboratory of Tropical Disease Control,Ministry of Education,Sun Yat-Sen University,Guangzhou,China

X.DingÁL.JiangÁK.CaoÁX.ZhuÁZ.HeÁL.LiuÁ

J.WuÁM.Li(&)

Department of Microbiology,Zhongshan School of Medicine, Sun Yat-Sen University,74Zhongshan Road II,Guangzhou, Guangdong510080,China

e-mail:[email protected]

C.KeÁX.YangÁY.ZhangÁP.Huang

Guangdong Province Center for Disease Control and Prevention, Guangzhou,China

Z.YangÁC.LeiÁX.Tang(&)

The8th People’s Hospital of Guangzhou,627Dongfengdong Road,Guangzhou,Guangdong510060,China

e-mail:[email protected] K.Cao

Research Centre for Clinical Laboratory Standard,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China J.Xu

School of Pharmaceutical Sciences,Sun Yat-Sen University, Guangzhou,China

L.Xu

Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

W.Huang

Department of Medical Genetics and Center for Genome Research,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,China

J.LiÁJ.Yuan

Department of Biochemistry,Zhongshan School of Medicine, Sun Yat-Sen University,Guangzhou,China

Virus Genes(2010)41:329–340 DOI10.1007/s11262-010-0526-z

HA Hemagglutinin

NP Nucleoprotein

NS Nonstructural protein

NA Neuraminidase

M Matrix protein

CFR Case fatality ratios

RBS Receptor-binding sites

PDB Protein data bank

S Serine

T Threonine

D Aspartic acid

G Glycine

E Glutamic acid

MOE Molecular operating environment

Introduction

In March2009,a novel H1N1swine-origin influenza A virus(IAV)wasfirst detected in Mexico.With the ability to spread human-to-human,it sparked a growing outbreak of illness globally.The level of influenza pandemic alert wasfinally raised to Phase6by WHO on June11,2009.As of November22,2009,there have been more than622,482 laboratory confirmed cases of infection of the pandemic influenza H1N12009virus(A/H1N1/09)and over7826 deaths reported to WHO[1],with the actual estimation of infections far exceeding the numbers of laboratory con-firmed cases and deaths due to incomplete reporting.By May14,2010,more than214countries and overseas ter-ritories or communities have reported laboratory confirmed cases of pandemic influenza H1N12009(A/H1N1/09), including at least18,036deaths reported to WHO[2].

IAVs cause epidemics and pandemics through antigenic drift and antigenic shift,respectively[3].Antigenic drift results from an accumulation of point mutations leading to minor and gradual antigenic changes,while antigenic shift involves major antigenic changes by introduction of new HA and/or NA subtype into human population.Although the current A/H1N1/09influenza virus remains to be of the H1N1subtype,it is obvious that the viral changes have reached the level of intra-subtypic antigenic shift that gives rise to a pandemic.

Since1918,three influenza pandemics,namely,the 1918–1919H1N1,the1957H2N2,and the1968H3N2 pandemics,have emerged in human,all of which are thought to have originated from non-human reservoirs [4–6].The current outbreak of A/H1N1/09pandemic,as revealed by recent studies,is caused by a novel influenza virus containing a combination of gene segments from different sources.Sequence analyses have demonstrated that the hemagglutinin(HA);polymerases PB1,PB2,and PA;nucleoprotein(NP);and nonstructural protein(NS) gene segments of the A/H1N1/09virus have the highest homologies with those derived from the swine triple reas-sortant lineage,which has been circulating in pigs in North America,and the neuraminidase(NA)and matrix protein (M)gene segments are most closely related to those of the Eurasian swine influenza viruses[7].These analyses sug-gest that the novel virus might have derived from reas-sortment events occurring between the North American and the Eurasian lineages.

It is of note that the A/H1N1/09pandemic initially has exhibited a relatively low mortality,with case fatality ratios(CFR)ranging from0.3to1.5%,indicating that the currently widespread virus probably have not mutated to support a most virulent phenotype.Whereas,a relatively high transmissibility,found by the clinical surveillance of the pandemic influenza H1N12009virus[8–12],suggests that the virus is able to escape protective immunity easily. Furthermore,it has been recently reported the A/H1N1/09 pandemic exhibits an unusual pattern of age-related mor-bidity and mortality,as it disproportionately affects chil-dren and young adults(ages4–25),compared with seasonal influenza viruses[9–12].The incidence of severe disease decreases with age,with the lowest occurrence in the population65years and older,suggesting preexisting immunity to the2009pandemic virus in people born before 1957[13].It is hence of great interest to identify the gene sites/mutations to which the highly transmissible feature of the novel virus is attributable.

The HA glycoprotein on the surface of IAV particles function as the receptor-binding ligand,mediating entry, and internalization of the virus into host cells and sub-sequent membrane-fusion events in the infected cells.The mature HA is a homotrimer of*220kD containing sev-eral glycosylation sites.Each HA molecule is synthesised as a single polypeptide precursor(HA0)and subsequently cleaved into HA1and HA2subunits by an endoprotease that targets a specific cleavage site in HA0[14].This event is a prerequisite condition for successful infection,and the generated HA1polypeptide bears the receptor-binding sites (RBS).Furthermore,major epitopes specific for protective immune response are also located in HA of IAV,as well as of influenza vaccines,as identified by previous studies as epitopes A through E[15].Recently,experimental data have shown that this A/H1N1/09influenza virus can bind to both2,3-and2,6-linked sialic acid receptors and repli-cate in the lower respiratory tracts of infected mammals [16].Interestingly,Krause et al.reported that the naturally occurring human monoclonal antibodies neutralize both 1918and2009pandemic influenza A(H1N1)viruses[17]. These features raised the concern that this new virus may possess virulence characteristics similar to those of the

highly pathogenic1918pandemic influenza viruses. Lately,Wei et al.defined the structural basis for cross-neutralization and protection between two distant pan-demic influenza viruses of the1918and2009pandemics, suggesting that specific N-glycans in HA may play a key role in modulating immune recognition and influencing on viral evolution[18].Accordingly,mutations in HA there-fore can contribute to changes in virulence and transmis-sibility of influenza viruses.

Influenza virus is subject to genetic mutation,mainly due to the lack of proof-reading activity of its polymerase. Mutations in influenza viral genes accumulate over time and are under selection pressure during epidemics or pan-demics.Thus,frequencies of mutations detected for a specific IAV strain may indicate a positive or negative selection,as well as the underlying biological or epide-miological factors,under which the virus evolves in the process of emergence and spreading among the host pop-ulation.For example,previous analysis on the evolution of the M gene have found that one and ten sites in M1and M2 regions,respectively,are under positive selection in human,and that the M1and M2regions are evolving independently under different selective pressure in differ-ent hosts.The study also identified potentially important sites that may be related to host tropism and immune responses[19].

In this study,we analyzed the sequences of A/H1N1/09 IAV with a focus on its HA protein using various analysis tools.Our results revealed the presence of mutations pos-sibly relevant to the pathogenesis and evolution of A/H1N1/09IAV,and therefore might be useful for further investigation of the pathogenic and immunogenic proper-ties of this rapidly spreading virus and future design of more effective vaccines.

Methods

Point mutation analysis

All the non-redundant HA sequences of the A/H1N1/09 virus in the GenBank(deposited as of April21,2010),with a total number of704(full-length only,collapse identical sequences,and including the information of exact collec-tion time),were downloaded and loaded into the ClustalW and the BioEdit programs for multiple alignment analysis, which led to the generation of a consensus sequence. Variations at each amino acid position along the HA pro-tein were identified among704downloaded sequences.

The HA genes of four IAVs(H1N1)that were circu-lating in humans and recommended by WHO as an influ-enza vaccine component between2001and2009were used to create a consensus sequence for human seasonal H1N1 influenza viruses.

Meanwhile,the protein sequences were compared between the A/H1N1/09and human seasonal IAV HA consensus sequences using multi-sequence alignment anal-ysis.Individual A/H1N1/09HA sequences,including A/California/04/2009(H1N1),A/Beijing/01/2009(H1N1), A/Sichun/1/2009(H1N1)and A/Guangdong/1/2009(H1N1), and seasonal IAV HA sequences,including A/New Caledonia/20/1999(H1N1),A/Solomon Islands/03/2006 (H1N1),A/Brisbane/59/2007(H1N1),and A/Washington/ 10/2008(H1N1),were also compared with one another and with both consensus HA sequences,to identify variations along the HA protein.The glycosylation sites of both consensus HA sequences were analyzed by NetNGlyc1.0 server[20].

Structural modeling

3-D structure of A/H1N1/09HA protein was modeled using the Modeller program to modify previously known H1N1HA protein,whose crystal structures have been determined,with altered amino acids identified in the HA of novel A/H1N1/09virus.Briefly,the A/H1N1/09HA consensus sequence wasfirst used to search the PDB[21] using BLAST tofind deposited IAV HA proteins with the highest similarity to it.This procedure led to the identifi-cation of three HA proteins in the PDB(PDB ID:1RUY, 1RVT,and1RVO),which were then downloaded as tem-plates for further modeling procedures.Subsequently,a homology model was created between A/H1N1/09HA and the selected templates(1RUY,1RVT,and1RVO),and the sequence of A/H1N1/09HA protein was modeled50times onto the selected templates.The best model resulting from the above modeling procedure was obtained and visualized in Jmol[22].

Site-by-site analysis

All the704coding sequences of the A/H1N1/09virus available in GenBank(deposited as of April21,2010)were taken for further site-by-site positive selection analysis using the HyPhy program[23]under the‘‘MG949HKY859 3_492_Rates’’model(4rate categories assigned).The ratios of non-synonymous(dN)and synonymous(dS)sub-stitutions were calculated for each site in all codons.All the calculated dN/dS values were then further tested with the empirical Bayes method[24,25],and when the Bayes factor (the ratio of posterior odds of an event and its prior odds)was significantly greater than1,it was considered that the

hypothesis of dN/dS[1or dN/dSwas true.Sites where dN/dS[1or dN/dSwere considered positively selec-ted or negatively selected,respectively.

Analysis for isolation time and geographic regions distribution of HA S220and T220

For further analysis of the distribution of different residues on position220of the HA protein(pandemic2009H1 numbering),all the704non-redundant HA protein sequences of the A/H1N1/09influenza virus in the NCBI influenza virus sequence database(deposited as of April 21,2010)were downloaded and placed into12groups according to their isolation times.Multi-sequence align-ment was performed.The frequency of each allele was calculated as function of isolation time to evaluate whether the frequency of a specific amino acid present at position 220changed over time.The trends of such changes were then tested by employing the Kendall test and linear model in the R[26]program,and P

高铁一响,黄金万两——杭黄高铁再擎区域价值高峰

荣盛君 荣盛康旅黄山国际康养度假区

全国范围内,最美高铁——杭黄,年内通车

自2014年6月30日开工建设

到2018年3月12日铺轨全线贯通

我们已等待了四年的杭黄高铁,即将登场

除了“一生痴绝处,无梦到徽州”

那些对徽州度假的无限渴望

和不断缩短的“黄山时间”

高铁,作为城际、省际出行的重要方式

其经济价值已然令投资者狂热

据悉,这条“颜值担当”的高铁线

不仅串起了山、湖、城的人间美景

更用时间拉近了上海、杭州与黄山的距离

此前,上海到黄山,最快高铁需4.5小时

此后,仅2.5小时,便能从国际的上海都市

来到云海、日出、迎客松间的黄山

荣盛·浦溪水镇

作为黄山北大门门户的荣盛·浦溪水镇

总占地2600亩,总投资60亿元

是集旅游、养老、文化、度假、休闲于一体的综合性文旅项目

也是市、区重点建设项目之一

建成后,势必成为黄山——九华山旅游线上的一颗明珠

亚洲最长索道——黄山太平索道口

是上下黄山的重要线路

浦溪水镇,在索道口畔

以商业、住宅、酒店,三大板块全业态链合

成就黄山北门旅游集散地

也将成为黄山景区,最具投资属性的“价值洼地”

有数据表明,2016年黄山进山游客约330万

2017年约400万,且在2018年

这个数字将突破500万

而当杭黄高铁通车后,去往黄山旅游度假的人数

必将呈现倍增的趋势

浦溪水镇,踞守黄山北大门

艺术家村落、盛典演艺中心、阿尔卡迪亚酒店

文化娱乐中心、河岸徽派商业街……

千亿级商业资源注入

大黄山旅游版图的未来价值

已随高铁,无可估量

.05was chosen to indicate that a trend was significant.The frequency of each allele was also calculated as function of geographic regions of isolation following grouping non-redundant A/H1N1/09 HA protein sequences according to the regions where they were isolated(Asia,Europe,North America,South America,and Oceania)and tested using the v2test. Binding energy analysis

The MOE(molecular operating environment)program[27] was used to calculate the binding energy between A/H1N1/ 09HA and its cellular receptor based on a known binding structure(PDB ID:3GBN,X-ray diffraction)composed of the A/South Carolina/1/1918HA and a receptor analog. First,we partially minimized the complex by relaxing the ligand and the side chains within10nm from the ligand, while keeping all other atomsfixed.

Following calculation of energies,factor analysis(FA) and multiple regression analysis(MRA)were employed to generate an LRE-like equation[28,29]:

D G b FEB

ðÞ¼x1D G b vdWþx2D G b eleþx3D G b solvþx4D G b n

D G b¼D G complexþD G proteinþD G ligand

In this equation,D G(FEB)stands for the free energy of binding,D G vdW,D G ele,D G solv,and D G n stand for the van der Walls contribution,the electrostatic contribution,the polar solvation contribution,and the nonpolar solvation contribution to the binding process,respectively,where w1, w2,w3,and w4are weight factors,and D G b represents binding energy(i.e.,energy difference between ligand/ receptor complex and free protein and ligand).Results

Point mutation analysis

To specify the differences between the HA molecules of the A/H1N1/09virus and recently circulating seasonal IAVs,we generated a consensus sequence from704 A/H1N1/09HA amino acid sequences deposited in the GenBank(as of April21,2010)and a consensus sequence from the HA proteins of4seasonal H1N1IAV strains that had been recommended by WHO for production of influ-enza vaccines,and compared these two groups of HA sequences by aligning both consensus sequences and sev-eral individual HA sequences from each group(Fig.1).

As shown in Fig.1and Table1,while the overall dif-ference between the consensus sequences of seasonal H1N1and A/H1N1/09HA proteins was as high as19.61% (111/566),the cleavage sites of HA,at which the HA0 protein is recognized by specific protease(s)and enzy-matically cleaved into HA1and HA2,thereby becoming activated in mediating the entry of IAVs into host cells, remain identical(PSIQSR;GLFGAI)among the strains analyzed.Meanwhile,the Asp204,Asp239,Gln240,and Gly242residues at the RBS that are responsible for the viral attachment to the host cell receptor,a critical step for viral entry,were also identical between the two consensus sequences.It is of note that the four positions(i.e.,204, 239,240,and242)have been found previously to be involved in the specific binding capacity of HA to the host cell receptor[30–32].Whereas,amino acid variations were found at a number of positions at the HA RBS among different seasonal H1N1IAVs and the A/H1N1/09viruses, including positions150,152,206,207,210,211,and212. Whether variations at these sites could impact the infec-tivity of an influenza virus to a specific host needs to be further investigated biologically.

To address the differences in the antigenic properties between A/H1N1/09and human seasonal IAVs,previously proposed antigenic epitope regions were comparatively analyzed.In this context,two groups of epitope regions, namely,the highly conserved regions and the highly vari-able regions[33],were analyzed,respectively.As shown in Fig.1and Table2,four highly conserved epitope regions (1–4),located in HA2,involving residues345–354, 359–376,394–411,and436–453,were all identical between A/H1N1/09isolates and seasonal H1N1viruses. In contrast,the highly variable regions,which lie in the HA1globular head and include sites(residues86–91),Sa (residues141–142,170–174,and176–181),Sb(residues 201–212),Ca1(residues183–187,220–222,and252–254) and Ca2(residues154–159,and238–239)[34],showed dramatic changes in A/H1N1/09isolates when compared with those in seasonal IAVs(H1N1).Such changes might

Fig.1Alignment of HA amino acid sequences of human seasonal IAVs and their consensus sequence in comparison with the pandemic A/H1N1/09IAVs and their consensus sequence.Residues different from the consensus HA amino acid sequence(top line)of human seasonal H1N1IAVs are shown,and dots stand for identical residues. The sequences were numbered according to pandemic2009H1 numbering.Boxed residues indicate the Asn-X-Thr/Ser motifs of glycosylation sites

Table1Comparison between the new A/H1N1/09isolates and seasonal H1N1IAVs at HA0cleavage site and RBS

Functional sites Virus Residues

The cleavage site Human seasonal PSIQSR;GLFGAI

A/H1N1/09PSIQSR;GLFGAI

Difference:0

RBS(in HA1)Residue positions(pandemic2009H1numbering)

149–152204–212235–242

Human seasonal V S A S DQ RA LY HTE PKVRDQEG

A/H1N1/09V T A A DQ QS LY QNA PKVRDQEG

Difference:2/4Difference:5/9Difference:0

provide a molecular explanation for the observed lack of cross-protection from previous infection or vaccination of seasonal IAVs against the novel A/H1N1/09virus.

Since point mutations could cause emergence or loss of Asn-X-Thr/Ser motifs and thereby,attachment or loss of N-glycans,respectively,leading to alteration of the anti-genicity and receptor specificity of HA,we analyzed the glycosylation sites on H1HAs by further examining both consensus sequences of the seasonal H1N1and the novel pandemic H1N1,which revealedfive identical glycosyla-tion sites(28,40,104,304,498,pandemic2009H1 numbering)between the two consensus sequences,as shown in Fig.1.In marked contrast,the A/H1N1/09stains contained amino acid mutations predicted to lose three

Table2Comparison between A/H1N1/09isolates and seasonal H1N1IAVs at antigenic epitope regions Epitope region Virus Residues and positions b

Highly conserved regions(in HA2)1345–354in HA2

Human seasonal GLFGAIAGFI

A/H1N1/09GLFGAIAGFI

Difference:0

2359–376in HA2

Human seasonal TGMVDGWYGYHHQNEQGS

A/H1N1/09TGMVDGWYGYHHQNEQGS

Difference:0

3394–411in HA2

Human seasonal NKVNSVIEKMNTQFTAVG

A/H1N1/09NKVNSVIEKMNTQFTAVG

Difference:0

4436–453in HA2

Human seasonal WTYNAELLVLLENERTLD

A/H1N1/09WTYNAELLVLLENERTLD

Difference:0

Highly variable regions(in HA1)a Cb86–91in HA1

Human seasonal L ISK(R)E S

A/H1N1L STAS S

Difference:4/6

Sa141–142in HA1170–174in HA1176–181in HA1 Human seasonal PN G K NGL P N LSKS

A/H1N1/09PN K K GNS P K LSKS

Difference:0Difference:4/5Difference:1/6 Sb201–212in HA1

Human seasonal NIG D(N)Q R(K/M)A(T)LY HT(K)E

A/H1N1/09TSA DQ QS LY QNA

Difference:8/12

Ca1183–187in HA1220–222in HA1252–254in HA1 Human seasonal A(V)N N K E SS H EPG

A/H1N1/09I N D K G T S R EPG

Difference:3/5Difference:1/3Difference:0 Ca2154–159in HA1238–239in HA1

Human seasonal S H N G E(K)S RD

A/H1N1/09P H A G AK RD

Difference:4/6Difference:0

a Amino acid sequences for all highly variable epitope regions listed in this table are given using the consensus sequences generated for A/H1N1/09isolates and seasonal H1N1IAVs,respectively,as presented in Fig.1.Variations in individual sequences as compared with the consensus sequence are given in parenthesis

b Numbering according to pandemic2009H1numbering

glycosylation sites at position71,142,and177.It is of note that the two highly conserved glycosylation sites(i.e.,142 and177)have been found previously to be involved in the antigenic properties of H1N1IAVs,which was within or around the Sa site.Meanwhile,we found that the A/H1N1/ 09stains carried amino acid mutations predicted to acquire one potential glycosylation site at position293,raising the issues whether such glycosylation does occur at the site and alters the antigenicity and receptor specificity of2009 influenza A(H1N1)virus.

Point-to-point analysis of mutations in the HA protein was performed among704isolates of the novel A/H1N1/09 virus.As shown in Tables3and4,13out of566positions in the entire HA molecule displayed variations among704 non-redundant A/H1N1/09HA sequences(variation fre-quency was higher than2),including5positions at RBS, and11positions in the highly variable epitopes,and all other variations were shown in the supplement data Table S1.Specifically,the amino acid change at position342 (Gln342Leu)was at the cleavage site of one A/H1N1/09 isolate[A/Guangdong/03/2009(H1N1)].Whether such a change would influence the interaction of HA0with pro-teolytic enzyme(s)and consequently,the cleavage activity, remains unclear.It requires further investigation to clarify whether mutation Asp239Glu in two isolates[A/Paris/ 2591/2009(H1N1)and A/New Jersey/01/2009(H1N1)] was relevant to host adaptation,since Glu239had been previously found to be associated with acquisition of SA a-2,6Gal binding specificity[30,35].Most noteworthily is the high frequency(72.02%)of Ser220Thr mutation in the Ca1epitope,strongly indicating existence of a positive selection or,at a lesser likelihood(due to lack of other high-frequency mutated positions in the HA),more than one origin of the new A/H1N1/09viruses.

Structural modeling

To better characterize the variations in A/H1N1/09HA,we sought to map the altered amino acids to a predicted three-dimensional(3-D)HA structure.Wefirst BLAST searched the PDB database for deposited HA molecules with the

highest sequence homologies to A/H1N1/09HA.Three sequences,together with their previously X-ray determined 3-D structures,of similarities higher than80%were selected,which were1RUY[HA of A/swine/Iowa/15/30 (H1N1)],1RVT[HA of A/swine/Iowa/15/30(H1N1) complexed with receptor analog LSTC]and1RV0[HA of A/swine/Iowa/15/30(H1N1)complexed with receptor analog LSTA][32].Using the selected molecules as tem-plates,onto which the new A/H1N1/09HA consensus sequence was modeled for50times,a predicted3-D structure of the A/H1N1/09HA was obtained,visualized with Jmol,and demonstrated in Fig.2.

As shown in Fig.2,the structure of A/H1N1/09HA monomer was similar to those of other published HAs,as expected,with a globular head containing the RBS,a cleavage site,a trans-membrane domain,and an a-helical stalk.Variations found in various isolates of the A/H1N1/ 09HA,as well as differences between the HA molecules of human seasonal IAVs and the new A/H1N1/09virus, and the glycosylation attachment sites were mapped on the3-D structure as shown in distinct colors in Fig.2. These variations and different residues were mainly located in the HA1fragment,most of which lied in antigenic epitopes.

Table3Amino acid variations in the HA protein sequence among A/H1N1/09isolates

Positions a Primary

residue

Frequency

(%)

Variation(s)Variation

frequency(%) HA136V97.73I 2.13

L0.14 49L96.45I 3.41

X0.14 103D97.17E0.14

G 2.41

M0.28 114D95.74N 4.12

X0.14 145S95.32M 4.40

X0.28 220S26.99A0.14

T72.02

X0.85 222R96.17G0.14

K 3.13

S0.28

T0.14

X0.14 239D88.78E 4.69

G 3.55

N0.99

X 1.99 310Q95.31H 4.26

X0.43 314P97.30S 2.70

338V94.88I 4.69

S0.43

HA2391E88.78G0.71

K10.51 428V97.30I 2.56

X0.14

a Positions listed according to pandemic2009H1numbering

b‘‘X’’stands for any amino acid

Site-by-site analysis

To address the significance of the mutations identified in A/ H1N1/09HA,we conducted positive selection analysis on a site-by-site basis,as positive natural selection could drive the increase in prevalence of advantageous traits,which may lead to a new pandemic virus.In this study,the ratio between non-synonymous(dN)and synonymous(dS)substitutions were used to indicate selective pressure on each codon.According to Yang et al.[24],when the dN/dS value on a certain codon is greater than1and tested to be significant by the Bayes test,the site is considered to be under positive selection;and in con-trast,the site would be recognized as being under negative selection in the case if the dN/dS value is significantly smaller than1.Using the HyPhy program[23],our positive selection analysis revealed that34codon sites showed dN/dS[1with statistical significance(Bayes factor[1)(Fig.3).To mini-mize possible errors such as those caused by biased sampling, we chose to use a very conservative strategy in the identifi-cation of the site mutations under positive selection by selecting only those codon sites with the highest Bayes fac-tors,which accounted for the leading5%of all codon sites with a Bayes factor[1.Consequently,these procedures led to identification of two sites,namely,positions220and239 (pandemic2009H1numbering,equivalent to206and225, respectively,according to the H3numbering)in the HA1 protein.Notably,the residue220lied in the Ca1epitope region in the head of HA1,and residue239was found to be included in the RBS of HA1.It is of interest that previous studies have suggested that both regions are relevant to the determination of the severity and transmissibility of an IAV [35],raising the question whether the mutations at these sites could favor the prevalence of the novel virus.

Variation at position220

Particularly noteworthy was the relatively high frequencies of S220and T220present in the HA of A/H1N1/09

Table4Amino acid variations in functional regions of A/H1N1/09 isolates

Functional region Residue positions a Variations a

The cleavage site Between HA1and HA2None

RBS(in HA1)149–152151

204–212204

235–242238,239,240

Highly conserved epitopes(in HA2)345–354None 359–376None 394–411None 436–453None

Highly variable epitopes(in HA1)

Cb86–9190

Sa141–142None

170–174172

176–181179

Sb201–212202,203,204 Ca1183–187None

220–222220,222

252–254252

Ca2154–159None

238–239238,239

a Positions listed according to pandemic2009H1

numbering

Fig.2Mapping of mutations onto the3-D structure of HA monomer. Side views of the HA monomers are shown.Each has the highly conserved epitope regions in HA2colored violet and the highly variable epitope regions in HA1colored cyan.a the reference HA monomer,and b,c the HA monomers with mutations mapped.In b,regions colored blue stand for mutations detected among the A/H1N1/ 09viruses;in c,regions colored yellow demonstrate differences between the human seasonal influenza virus and this novel virus,and the amino acid variations at residues220and239and the glycosylation sites were numbered(Colorfigure online)

isolates,prompting us to ask whether they were a result of selection during the course of evolution.To address this issue,we further analyzed the frequency distribution of S220and T220in the HA among isolates of the novel A/H1N1/09virus as a function of isolation time and geo-graphic regions.When 704HA sequences derived from A/H1N1/09viruses isolated between the period of March 30,2009and April 21,2010,were grouped according to their isolation time,a total of 507isolates (72%)with T220were identified,and the percentage of S present at position 220was found to gradually decline,while the frequency of T220increased over time (Fig.4).To test the significance of the descending trend of S220,Kendall test and a linear model were used,and results revealed a Kendall test

P value of 2.503910-5and a descending rate of 0.06655with a P value of 9.84910-5in the linear model analysis.Both tests confirmed the significant descending trend of the residue S at position 220,with T220gradually becoming prevalent in the infected population.In contrast,no sig-nificant difference in the frequency distribution of S220versus T220among HA molecules derived from viruses isolated from Asia,Europe,North America,Oceania,and South America,with a v 2test P value of 0.2801,indicating that the changes in the frequencies of S220versus T220found in the above study probably were not geographic region-specific and might have been occurring worldwide.We next sought to investigate whether S220and T220were present in the HA molecules of previously isolated swine IAVs and seasonal human H1N1viruses.We down-loaded 272non-redundant HA protein sequences of North American classical swine flu virus (H1subtype)from the NCBI influenza virus sequence database and analyzed the frequency of amino acids present at position 220.Interest-ingly,251out of the 272sequences were found to have S220,as opposed to that only 20isolates carried T at the position 220.Moreover,all of the 20strains with T220were H1N1subtype swine influenza A viruses,among which 14were isolated from Tennessee in 1976,1977,and 1978,and the remaining six strains were isolated sporadically from several other US states.On the other hand,the frequency distribu-tion in 1767HA sequences derived from human-infected H1subtype IAVs isolated before the 2009pandemic showed an S220:T220ratio of 1760:7.Variation at position 239

Since our point mutation analysis found a notable vari-ability of position 239in HA,we analyzed the

frequencies

of different residues at the position in HA of704A/H1N1/09 isolates.Our analysis found that aspartic acid(D)was present at a frequency of88.78%(623/704),glycine(G)at 3.55%(25/704),and glutamic acid(E)at4.69%(33/704), with the remaining residues undetermined.Possible impact of mutations at this site on receptor binding was examined by calculating the minimal energy using the MOE(Molec-ular Operating Environment)program,and the result showed that the binding energy between receptor analog and wild type HA(D239)was-29.506kcal/mol,while for mutants G239and E239,it was-17.027and-16.445kcal/ mol,respectively,indicating possibly weakened receptor-binding capacities of the mutants.

Discussion

Our current study analyzed the site variations in the HA protein sequences among the novel A/H1N1/09viral iso-lates,as well as those between human seasonal influenza viruses and A/H1N1/09.3-D structure of the HA was also modeled,and identified point mutations were analyzed to predict their potential significance in molecular evolution and function of the pandemic virus.

In this study,the point mutation analysis showed that the A/H1N1/09strains carry amino acid mutations that might lead to acquisition of one glycosylation site(position293) and loss of three glycosylation sites(position71,142,and 177).It has been reported that variation in glycosylation is used by influenza viruses to interfere with surveillance by the host immune system.Acquisition of a glycosylation site masks the protein surface from antibody recognition because the glycans themselves are host-derived,and hence considered as‘‘self’’by the human immune system[36,37] Meanwhile,upon the addition of glycosylation to this region has been thought to slow down yearly antigenic drift,pre-sumably because glycosylation shielded this region from the antigenic pressure of antibodies[18,36].Recently,Wei et al.and Xu et al.have evaluated the cross-neutralization of pandemic1918and2009H1N1influenza viruses and found that they were both resistant to antisera directed to a rela-tively recent seasonal influenza virus of the same subtype [18,38].Interestingly,pandemic2009H1N1influenza virus (A/California/04/2009),like A/South Carolina/1/1918virus, does not have any glycosylation in or around the Sa site in HA and hence,the epitope is exposed for antibody recog-nition.They suggest that these N-glycans of the RBD and antigenic epitope regions may play roles in evading the human immune response and viral evolution in humans[18]. The probability of glycosylation of the predicted asparagine residues293above mentioned in our current study,and the role of the acquisition(position293)or loss of glycosylation (position71)in modulating immune recognition and its influence on viral evolution,is under investigation in our laboratory.

By mapping the variations in the HA protein sequence, we identified that the distinction between the HA antigenic specificities of the human seasonal influenza virus and the novel A/H1N1/09virus mainly lie in the HA1epitope regions,which might explain the lack of effective cross-protection by previous seasonal IAV vaccines or infections and the highly transmissible property of the A/H1N1/09 virus.

A key notion derived from this study is the possibility that two HA1sites in the novel A/H1N1/09virus,namely, residues220and239,might be positively selected during its evolution.Interestingly,residue239lies in a region possibly involved in both receptor binding and determi-nation of antigenic specificity[30–32,34].It has been previously reported that when residue239changes from Gly to Glu,the virus becomes more adaptive to human host [30].Recently,Chen et al.reported the D225G(H3num-bering,D239G according to2009pandemic H1number-ing)substitution in7(12.5%)of57patients with severe disease,and in0(0%)of60patients with mild disease,and the D225E mutation was identified in one patient with severe disease,by direct analysis of polymorphisms in126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N12009virus from117 clinical specimens in Hong Kong[39].Our current study revealed that the vast majority of pandemic A/H1N1/09 isolates carried D239(88.78%)and yet a small fraction of the A/H1N1/09isolates had glutamic acid(E,4.69%)and glycine(G,3.55%)at this position.Free binding energy analysis suggested that a D239E/G mutation tended to decrease the affinity of the H1subtype IAV to the sialic acid receptor.It remains to be determined,whether the identified positive selection of the D239E/G mutation is functionally significant during the spreading of the A/H1N1/09virus.

Residue220lies in the Ca1epitope region.In all A/H1N1/09isolates obtained as of August2009,T220was present at a frequency of72.02%,whereas the frequency of S220was26.99%.It is of note that both T220and S220 were present in H1subtype classical swine IAVs,from which the HA of the pandemic A/H1N1/09was originated, and that the frequency of T220was far lower than that of S220in these swine IAVs.Our analysis on the dynamic change of T220versus S220demonstrated an ascending trend of the percentage of T220and a descending curve of S220during the course of the pandemic spreading,sug-gesting that T220might have been positively selected over S220.Speculatively,T220was thenfixed through natural or other selection at the peak of this pandemic and favored the transmissibility of the new virus.Interestingly,com-pared with serine,amino acid threonine carries an extra

methyl group and therefore displays a different polarity than serine.Whether such a difference in the polarity could contribute to altering recognition and binding capacity between the antigenic epitope and specific antibodies and thereby should be taken into consideration when develop-ing more effective vaccines and therapeutic drugs remains to be further investigated.

Acknowledgments This study was supported by the National Natural Science Foundation of China-Guangdong Province joint grant (U0632002),a grant from the State Major Infectious Disease Research Program(China Central Government,2009ZX10004-213), a Key Science and Technique Research Project of Guangdong Province(2009A020101006,2009B020600001),National High-Tech R&D Program(863Program)(Ministry of Science and Technology, China,2006AA02A223,2007AA09Z448,2007AA09Z431),National Science and Technique Research Program for public welfare appli-cations(201005022),and a Key Project of Science&Technology Planning of Guangdong Province(2007A03260001). References

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如对您有帮助,可购买打赏,谢谢

生活常识分享孕妇可以吃花生酥吗

导语:花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是

花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是一种高热量的饮食。生活中很多的孕妇对于花生酥也是情有独钟,但是考虑到孕妇身体的特殊性使得人们比较的重视,那么,孕妇可以吃花生酥吗?

花生酥香甜而不腻,具有极高的营养价值,可以与鸡蛋、鱼肉等相媲美,孕妇在生活中可以适当的吃一些花生酥,补充身体所需要的能量,但是不可以多吃,以免对身体不利。

孕妇可以吃花生酥吗?花生酥的营养价值比粮食高,可以与鸡蛋、牛奶、肉类等一些动物性食物媲美。它含有大量的蛋白质和脂肪,特别是不饱和脂肪酸的含量很高,很适宜制造各种营养食品。

孕妈妈常吃花生酥能够预防产后缺乳,而且花生衣中含有止血成分,可以对抗纤维蛋白溶解,增强骨髓制造血小板的功能,缩短出血时间,提高血小板量,改善血小板质,加强毛细血管的收缩功能.是孕妇防治再生障碍性贫血的最佳选择。此外,用新鲜花生叶煎水代茶饮,还能够有效防治妊娠高血压综合征。

以上的内容就是对于孕妇可以吃花生酥吗的介绍,希望能够给您带去一定的帮助。在一般的情况下人们都是可以食用花生酥的,但是对于孕妇来说不可过量的食用,花生酥中的糖分会引起妊娠高血糖等病变,对孕妇和胎儿的健康不利,我们需要避免。

。

普吉岛甲米蒂瓦娜广场酒店(DeevanaPlazaKrabi)

舒适度作为普吉岛甲米蒂瓦娜广场酒店所有的客房首要标准,一切设施都以此为目标,一定不会让您失望。酒店宽敞的客房,配有住客评分分数来自522条评语等设施,让您瞬间忘记旅途的疲倦。

中文名称普吉岛甲米蒂瓦娜广场酒店英文名称DeevanaPlazaKrabi酒店星级4星级房间数量213酒店地址186Moo3,AonangSoi8,Aonang,Muang,81180奥南海滩,泰国

【好巧网解读】4大卖点

1.很安全2.员工热情有礼貌,一部分客人带着两岁多的女儿,他们对小孩也很好3.酒店还设有池畔酒吧和24小时客房服务4.床也很舒服

酒店的图片

酒店房型房价介绍

每个客房都配有阳台,电话,DVD播放机,视频游戏,有线频道,平面电视,保险箱,空调,熨斗,书桌,熨衣设备,客厅角,瓷砖/大理石地板,衣柜/衣橱,淋浴,吹风机,浴袍,免费洗浴用品,卫生间,浴室,拖鞋,迷你吧,冰箱,电烧水壶,唤醒服务,希望能让客户在入住时更加愉快惬意。酒店的房型有多种选择,提供了豪华特大号床间-可使用游泳池(3位成人)、高级双床间(2位成年人、家庭间、豪华双床间-可直通泳池(3位成人)、高级双床间(3名成人)、豪华特大大床房(3人)、家庭间(3位成人)、豪华双床间(2位成人)、高级特大号床间(3位成人)、豪华双床间(3位成人)、豪华双床间-可使用游泳池(2位成人),房间布置都到位,服务员也很热情。简而言之,客人在普吉岛甲米蒂瓦娜广场酒店享受的服务与设施会有宾至如归的感觉。再讲究的客人也能在酒店得到满意的服务。

相关条款

入住时间从14:00时退房时间12:00时之前预订取消/预付政策不同类型的客房附带不同的取消预订和预先付费政策请输入您的入住日期并参阅您所需的客房的条款。儿童和加床允许客人携带儿童入住。允许1名12岁以下的儿童,使用现有床铺的收费是每人每晚THB200。允许1名2岁以下的儿童,加1张婴儿床,免费。允许1名年龄较大的儿童或者成人,一张加床收费:每人每晚THB1070。最多容纳:每间客房1张加床/婴儿床。所提出的任何加床或婴儿床的要求均需获得酒店的确认。附加费用不会自动计算在总价中,您需在入住时另行支付。宠物不允许携带宠物入住。团体如果预订客房数超过7间,住宿方将采用不同的政策和额外补充规定。酒店接受的银行卡类型将鼠标悬停在卡片标志上,即可查看更多信息。

好巧网酒店评价分

非常好,88分

酒店优缺点

游客提到的优点:安全适合带小孩,服务好餐厅很棒床铺好干净游客提到的缺点:浴缸没有那么好晚上有蚊子

备注:好巧网综合打分是基于100多个酒店预订网站的评分综合算出。以上是该酒店在几家酒店预订网站上的评分,供您参考。好巧网对酒店特色的分析数据,来自主流的酒店预订网站、旅游社区网站提到该酒店的评论,尽量展示客观中立的分析。查询更多用户评论信息,请访问http://www.haoqiao.cn/Phuket_c3/274779.html

现因借款人__________身份证号:__________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。借款种类为现金,借款日期为_____年____月____日,还款日期为______年____月____日前, 特立此据为凭。

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因___________(写借款用途)需向贷款人________借款人民币_______________元整(小写_____元整)。借得款种类为现金,借得款日期为_____年____月____日,还款日期为____年____月____日前, 按时一次性偿还清¥:____________借款加利息。借款利息为: ___%(年利率),特立此据为凭。

借款人:__________(亲笔签名并按手印)

贷款人:__________ (亲笔签名并按手印)

见证人1:_________ (亲笔签名并按手印)

见证人2:__________ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。(注:因借款人超过还款日期不守信用赖账的,需在省级权威媒体向贷款人道歉,借款人按借得款两倍偿还给贷款人。)

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

。

  中新网8月20日电 据美国中文网报道,美国康州、佛州和俄亥俄州当局表示,最近挫败了3起独立的大规模枪击图谋,3名白人男子被捕。

  据报道,公众线报帮助破获了这3起事件,三州警方在15和16日将3名嫌犯逮捕。当局称,3名嫌犯均是20出头的白人男性,他们或在网上发帖、或发短信进行大规模枪击威胁。近日,加州、得州和俄亥俄州相继发生大规模枪击事件,最近几周,多次误报和枪击威胁恶作剧让美国民众心神不宁,要求变更枪支立法的呼声迭起。

  康州

  诺沃克(Narwalk)市警部门称,22岁瓦格肖(Brandon Wagshol)被捕。瓦格肖在诺沃克警方和FBI的联合调查后被捕,FBI收到线报,据称他试图从外州购买大容量的步枪弹夹。

  警方表示,瓦格肖在网上购买了步枪零件,打算自己制造武器,并在“脸书”上展示出“他对进行大规模枪击的兴趣”。诺沃克警方说,在执行搜查令时,警方发现两把注册在瓦格肖父亲名下的枪,多发弹药,防弹衣和其他战术装备。

  佛州

  沃卢西亚郡(Volusia)治安官办公室称,25岁代托纳比奇(Daytona Beach)市居民威克斯(Scott Wix)16日被捕,被控威胁要进行大规模射击。警方接到多条线报,据称威克斯发送了多条短信,阐述了他进行大规模枪击的计划。警方没有说明威克斯向谁发送了这些短信。

  警长办公室描述威克斯的短信时称,“学校是个弱的目标……我更有可能向3英里外的一大群人开枪……我想打破连续杀人时间最长的世界纪录。”据称另一条短信写道:“但是要能杀死100人会很好。我已经有一个地点选择(笑出眼泪的emoji)很坏吗?”

  警方在一份声明中表示,威克斯称他并未拥有枪支,但“对大规模枪击事件着迷”。警方同时发布了威克斯被捕时的视频。

  俄亥俄州

  FBI称,警方接到一个线报,一名男子在网上发视频称,自己是犹太社区中心枪击事件的枪手。虽然该枪击事件并未发生,但警方将该男子逮捕,他是20岁的里尔顿(James P. Reardon)。FBI克利夫兰分部表示,里尔顿于16日被捕,涉嫌电信骚扰和加重恐吓。

  警方称,里尔顿在“图片墙”(Instagram)上发布的视频帖子,标记了扬斯敦犹太社区中心。警方收到该视频线报的当天,在里尔顿父母家执行了搜查令。他随后顺利被警方逮捕,警方还在现场发现了弹药,半自动武器和反犹信息。

。

 

联邦快递明年1月起每周7天都提供快递服务

(综合三十日电)为了满足电子商务市场持续增加的送货需求,美国联邦快递公司(FedEx)将从明年开始,每周送货七天,而且不会收取额外的费用。联邦快递明年1月起每周七天都提供快递服务,以试图跟上网购的繁荣景气。该公司也将取回目前由邮局处理的每天近200万件包裹快递能量,并表示,将SmartPost包裹转移回自有快递网络将使送货司机能将更多的包裹紧密安排规画,提高送货效率。除了每周增加一天送货服务,联邦快递公司还计划配合陆运路线,将更多包裹送到客户的家门口,以降低成本。此外,该公司明年将会有大约200万个包裹,不再委托美国邮局运送。「消费者每周七天在线购物」,联邦快递总裁兼首席运营官苏伯拉曼尼(Raj Subramaniam)表示,因此,网购者以及电商业者对每周七天送货服务的需求不断增加。他强调,每周增加一天送货服务,不会提高收费,也不是针对特定电商业者,而是对小型托运者到大型零售商都一视同仁,提供全面送货服务。联邦快递公司估计,到了2026年,美国小包裹出货量将翻倍,该公司必须提前准备。联邦快递和其竞争对手联合包裹服务公司(UPS)近年来投入资金改善送货管理,并且将所谓的「最后一英里」(Last-Mile,即送到客户家门口)利润较低的送货,外包给美国邮局(USPS)或其它业者。然而,随著网购增加,包裹数量也与日俱增,全美每天国内包裹达到5,000万件。为了因应这个趋势,FedEx及UPS正在调整业务,以增加市场份额及处理周末的送货需求。与此同时,由于亚马逊及沃尔玛等零售商在全美增建仓库,以缩短送货距离,导致联邦快递在全美或全球范围内的空运快递业务量逐渐萎缩。此外,多家零售商建立自己的送货系统,销售订单管理软件公司OrderDynamics营销副总裁迪莫夫(Charles Dimov)说:「联邦快递如果不提供周日送货服务,很有可能被市场淘汰。」UPS目前是每周送货六天,发言人扎卡拉(Glenn Zaccara)表示,该公司「持续评估扩大服务的时机」。美国邮局正在考虑将包裹递送服务扩大到每周七天,目前邮局在周日为亚马逊提供送货服务,并在假日期间为其它业者提供送货服务。

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猜对了吗

教学内容

大象版小学科学三年级(下)第一单元第2课。

教学目标

1、利用实验验证的方法,明白猜想只是一种可能的答案,它和事实并不总是一致。

2、学会使用酒精灯加热的技能,了解材料传热的性能。

3、培养重视实验和证明的科学态度。

教学重点

了解猜想、假设和事实的区别。

教学难点

培养学生重视实验和证明的科学态度。

实验器材

酒精灯、铁架台、纸杯、火柴、水槽、烧杯、镊子、铁棒、纸团、纸条等。教学过程

一、激趣导入

同学们,你们喜欢猜谜语吗?(喜欢)老师今天给大家带来了几个谜语,请大家猜一猜,看谁最聪明。(课件出示谜语)

1、上边毛,下边毛,中间一个黑葡萄。生:眼睛。

2、麻屋子,红帐子,里面住着白胖子。生:花生。

3、身体生来瘦又长,五彩衣裳黑心肠,嘴儿尖尖说黑话,只见短来不见长。生:铅笔。

4、小铁驴,真是好,又不踢,又不咬,屁股后面把烟冒,突突突叫着跑。生:摩托车。这几则谜语你猜对了吗?生:猜对了。

二、猜想和验证

1、大胆猜想

你们想挑战更难的吗?(想)请同学们来猜想。(课件出示猜想问题图)

用酒精灯烧空纸杯的底部,纸杯会烧着吗?

把空纸杯倒扣在酒精灯上,纸杯会烧着吗?

用酒精灯烧装水的纸杯底部,谁能烧开吗?

生把猜想填入表格,指明说说自己猜想的根据。

2、学习使用酒精灯

我们进行了大胆的猜想,猜对了吗?我们要设法验证

自己的猜想。要验证自己的猜想,就必须学会安全使用酒精灯。请同学们打开书第6页,阅读左下角的“安全使用酒精灯。”

生阅读。

请同学们在小组内互相说一说怎样安全使用酒精灯。生在小组内互说,指明说一说怎样安全使用酒精灯。

师示范安全使用酒精灯。

3、验证

小组讨论所用实验材料。小组组长到材料超市选取实验材料,分组实验,是巡视指导,生填写实验报告单。汇报实验结果。

4、我进步我成功

课件出示:在几次猜想中,我的猜想与实验结果:

总是不一样()嘿,没关系!

有时不一样()呵,再努力!

总是一样的()瞧,我真棒!

指名说,是鼓励。

5、你在猜想与验证活动中取得了哪些收获?

师:猜想只是一种可能的答案,它和事实并不总是一

样的,要想知道猜想是否正确,必须设法验证。

三、课外延伸

我们再来进行两个猜想。

杯子竖直扣入水底,塞在杯底的纸团会湿吗?

用一张普通的纸,裁成长条,以螺旋状紧绕在一根铁棒上,然后火柴去烧铁棒上的纸条,纸条会被烧着吗?

想知道你猜对了吗?(想)怎么办?

。

  原标题:印尼狮航空难一周年 美国对波音的调查怎么样了?

  参考消息网10月29日报道 外媒称,10月29日是印尼狮航737 MAX客机坠毁一周年纪念日,美国议员表示,在“99.9%的美国公众”和决策者确信客机是安全的之前,波音该机型客机不会复飞。

  新加坡《联合早报》网站10月28日援引路透社报道称,波音公司首席执行长米伦伯格将连续两天出席听证会。此前,有多份报告发现,波音在设计737 MAX客机时,没有充分考虑飞行员应该如何应对驾驶舱紧急情况。

  据报道,美国联邦航空管理局已经花了数月时间,对波音提交的关于关键安全系统软件的升级、培训以及系统变化等进行评估。但报道称,预计最早要到12月才可能让该机型飞机复飞。

  美国参院商业委员会主席、共和党参议员威克说,“除非99.9%的美国公众和政策制定者相信,它(737 MAX客机)是绝对安全的,否则这一型号飞机不会复飞。”

  威克称,他还计划在推进调查期间,加强波音与美国联邦航空管理局的沟通,并在听证会期间加强“监管机构与制造商之间的关系”。

。

  原标题:川普催促美国实行负利率 美联储官员不乐意

  中新网11月21日电 综合报道,尽管美国总统川普一再呼吁在美国实行负利率政策,但20日公布的一项会议记录显示,美联储的官员们并不乐意通过这种方式来刺激经济。

资料图:美联储主席鲍威尔中新社记者 陈孟统 摄资料图:美联储主席鲍威尔中新社记者 陈孟统 摄

  据报道,一直以来,川普声称欧洲与其他地区采取的负利率措施,为这些国家带来了竞争优势,于美国不利,因而呼吁美联储降息。

  然而,这份美联储10月份的会议记录明确显示,美联储不仅在当前美国经济仍在增长的情况下不愿采用负利率,也对经济陷入衰退时采用负利率手段刺激经济深表怀疑。

  根据会议记录,美联储的17位决策委员会成员都认同,将借款成本推低至低于零“在美国似乎并不是有吸引力的货币政策工具”。

  决策者们也不热衷于通过购买国债来控制长期利率,一些决策者认为这样做会被视为干涉财政部对国家债务的管理,其他决策者则担心这会使美联储资产负债表膨胀。

  美联储公开市场委员会(FOMC)利率决策委员在会中陈述,在一些已尝试执行负利率的国家,证据显示效果好坏并存。因此委员们认为,近期内进一步降息“并无必要”,除非迎来重大变化。

  据悉,自2019年7月以来,美联储已3次降息,但此前从2015年末开始连续9次加息。川普曾多次指责美联储没能更大幅度地降息,影响了他任期内的经济增长。

 

HTML5取代Android和iOS应用程序?

大量新生移动设备的兴起,改变了互联网的未来。在技术的发展上,HTML5会取代App应用吗?或者说能够在多大程度上取代呢?在HTML5规范中,已经加入了相机、磁力罗盘、GPS信息的支持。很多新兴浏览器也已经开始支持这些新特性。能否用一个统一的HTML5来替代android和ios并行开发的双重成本呢?以下译自MichaelMahemoff的一篇文章,详细分析了HTML5能否取代Android和iOS应用程序。

介绍

移动应用程序(App)和HTML5都是目前最火的技术,二者之间也有不少重叠之处。在移动设备浏览器里运行的html5的web页面,也可以重新打包成不同平台上运行的app。目前很多浏览器都有很好的跨平台支持,(译注:firefox居然可以在android中使用和windows下同样的浏览器内核),HTML5的web方案,对开发者来说更为方便。完成一次,即可多平台使用。但这确实可行吗?仍然有许多必要原因,使得开发者选择了app开发。很明显,很多人已经在这么做了。本文将详细分析两种方案的优劣。

功能丰富

正方:App里可以开发出更丰富的功能

我们把移动功能分成两类。程序本身和程序与系统的结合。比如android里,加入widget图标或者通知提醒之类的。App对这两者都没问题。不用多说,这是肯定的。

反方:APP是挺强,但Web也正在迎头跟进

确实很多原生app实现的功能是HTML5望尘莫及的。不管你的web做的再牛,如果停留在一个没有摄像头支持的沙盒中,很多场合还是玩不转。幸运的是,现在没有这样的沙盒限制了。如果你需要你的web照相片,可以做一个负责照像的app,再把你的web打包进这个应用里面。开源的PhoneGap框架是这么干的。这样widget,手机提醒也都没问题了。

但这种混合开发的问题在于,增加了复杂性,而且不象传统web那样可以直接在浏览器里运行。这个问题短时间内恐怕没辙。好在现在网络标准在不断的高速扩充,先进的浏览器也在一直跟进。Android3.1已经支持camera了。iOS浏览器也支持WebSocket和设备方向检测了。

总得来说,移动设备在发展,而web也同样在快速变化。桌面浏览器本身,有5家主要浏览器开发商在改进现有标准,丰富新的功能。所以原生App在快速前进,同时,web也在缩小差距。

运行效率

正方:原生APP速度更快

原生APP没有瓶颈,而且可以直接调用GPU加速、使用多线程。

反方:现如今Web已经快多了,而且多数应用也用不着那么快。

这说法有点落伍了。Chrome发布之时带来的JavascriptV8,给Web速度带来的飞跃。而现在,计算速度变得更快了:

图片处理引擎已经使用web加速。现在硬件加速也已经开始应用了。

要开发3D游戏的就不用抬杠了,但对于平而来说,新闻、邮件、时间管理、社交网络,这些用Web都够用了。试试SteveSouders的手机性能测试工具。另外,越来越多的框架结合WebGL,可以发挥OpenGL的优势了。比如ImpactJS,帮助开发JS游戏。

开发感受

正方:原生APP好写

原生APP使用强壮的程序语言(Java,ObjectiveC,C++)。适合写复杂程序,经过历史验证,API丰富。在桌面环境可以方便的用模拟器测试。而Web程序的runtimes和乱七八糟的各路浏览器让人头大。

反方:一般都是Web更简单,特别是需要兼容不同设备的时候。

Web最初的功能只限于文档展示,而不是程序应用,貌似最近俩星期才有了JS。但有了JS后,web的世界马上就不一样了。更何况web不只是静止的,HTML5,CSS3,EcmaScriptHarmony(谁知道这是什么?)都给开发者极大帮助。你是喜欢C++,java,JavaScript,那你的个人爱好,也是基于你已经攒下的代码。但是现在没人能否认JavaScript也和前者站在同一擂台上。

浏览器/runtime的互不兼容(碎片),反过来看做APP也是一样。用Java写了Androidapp,然后又要面对iOS的ObjectiveC。如果能写一个程序,马上能在Android和iOS上运行,多省事啊。这咱还没提WebOS,BlackBerry,WindowsMobile呢。当然,这是理论上的。要是想让程序在每个平台都跑得很漂亮,得做不少调试和妥协。这对很多原生APP也是一样的。不同OS版本,不同的设备。。

所谓的Web碎片化,一直都是如此。但好消息是现在已经有很多不错的解决办法。Modernizr库,用得好的话,可以帮你兼容一大批主流设备,不管是啥系统,哪个牌子的。看看我们2011年的GoogleIO演示。

用户体验

正方:原生APP更切合原有平台

操作感受的定义之一,就是用户希望在你的程序里,用与系统连贯统一的方式来操作。不同的平台,都有一些约定俗成的习惯。比如长按按钮会有啥反应。你不能指望用一套统一的HTML5App去满足所有用户。

此外,整个平台的操作感受都由用平台自有的软件库协调。直接调用平台工具包就能直接免费获得完整支持。

反方:我们Web有自己的传统,你要特想做原有平台那种感觉的web,也一样能做出来

前面说了,Web开发的方式,是先做一个大体适合所有平台的版本,然后再针对不同平台不断改进。当这些改进主要是针对功能时,你可以选择几个你最关心的平台做优化。类似于浏览器检测。技术论坛里的悲催技术员们,经常抱怨这事。太多不同的浏览器版本了。不过如果你优先关注两三种主流平台,是值得为他们多花点时间做做优化。

web本来就有自己的操作感受。我们也可以说,不同的默认浏览器以及运行环境造就了独特的”Web感受”。从更广的角度看,这本身就是一种用户公认的方式。此外,还有很多成功的案例并不遵循移动设备的原生操作习惯,人家也成功了。想想你最喜欢的手机游戏的界面?很多更传统的app也是一样,比如Twitter客户端。

传播途径

正方:原生应用更容易接触客户

象GooglePlay和AppleStore这样的app发布机制这几年势不可挡,推动了整个移动行业。每个程序员都能在市场里发布自己的应用。用户都挤在市场里浏览,搜索,接受推荐。不仅如此,只要你的程序够好,现有用户的打分会帮助你说服更多新的客户。

反方:其实web才容易接触到客户

。

【侨报讯】参议院多数党领袖米奇·麦康奈尔(Mitch McConnell)针对参议院弹劾审判程序做出了详细计划。众议院弹劾经理团队和川普总统法律团队将各自拥有24小时进行公开辩论,分为两天进行。这一举动表明,参议院共和党人希望审判能够尽快完成,至少在总统2月4日发表国情咨文之前。

据CNN报道,肯塔基州共和党人的弹劾审判程序决议文件中记载了上述归责,这也是对比尔·克林顿总统弹劾案审判程序的另一个突破,在克林顿的审判程序中,24小时的开庭陈述时间被分在4天进行。

民主党人则反对麦康奈尔的日程安排,他们表示这是为“总统的不当行为盖上遮羞布”。

参议院少数党领袖查克·舒默(Chuck Schumer)在一份声明中说:“很明显,麦康奈尔参议员已经下定决心要加快庭审进程、并尽可能阻止证人和证物出庭,在弹劾这样重要的问题上,麦康奈尔参议员提出的程序决议简直是国家的耻辱。”

而且根据时间表来看,庭审很可能进入深夜,因为每天的审判可以持续12个小时,但审判开始于东部时间的下午1时。

麦康奈尔小组决议推迟证人出庭的问题,双方在完成辩论后,参议员有16个小时的时间可以提出问题,他们可以通过主持审判的首席大法官约翰·罗伯兹(John Roberts)对证人问题作出裁定。

而且程序决议中还包含一项提议,即在上述时间点上,参议院将对一项提议进行表决,即“为了更好的考量和辩论,根据弹劾规则是否可以提出传唤人证或物证的动议”。

如果参议院的投票结果是否定的,那么民主党弹劾经理团队、总统法律团队或参议员都得传唤人证或物证。如果参议院批准这项提议,那么双方都可以提出传唤证人的动议,届时参议院将对证人进行辩论和投票。

在出台上述提议前,麦康奈尔办公室与参议院共和党温和派进行了详尽的谈判,即将连任的缅因州参议员苏珊·柯林斯(Susan Collins)、犹他州参议员罗姆尼(Mitt Romney)、田纳西州参议员拉马尔·亚历山大(Lamar Alexander)和阿拉斯加州参议员丽莎·穆尔科斯基(Lisa Murkowski)参与了谈判。

熟悉该场谈判的工作人员表示,在场人士逐字逐句地通过了这项决议,仔细分析了什么样的表述能够使麦康奈尔在获得共和党支持的情况下,还能得到温和派选票。

白宫立法主任尤兰(Eric Ueland)在一份声明中说,白宫“对决议草案能够保护总统获得公平审判感到满意”。他说:“我们希望能够尽快按照程序还原事实,为总统进行有力辩护并尽快让总统得到无罪的判决结果。”

而舒默预计将会在庭审开始时尝试强行公布证人和相关物证文件,对麦康奈尔的决议提出修正案。舒默和其他参议院民主党人敦促参议院听取4名证人的证词,其中包括前国家安全顾问约翰·博尔顿(John Bolton)和白宫代理幕僚长马尔瓦尼(Mick Mulvaney)。

但麦康奈尔说,哪怕没有民主党的支持,共和党的票数已经足够支持这项决议。(朱三景编译)

。.05was chosen to indicate that a trend was significant.The frequency of each allele was also calculated as function of geographic regions of isolation following grouping non-redundant A/H1N1/09 HA protein sequences according to the regions where they were isolated(Asia,Europe,North America,South America,and Oceania)and tested using the v2test. Binding energy analysis

The MOE(molecular operating environment)program[27] was used to calculate the binding energy between A/H1N1/ 09HA and its cellular receptor based on a known binding structure(PDB ID:3GBN,X-ray diffraction)composed of the A/South Carolina/1/1918HA and a receptor analog. First,we partially minimized the complex by relaxing the ligand and the side chains within10nm from the ligand, while keeping all other atomsfixed.

Following calculation of energies,factor analysis(FA) and multiple regression analysis(MRA)were employed to generate an LRE-like equation[28,29]:

D G b FEB

ðÞ¼x1D G b vdWþx2D G b eleþx3D G b solvþx4D G b n

D G b¼D G complexþD G proteinþD G ligand

In this equation,D G(FEB)stands for the free energy of binding,D G vdW,D G ele,D G solv,and D G n stand for the van der Walls contribution,the electrostatic contribution,the polar solvation contribution,and the nonpolar solvation contribution to the binding process,respectively,where w1, w2,w3,and w4are weight factors,and D G b represents binding energy(i.e.,energy difference between ligand/ receptor complex and free protein and ligand).Results

Point mutation analysis

To specify the differences between the HA molecules of the A/H1N1/09virus and recently circulating seasonal IAVs,we generated a consensus sequence from704 A/H1N1/09HA amino acid sequences deposited in the GenBank(as of April21,2010)and a consensus sequence from the HA proteins of4seasonal H1N1IAV strains that had been recommended by WHO for production of influ-enza vaccines,and compared these two groups of HA sequences by aligning both consensus sequences and sev-eral individual HA sequences from each group(Fig.1).

As shown in Fig.1and Table1,while the overall dif-ference between the consensus sequences of seasonal H1N1and A/H1N1/09HA proteins was as high as19.61% (111/566),the cleavage sites of HA,at which the HA0 protein is recognized by specific protease(s)and enzy-matically cleaved into HA1and HA2,thereby becoming activated in mediating the entry of IAVs into host cells, remain identical(PSIQSR;GLFGAI)among the strains analyzed.Meanwhile,the Asp204,Asp239,Gln240,and Gly242residues at the RBS that are responsible for the viral attachment to the host cell receptor,a critical step for viral entry,were also identical between the two consensus sequences.It is of note that the four positions(i.e.,204, 239,240,and242)have been found previously to be involved in the specific binding capacity of HA to the host cell receptor[30–32].Whereas,amino acid variations were found at a number of positions at the HA RBS among different seasonal H1N1IAVs and the A/H1N1/09viruses, including positions150,152,206,207,210,211,and212. Whether variations at these sites could impact the infec-tivity of an influenza virus to a specific host needs to be further investigated biologically.

To address the differences in the antigenic properties between A/H1N1/09and human seasonal IAVs,previously proposed antigenic epitope regions were comparatively analyzed.In this context,two groups of epitope regions, namely,the highly conserved regions and the highly vari-able regions[33],were analyzed,respectively.As shown in Fig.1and Table2,four highly conserved epitope regions (1–4),located in HA2,involving residues345–354, 359–376,394–411,and436–453,were all identical between A/H1N1/09isolates and seasonal H1N1viruses. In contrast,the highly variable regions,which lie in the HA1globular head and include sites(residues86–91),Sa (residues141–142,170–174,and176–181),Sb(residues 201–212),Ca1(residues183–187,220–222,and252–254) and Ca2(residues154–159,and238–239)[34],showed dramatic changes in A/H1N1/09isolates when compared with those in seasonal IAVs(H1N1).Such changes might

Fig.1Alignment of HA amino acid sequences of human seasonal IAVs and their consensus sequence in comparison with the pandemic A/H1N1/09IAVs and their consensus sequence.Residues different from the consensus HA amino acid sequence(top line)of human seasonal H1N1IAVs are shown,and dots stand for identical residues. The sequences were numbered according to pandemic2009H1 numbering.Boxed residues indicate the Asn-X-Thr/Ser motifs of glycosylation sites

Table1Comparison between the new A/H1N1/09isolates and seasonal H1N1IAVs at HA0cleavage site and RBS

Functional sites Virus Residues

The cleavage site Human seasonal PSIQSR;GLFGAI

A/H1N1/09PSIQSR;GLFGAI

Difference:0

RBS(in HA1)Residue positions(pandemic2009H1numbering)

149–152204–212235–242

Human seasonal V S A S DQ RA LY HTE PKVRDQEG

A/H1N1/09V T A A DQ QS LY QNA PKVRDQEG

Difference:2/4Difference:5/9Difference:0

provide a molecular explanation for the observed lack of cross-protection from previous infection or vaccination of seasonal IAVs against the novel A/H1N1/09virus.

Since point mutations could cause emergence or loss of Asn-X-Thr/Ser motifs and thereby,attachment or loss of N-glycans,respectively,leading to alteration of the anti-genicity and receptor specificity of HA,we analyzed the glycosylation sites on H1HAs by further examining both consensus sequences of the seasonal H1N1and the novel pandemic H1N1,which revealedfive identical glycosyla-tion sites(28,40,104,304,498,pandemic2009H1 numbering)between the two consensus sequences,as shown in Fig.1.In marked contrast,the A/H1N1/09stains contained amino acid mutations predicted to lose three

Table2Comparison between A/H1N1/09isolates and seasonal H1N1IAVs at antigenic epitope regions Epitope region Virus Residues and positions b

Highly conserved regions(in HA2)1345–354in HA2

Human seasonal GLFGAIAGFI

A/H1N1/09GLFGAIAGFI

Difference:0

2359–376in HA2

Human seasonal TGMVDGWYGYHHQNEQGS

A/H1N1/09TGMVDGWYGYHHQNEQGS

Difference:0

3394–411in HA2

Human seasonal NKVNSVIEKMNTQFTAVG

A/H1N1/09NKVNSVIEKMNTQFTAVG

Difference:0

4436–453in HA2

Human seasonal WTYNAELLVLLENERTLD

A/H1N1/09WTYNAELLVLLENERTLD

Difference:0

Highly variable regions(in HA1)a Cb86–91in HA1

Human seasonal L ISK(R)E S

A/H1N1L STAS S

Difference:4/6

Sa141–142in HA1170–174in HA1176–181in HA1 Human seasonal PN G K NGL P N LSKS

A/H1N1/09PN K K GNS P K LSKS

Difference:0Difference:4/5Difference:1/6 Sb201–212in HA1

Human seasonal NIG D(N)Q R(K/M)A(T)LY HT(K)E

A/H1N1/09TSA DQ QS LY QNA

Difference:8/12

Ca1183–187in HA1220–222in HA1252–254in HA1 Human seasonal A(V)N N K E SS H EPG

A/H1N1/09I N D K G T S R EPG

Difference:3/5Difference:1/3Difference:0 Ca2154–159in HA1238–239in HA1

Human seasonal S H N G E(K)S RD

A/H1N1/09P H A G AK RD

Difference:4/6Difference:0

a Amino acid sequences for all highly variable epitope regions listed in this table are given using the consensus sequences generated for A/H1N1/09isolates and seasonal H1N1IAVs,respectively,as presented in Fig.1.Variations in individual sequences as compared with the consensus sequence are given in parenthesis

b Numbering according to pandemic2009H1numbering

glycosylation sites at position71,142,and177.It is of note that the two highly conserved glycosylation sites(i.e.,142 and177)have been found previously to be involved in the antigenic properties of H1N1IAVs,which was within or around the Sa site.Meanwhile,we found that the A/H1N1/ 09stains carried amino acid mutations predicted to acquire one potential glycosylation site at position293,raising the issues whether such glycosylation does occur at the site and alters the antigenicity and receptor specificity of2009 influenza A(H1N1)virus.

Point-to-point analysis of mutations in the HA protein was performed among704isolates of the novel A/H1N1/09 virus.As shown in Tables3and4,13out of566positions in the entire HA molecule displayed variations among704 non-redundant A/H1N1/09HA sequences(variation fre-quency was higher than2),including5positions at RBS, and11positions in the highly variable epitopes,and all other variations were shown in the supplement data Table S1.Specifically,the amino acid change at position342 (Gln342Leu)was at the cleavage site of one A/H1N1/09 isolate[A/Guangdong/03/2009(H1N1)].Whether such a change would influence the interaction of HA0with pro-teolytic enzyme(s)and consequently,the cleavage activity, remains unclear.It requires further investigation to clarify whether mutation Asp239Glu in two isolates[A/Paris/ 2591/2009(H1N1)and A/New Jersey/01/2009(H1N1)] was relevant to host adaptation,since Glu239had been previously found to be associated with acquisition of SA a-2,6Gal binding specificity[30,35].Most noteworthily is the high frequency(72.02%)of Ser220Thr mutation in the Ca1epitope,strongly indicating existence of a positive selection or,at a lesser likelihood(due to lack of other high-frequency mutated positions in the HA),more than one origin of the new A/H1N1/09viruses.

Structural modeling

To better characterize the variations in A/H1N1/09HA,we sought to map the altered amino acids to a predicted three-dimensional(3-D)HA structure.Wefirst BLAST searched the PDB database for deposited HA molecules with the

highest sequence homologies to A/H1N1/09HA.Three sequences,together with their previously X-ray determined 3-D structures,of similarities higher than80%were selected,which were1RUY[HA of A/swine/Iowa/15/30 (H1N1)],1RVT[HA of A/swine/Iowa/15/30(H1N1) complexed with receptor analog LSTC]and1RV0[HA of A/swine/Iowa/15/30(H1N1)complexed with receptor analog LSTA][32].Using the selected molecules as tem-plates,onto which the new A/H1N1/09HA consensus sequence was modeled for50times,a predicted3-D structure of the A/H1N1/09HA was obtained,visualized with Jmol,and demonstrated in Fig.2.

As shown in Fig.2,the structure of A/H1N1/09HA monomer was similar to those of other published HAs,as expected,with a globular head containing the RBS,a cleavage site,a trans-membrane domain,and an a-helical stalk.Variations found in various isolates of the A/H1N1/ 09HA,as well as differences between the HA molecules of human seasonal IAVs and the new A/H1N1/09virus, and the glycosylation attachment sites were mapped on the3-D structure as shown in distinct colors in Fig.2. These variations and different residues were mainly located in the HA1fragment,most of which lied in antigenic epitopes.

Table3Amino acid variations in the HA protein sequence among A/H1N1/09isolates

Positions a Primary

residue

Frequency

(%)

Variation(s)Variation

frequency(%) HA136V97.73I 2.13

L0.14 49L96.45I 3.41

X0.14 103D97.17E0.14

G 2.41

M0.28 114D95.74N 4.12

X0.14 145S95.32M 4.40

X0.28 220S26.99A0.14

T72.02

X0.85 222R96.17G0.14

K 3.13

S0.28

T0.14

X0.14 239D88.78E 4.69

G 3.55

N0.99

X 1.99 310Q95.31H 4.26

X0.43 314P97.30S 2.70

338V94.88I 4.69

S0.43

HA2391E88.78G0.71

K10.51 428V97.30I 2.56

X0.14

a Positions listed according to pandemic2009H1numbering

b‘‘X’’stands for any amino acid

Site-by-site analysis

To address the significance of the mutations identified in A/ H1N1/09HA,we conducted positive selection analysis on a site-by-site basis,as positive natural selection could drive the increase in prevalence of advantageous traits,which may lead to a new pandemic virus.In this study,the ratio between non-synonymous(dN)and synonymous(dS)substitutions were used to indicate selective pressure on each codon.According to Yang et al.[24],when the dN/dS value on a certain codon is greater than1and tested to be significant by the Bayes test,the site is considered to be under positive selection;and in con-trast,the site would be recognized as being under negative selection in the case if the dN/dS value is significantly smaller than1.Using the HyPhy program[23],our positive selection analysis revealed that34codon sites showed dN/dS[1with statistical significance(Bayes factor[1)(Fig.3).To mini-mize possible errors such as those caused by biased sampling, we chose to use a very conservative strategy in the identifi-cation of the site mutations under positive selection by selecting only those codon sites with the highest Bayes fac-tors,which accounted for the leading5%of all codon sites with a Bayes factor[1.Consequently,these procedures led to identification of two sites,namely,positions220and239 (pandemic2009H1numbering,equivalent to206and225, respectively,according to the H3numbering)in the HA1 protein.Notably,the residue220lied in the Ca1epitope region in the head of HA1,and residue239was found to be included in the RBS of HA1.It is of interest that previous studies have suggested that both regions are relevant to the determination of the severity and transmissibility of an IAV [35],raising the question whether the mutations at these sites could favor the prevalence of the novel virus.

Variation at position220

Particularly noteworthy was the relatively high frequencies of S220and T220present in the HA of A/H1N1/09

Table4Amino acid variations in functional regions of A/H1N1/09 isolates

Functional region Residue positions a Variations a

The cleavage site Between HA1and HA2None

RBS(in HA1)149–152151

204–212204

235–242238,239,240

Highly conserved epitopes(in HA2)345–354None 359–376None 394–411None 436–453None

Highly variable epitopes(in HA1)

Cb86–9190

Sa141–142None

170–174172

176–181179

Sb201–212202,203,204 Ca1183–187None

220–222220,222

252–254252

Ca2154–159None

238–239238,239

a Positions listed according to pandemic2009H1

numbering

Fig.2Mapping of mutations onto the3-D structure of HA monomer. Side views of the HA monomers are shown.Each has the highly conserved epitope regions in HA2colored violet and the highly variable epitope regions in HA1colored cyan.a the reference HA monomer,and b,c the HA monomers with mutations mapped.In b,regions colored blue stand for mutations detected among the A/H1N1/ 09viruses;in c,regions colored yellow demonstrate differences between the human seasonal influenza virus and this novel virus,and the amino acid variations at residues220and239and the glycosylation sites were numbered(Colorfigure online)

isolates,prompting us to ask whether they were a result of selection during the course of evolution.To address this issue,we further analyzed the frequency distribution of S220and T220in the HA among isolates of the novel A/H1N1/09virus as a function of isolation time and geo-graphic regions.When 704HA sequences derived from A/H1N1/09viruses isolated between the period of March 30,2009and April 21,2010,were grouped according to their isolation time,a total of 507isolates (72%)with T220were identified,and the percentage of S present at position 220was found to gradually decline,while the frequency of T220increased over time (Fig.4).To test the significance of the descending trend of S220,Kendall test and a linear model were used,and results revealed a Kendall test

P value of 2.503910-5and a descending rate of 0.06655with a P value of 9.84910-5in the linear model analysis.Both tests confirmed the significant descending trend of the residue S at position 220,with T220gradually becoming prevalent in the infected population.In contrast,no sig-nificant difference in the frequency distribution of S220versus T220among HA molecules derived from viruses isolated from Asia,Europe,North America,Oceania,and South America,with a v 2test P value of 0.2801,indicating that the changes in the frequencies of S220versus T220found in the above study probably were not geographic region-specific and might have been occurring worldwide.We next sought to investigate whether S220and T220were present in the HA molecules of previously isolated swine IAVs and seasonal human H1N1viruses.We down-loaded 272non-redundant HA protein sequences of North American classical swine flu virus (H1subtype)from the NCBI influenza virus sequence database and analyzed the frequency of amino acids present at position 220.Interest-ingly,251out of the 272sequences were found to have S220,as opposed to that only 20isolates carried T at the position 220.Moreover,all of the 20strains with T220were H1N1subtype swine influenza A viruses,among which 14were isolated from Tennessee in 1976,1977,and 1978,and the remaining six strains were isolated sporadically from several other US states.On the other hand,the frequency distribu-tion in 1767HA sequences derived from human-infected H1subtype IAVs isolated before the 2009pandemic showed an S220:T220ratio of 1760:7.Variation at position 239

Since our point mutation analysis found a notable vari-ability of position 239in HA,we analyzed the

frequencies

of different residues at the position in HA of704A/H1N1/09 isolates.Our analysis found that aspartic acid(D)was present at a frequency of88.78%(623/704),glycine(G)at 3.55%(25/704),and glutamic acid(E)at4.69%(33/704), with the remaining residues undetermined.Possible impact of mutations at this site on receptor binding was examined by calculating the minimal energy using the MOE(Molec-ular Operating Environment)program,and the result showed that the binding energy between receptor analog and wild type HA(D239)was-29.506kcal/mol,while for mutants G239and E239,it was-17.027and-16.445kcal/ mol,respectively,indicating possibly weakened receptor-binding capacities of the mutants.

Discussion

Our current study analyzed the site variations in the HA protein sequences among the novel A/H1N1/09viral iso-lates,as well as those between human seasonal influenza viruses and A/H1N1/09.3-D structure of the HA was also modeled,and identified point mutations were analyzed to predict their potential significance in molecular evolution and function of the pandemic virus.

In this study,the point mutation analysis showed that the A/H1N1/09strains carry amino acid mutations that might lead to acquisition of one glycosylation site(position293) and loss of three glycosylation sites(position71,142,and 177).It has been reported that variation in glycosylation is used by influenza viruses to interfere with surveillance by the host immune system.Acquisition of a glycosylation site masks the protein surface from antibody recognition because the glycans themselves are host-derived,and hence considered as‘‘self’’by the human immune system[36,37] Meanwhile,upon the addition of glycosylation to this region has been thought to slow down yearly antigenic drift,pre-sumably because glycosylation shielded this region from the antigenic pressure of antibodies[18,36].Recently,Wei et al.and Xu et al.have evaluated the cross-neutralization of pandemic1918and2009H1N1influenza viruses and found that they were both resistant to antisera directed to a rela-tively recent seasonal influenza virus of the same subtype [18,38].Interestingly,pandemic2009H1N1influenza virus (A/California/04/2009),like A/South Carolina/1/1918virus, does not have any glycosylation in or around the Sa site in HA and hence,the epitope is exposed for antibody recog-nition.They suggest that these N-glycans of the RBD and antigenic epitope regions may play roles in evading the human immune response and viral evolution in humans[18]. The probability of glycosylation of the predicted asparagine residues293above mentioned in our current study,and the role of the acquisition(position293)or loss of glycosylation (position71)in modulating immune recognition and its influence on viral evolution,is under investigation in our laboratory.

By mapping the variations in the HA protein sequence, we identified that the distinction between the HA antigenic specificities of the human seasonal influenza virus and the novel A/H1N1/09virus mainly lie in the HA1epitope regions,which might explain the lack of effective cross-protection by previous seasonal IAV vaccines or infections and the highly transmissible property of the A/H1N1/09 virus.

A key notion derived from this study is the possibility that two HA1sites in the novel A/H1N1/09virus,namely, residues220and239,might be positively selected during its evolution.Interestingly,residue239lies in a region possibly involved in both receptor binding and determi-nation of antigenic specificity[30–32,34].It has been previously reported that when residue239changes from Gly to Glu,the virus becomes more adaptive to human host [30].Recently,Chen et al.reported the D225G(H3num-bering,D239G according to2009pandemic H1number-ing)substitution in7(12.5%)of57patients with severe disease,and in0(0%)of60patients with mild disease,and the D225E mutation was identified in one patient with severe disease,by direct analysis of polymorphisms in126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N12009virus from117 clinical specimens in Hong Kong[39].Our current study revealed that the vast majority of pandemic A/H1N1/09 isolates carried D239(88.78%)and yet a small fraction of the A/H1N1/09isolates had glutamic acid(E,4.69%)and glycine(G,3.55%)at this position.Free binding energy analysis suggested that a D239E/G mutation tended to decrease the affinity of the H1subtype IAV to the sialic acid receptor.It remains to be determined,whether the identified positive selection of the D239E/G mutation is functionally significant during the spreading of the A/H1N1/09virus.

Residue220lies in the Ca1epitope region.In all A/H1N1/09isolates obtained as of August2009,T220was present at a frequency of72.02%,whereas the frequency of S220was26.99%.It is of note that both T220and S220 were present in H1subtype classical swine IAVs,from which the HA of the pandemic A/H1N1/09was originated, and that the frequency of T220was far lower than that of S220in these swine IAVs.Our analysis on the dynamic change of T220versus S220demonstrated an ascending trend of the percentage of T220and a descending curve of S220during the course of the pandemic spreading,sug-gesting that T220might have been positively selected over S220.Speculatively,T220was thenfixed through natural or other selection at the peak of this pandemic and favored the transmissibility of the new virus.Interestingly,com-pared with serine,amino acid threonine carries an extra

methyl group and therefore displays a different polarity than serine.Whether such a difference in the polarity could contribute to altering recognition and binding capacity between the antigenic epitope and specific antibodies and thereby should be taken into consideration when develop-ing more effective vaccines and therapeutic drugs remains to be further investigated.

Acknowledgments This study was supported by the National Natural Science Foundation of China-Guangdong Province joint grant (U0632002),a grant from the State Major Infectious Disease Research Program(China Central Government,2009ZX10004-213), a Key Science and Technique Research Project of Guangdong Province(2009A020101006,2009B020600001),National High-Tech R&D Program(863Program)(Ministry of Science and Technology, China,2006AA02A223,2007AA09Z448,2007AA09Z431),National Science and Technique Research Program for public welfare appli-cations(201005022),and a Key Project of Science&Technology Planning of Guangdong Province(2007A03260001). References

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如对您有帮助,可购买打赏,谢谢

生活常识分享孕妇可以吃花生酥吗

导语:花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是

花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是一种高热量的饮食。生活中很多的孕妇对于花生酥也是情有独钟,但是考虑到孕妇身体的特殊性使得人们比较的重视,那么,孕妇可以吃花生酥吗?

花生酥香甜而不腻,具有极高的营养价值,可以与鸡蛋、鱼肉等相媲美,孕妇在生活中可以适当的吃一些花生酥,补充身体所需要的能量,但是不可以多吃,以免对身体不利。

孕妇可以吃花生酥吗?花生酥的营养价值比粮食高,可以与鸡蛋、牛奶、肉类等一些动物性食物媲美。它含有大量的蛋白质和脂肪,特别是不饱和脂肪酸的含量很高,很适宜制造各种营养食品。

孕妈妈常吃花生酥能够预防产后缺乳,而且花生衣中含有止血成分,可以对抗纤维蛋白溶解,增强骨髓制造血小板的功能,缩短出血时间,提高血小板量,改善血小板质,加强毛细血管的收缩功能.是孕妇防治再生障碍性贫血的最佳选择。此外,用新鲜花生叶煎水代茶饮,还能够有效防治妊娠高血压综合征。

以上的内容就是对于孕妇可以吃花生酥吗的介绍,希望能够给您带去一定的帮助。在一般的情况下人们都是可以食用花生酥的,但是对于孕妇来说不可过量的食用,花生酥中的糖分会引起妊娠高血糖等病变,对孕妇和胎儿的健康不利,我们需要避免。

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普吉岛甲米蒂瓦娜广场酒店(DeevanaPlazaKrabi)

舒适度作为普吉岛甲米蒂瓦娜广场酒店所有的客房首要标准,一切设施都以此为目标,一定不会让您失望。酒店宽敞的客房,配有住客评分分数来自522条评语等设施,让您瞬间忘记旅途的疲倦。

中文名称普吉岛甲米蒂瓦娜广场酒店英文名称DeevanaPlazaKrabi酒店星级4星级房间数量213酒店地址186Moo3,AonangSoi8,Aonang,Muang,81180奥南海滩,泰国

【好巧网解读】4大卖点

1.很安全2.员工热情有礼貌,一部分客人带着两岁多的女儿,他们对小孩也很好3.酒店还设有池畔酒吧和24小时客房服务4.床也很舒服

酒店的图片

酒店房型房价介绍

每个客房都配有阳台,电话,DVD播放机,视频游戏,有线频道,平面电视,保险箱,空调,熨斗,书桌,熨衣设备,客厅角,瓷砖/大理石地板,衣柜/衣橱,淋浴,吹风机,浴袍,免费洗浴用品,卫生间,浴室,拖鞋,迷你吧,冰箱,电烧水壶,唤醒服务,希望能让客户在入住时更加愉快惬意。酒店的房型有多种选择,提供了豪华特大号床间-可使用游泳池(3位成人)、高级双床间(2位成年人、家庭间、豪华双床间-可直通泳池(3位成人)、高级双床间(3名成人)、豪华特大大床房(3人)、家庭间(3位成人)、豪华双床间(2位成人)、高级特大号床间(3位成人)、豪华双床间(3位成人)、豪华双床间-可使用游泳池(2位成人),房间布置都到位,服务员也很热情。简而言之,客人在普吉岛甲米蒂瓦娜广场酒店享受的服务与设施会有宾至如归的感觉。再讲究的客人也能在酒店得到满意的服务。

相关条款

入住时间从14:00时退房时间12:00时之前预订取消/预付政策不同类型的客房附带不同的取消预订和预先付费政策请输入您的入住日期并参阅您所需的客房的条款。儿童和加床允许客人携带儿童入住。允许1名12岁以下的儿童,使用现有床铺的收费是每人每晚THB200。允许1名2岁以下的儿童,加1张婴儿床,免费。允许1名年龄较大的儿童或者成人,一张加床收费:每人每晚THB1070。最多容纳:每间客房1张加床/婴儿床。所提出的任何加床或婴儿床的要求均需获得酒店的确认。附加费用不会自动计算在总价中,您需在入住时另行支付。宠物不允许携带宠物入住。团体如果预订客房数超过7间,住宿方将采用不同的政策和额外补充规定。酒店接受的银行卡类型将鼠标悬停在卡片标志上,即可查看更多信息。

好巧网酒店评价分

非常好,88分

酒店优缺点

游客提到的优点:安全适合带小孩,服务好餐厅很棒床铺好干净游客提到的缺点:浴缸没有那么好晚上有蚊子

备注:好巧网综合打分是基于100多个酒店预订网站的评分综合算出。以上是该酒店在几家酒店预订网站上的评分,供您参考。好巧网对酒店特色的分析数据,来自主流的酒店预订网站、旅游社区网站提到该酒店的评论,尽量展示客观中立的分析。查询更多用户评论信息,请访问http://www.haoqiao.cn/Phuket_c3/274779.html

。

现因借款人__________身份证号:__________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。借款种类为现金,借款日期为_____年____月____日,还款日期为______年____月____日前, 特立此据为凭。

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因___________(写借款用途)需向贷款人________借款人民币_______________元整(小写_____元整)。借得款种类为现金,借得款日期为_____年____月____日,还款日期为____年____月____日前, 按时一次性偿还清¥:____________借款加利息。借款利息为: ___%(年利率),特立此据为凭。

借款人:__________(亲笔签名并按手印)

贷款人:__________ (亲笔签名并按手印)

见证人1:_________ (亲笔签名并按手印)

见证人2:__________ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。(注:因借款人超过还款日期不守信用赖账的,需在省级权威媒体向贷款人道歉,借款人按借得款两倍偿还给贷款人。)

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

。

  中新网8月20日电 据美国中文网报道,美国康州、佛州和俄亥俄州当局表示,最近挫败了3起独立的大规模枪击图谋,3名白人男子被捕。

  据报道,公众线报帮助破获了这3起事件,三州警方在15和16日将3名嫌犯逮捕。当局称,3名嫌犯均是20出头的白人男性,他们或在网上发帖、或发短信进行大规模枪击威胁。近日,加州、得州和俄亥俄州相继发生大规模枪击事件,最近几周,多次误报和枪击威胁恶作剧让美国民众心神不宁,要求变更枪支立法的呼声迭起。

  康州

  诺沃克(Narwalk)市警部门称,22岁瓦格肖(Brandon Wagshol)被捕。瓦格肖在诺沃克警方和FBI的联合调查后被捕,FBI收到线报,据称他试图从外州购买大容量的步枪弹夹。

  警方表示,瓦格肖在网上购买了步枪零件,打算自己制造武器,并在“脸书”上展示出“他对进行大规模枪击的兴趣”。诺沃克警方说,在执行搜查令时,警方发现两把注册在瓦格肖父亲名下的枪,多发弹药,防弹衣和其他战术装备。

  佛州

  沃卢西亚郡(Volusia)治安官办公室称,25岁代托纳比奇(Daytona Beach)市居民威克斯(Scott Wix)16日被捕,被控威胁要进行大规模射击。警方接到多条线报,据称威克斯发送了多条短信,阐述了他进行大规模枪击的计划。警方没有说明威克斯向谁发送了这些短信。

  警长办公室描述威克斯的短信时称,“学校是个弱的目标……我更有可能向3英里外的一大群人开枪……我想打破连续杀人时间最长的世界纪录。”据称另一条短信写道:“但是要能杀死100人会很好。我已经有一个地点选择(笑出眼泪的emoji)很坏吗?”

  警方在一份声明中表示,威克斯称他并未拥有枪支,但“对大规模枪击事件着迷”。警方同时发布了威克斯被捕时的视频。

  俄亥俄州

  FBI称,警方接到一个线报,一名男子在网上发视频称,自己是犹太社区中心枪击事件的枪手。虽然该枪击事件并未发生,但警方将该男子逮捕,他是20岁的里尔顿(James P. Reardon)。FBI克利夫兰分部表示,里尔顿于16日被捕,涉嫌电信骚扰和加重恐吓。

  警方称,里尔顿在“图片墙”(Instagram)上发布的视频帖子,标记了扬斯敦犹太社区中心。警方收到该视频线报的当天,在里尔顿父母家执行了搜查令。他随后顺利被警方逮捕,警方还在现场发现了弹药,半自动武器和反犹信息。

。

 

联邦快递明年1月起每周7天都提供快递服务

(综合三十日电)为了满足电子商务市场持续增加的送货需求,美国联邦快递公司(FedEx)将从明年开始,每周送货七天,而且不会收取额外的费用。联邦快递明年1月起每周七天都提供快递服务,以试图跟上网购的繁荣景气。该公司也将取回目前由邮局处理的每天近200万件包裹快递能量,并表示,将SmartPost包裹转移回自有快递网络将使送货司机能将更多的包裹紧密安排规画,提高送货效率。除了每周增加一天送货服务,联邦快递公司还计划配合陆运路线,将更多包裹送到客户的家门口,以降低成本。此外,该公司明年将会有大约200万个包裹,不再委托美国邮局运送。「消费者每周七天在线购物」,联邦快递总裁兼首席运营官苏伯拉曼尼(Raj Subramaniam)表示,因此,网购者以及电商业者对每周七天送货服务的需求不断增加。他强调,每周增加一天送货服务,不会提高收费,也不是针对特定电商业者,而是对小型托运者到大型零售商都一视同仁,提供全面送货服务。联邦快递公司估计,到了2026年,美国小包裹出货量将翻倍,该公司必须提前准备。联邦快递和其竞争对手联合包裹服务公司(UPS)近年来投入资金改善送货管理,并且将所谓的「最后一英里」(Last-Mile,即送到客户家门口)利润较低的送货,外包给美国邮局(USPS)或其它业者。然而,随著网购增加,包裹数量也与日俱增,全美每天国内包裹达到5,000万件。为了因应这个趋势,FedEx及UPS正在调整业务,以增加市场份额及处理周末的送货需求。与此同时,由于亚马逊及沃尔玛等零售商在全美增建仓库,以缩短送货距离,导致联邦快递在全美或全球范围内的空运快递业务量逐渐萎缩。此外,多家零售商建立自己的送货系统,销售订单管理软件公司OrderDynamics营销副总裁迪莫夫(Charles Dimov)说:「联邦快递如果不提供周日送货服务,很有可能被市场淘汰。」UPS目前是每周送货六天,发言人扎卡拉(Glenn Zaccara)表示,该公司「持续评估扩大服务的时机」。美国邮局正在考虑将包裹递送服务扩大到每周七天,目前邮局在周日为亚马逊提供送货服务,并在假日期间为其它业者提供送货服务。

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猜对了吗

教学内容

大象版小学科学三年级(下)第一单元第2课。

教学目标

1、利用实验验证的方法,明白猜想只是一种可能的答案,它和事实并不总是一致。

2、学会使用酒精灯加热的技能,了解材料传热的性能。

3、培养重视实验和证明的科学态度。

教学重点

了解猜想、假设和事实的区别。

教学难点

培养学生重视实验和证明的科学态度。

实验器材

酒精灯、铁架台、纸杯、火柴、水槽、烧杯、镊子、铁棒、纸团、纸条等。教学过程

一、激趣导入

同学们,你们喜欢猜谜语吗?(喜欢)老师今天给大家带来了几个谜语,请大家猜一猜,看谁最聪明。(课件出示谜语)

1、上边毛,下边毛,中间一个黑葡萄。生:眼睛。

2、麻屋子,红帐子,里面住着白胖子。生:花生。

3、身体生来瘦又长,五彩衣裳黑心肠,嘴儿尖尖说黑话,只见短来不见长。生:铅笔。

4、小铁驴,真是好,又不踢,又不咬,屁股后面把烟冒,突突突叫着跑。生:摩托车。这几则谜语你猜对了吗?生:猜对了。

二、猜想和验证

1、大胆猜想

你们想挑战更难的吗?(想)请同学们来猜想。(课件出示猜想问题图)

用酒精灯烧空纸杯的底部,纸杯会烧着吗?

把空纸杯倒扣在酒精灯上,纸杯会烧着吗?

用酒精灯烧装水的纸杯底部,谁能烧开吗?

生把猜想填入表格,指明说说自己猜想的根据。

2、学习使用酒精灯

我们进行了大胆的猜想,猜对了吗?我们要设法验证

自己的猜想。要验证自己的猜想,就必须学会安全使用酒精灯。请同学们打开书第6页,阅读左下角的“安全使用酒精灯。”

生阅读。

请同学们在小组内互相说一说怎样安全使用酒精灯。生在小组内互说,指明说一说怎样安全使用酒精灯。

师示范安全使用酒精灯。

3、验证

小组讨论所用实验材料。小组组长到材料超市选取实验材料,分组实验,是巡视指导,生填写实验报告单。汇报实验结果。

4、我进步我成功

课件出示:在几次猜想中,我的猜想与实验结果:

总是不一样()嘿,没关系!

有时不一样()呵,再努力!

总是一样的()瞧,我真棒!

指名说,是鼓励。

5、你在猜想与验证活动中取得了哪些收获?

师:猜想只是一种可能的答案,它和事实并不总是一

样的,要想知道猜想是否正确,必须设法验证。

三、课外延伸

我们再来进行两个猜想。

杯子竖直扣入水底,塞在杯底的纸团会湿吗?

用一张普通的纸,裁成长条,以螺旋状紧绕在一根铁棒上,然后火柴去烧铁棒上的纸条,纸条会被烧着吗?

想知道你猜对了吗?(想)怎么办?

。

  原标题:印尼狮航空难一周年 美国对波音的调查怎么样了?

  参考消息网10月29日报道 外媒称,10月29日是印尼狮航737 MAX客机坠毁一周年纪念日,美国议员表示,在“99.9%的美国公众”和决策者确信客机是安全的之前,波音该机型客机不会复飞。

  新加坡《联合早报》网站10月28日援引路透社报道称,波音公司首席执行长米伦伯格将连续两天出席听证会。此前,有多份报告发现,波音在设计737 MAX客机时,没有充分考虑飞行员应该如何应对驾驶舱紧急情况。

  据报道,美国联邦航空管理局已经花了数月时间,对波音提交的关于关键安全系统软件的升级、培训以及系统变化等进行评估。但报道称,预计最早要到12月才可能让该机型飞机复飞。

  美国参院商业委员会主席、共和党参议员威克说,“除非99.9%的美国公众和政策制定者相信,它(737 MAX客机)是绝对安全的,否则这一型号飞机不会复飞。”

  威克称,他还计划在推进调查期间,加强波音与美国联邦航空管理局的沟通,并在听证会期间加强“监管机构与制造商之间的关系”。

。

  原标题:川普催促美国实行负利率 美联储官员不乐意

  中新网11月21日电 综合报道,尽管美国总统川普一再呼吁在美国实行负利率政策,但20日公布的一项会议记录显示,美联储的官员们并不乐意通过这种方式来刺激经济。

资料图�:美联储主席鲍威尔中新社记者 陈孟统 摄资料图:美联储主席鲍威尔中新社记者 陈孟统 摄

  据报道,一直以来,川普声称欧洲与其他地区采取的负利率措施,为这些国家带来了竞争优势,于美国不利,因而呼吁美联储降息。

  然而,这份美联储10月份的会议记录明确显示,美联储不仅在当前美国经济仍在增长的情况下不愿采用负利率,也对经济陷入衰退时采用负利率手段刺激经济深表怀疑。

  根据会议记录,美联储的17位决策委员会成员都认同,将借款成本推低至低于零“在美国似乎并不是有吸引力的货币政策工具”。

  决策者们也不热衷于通过购买国债来控制长期利率,一些决策者认为这样做会被视为干涉财政部对国家债务的管理,其他决策者则担心这会使美联储资产负债表膨胀。

  美联储公开市场委员会(FOMC)利率决策委员在会中陈述,在一些已尝试执行负利率的国家,证据显示效果好坏并存。因此委员们认为,近期内进一步降息“并无必要”,除非迎来重大变化。

  据悉,自2019年7月以来,美联储已3次降息,但此前从2015年末开始连续9次加息。川普曾多次指责美联储没能更大幅度地降息,影响了他任期内的经济增长。

 

。

HTML5取代Android和iOS应用程序?

大量新生移动设备的兴起,改变了互联网的未来。在技术的发展上,HTML5会取代App应用吗?或者说能够在多大程度上取代呢?在HTML5规范中,已经加入了相机、磁力罗盘、GPS信息的支持。很多新兴浏览器也已经开始支持这些新特性。能否用一个统一的HTML5来替代android和ios并行开发的双重成本呢?以下译自MichaelMahemoff的一篇文章,详细分析了HTML5能否取代Android和iOS应用程序。

介绍

移动应用程序(App)和HTML5都是目前最火的技术,二者之间也有不少重叠之处。在移动设备浏览器里运行的html5的web页面,也可以重新打包成不同平台上运行的app。目前很多浏览器都有很好的跨平台支持,(译注:firefox居然可以在android中使用和windows下同样的浏览器内核),HTML5的web方案,对开发者来说更为方便。完成一次,即可多平台使用。但这确实可行吗?仍然有许多必要原因,使得开发者选择了app开发。很明显,很多人已经在这么做了。本文将详细分析两种方案的优劣。

功能丰富

正方:App里可以开发出更丰富的功能

我们把移动功能分成两类。程序本身和程序与系统的结合。比如android里,加入widget图标或者通知提醒之类的。App对这两者都没问题。不用多说,这是肯定的。

反方:APP是挺强,但Web也正在迎头跟进

确实很多原生app实现的功能是HTML5望尘莫及的。不管你的web做的再牛,如果停留在一个没有摄像头支持的沙盒中,很多场合还是玩不转。幸运的是,现在没有这样的沙盒限制了。如果你需要你的web照相片,可以做一个负责照像的app,再把你的web打包进这个应用里面。开源的PhoneGap框架是这么干的。这样widget,手机提醒也都没问题了。

但这种混合开发的问题在于,增加了复杂性,而且不象传统web那样可以直接在浏览器里运行。这个问题短时间内恐怕没辙。好在现在网络标准在不断的高速扩充,先进的浏览器也在一直跟进。Android3.1已经支持camera了。iOS浏览器也支持WebSocket和设备方向检测了。

总得来说,移动设备在发展,而web也同样在快速变化。桌面浏览器本身,有5家主要浏览器开发商在改进现有标准,丰富新的功能。所以原生App在快速前进,同时,web也在缩小差距。

运行效率

正方:原生APP速度更快

原生APP没有瓶颈,而且可以直接调用GPU加速、使用多线程。

反方:现如今Web已经快多了,而且多数应用也用不着那么快。

这说法有点落伍了。Chrome发布之时带来的JavascriptV8,给Web速度带来的飞跃。而现在,计算速度变得更快了:

图片处理引擎已经使用web加速。现在硬件加速也已经开始应用了。

要开发3D游戏的就不用抬杠了,但对于平而来说,新闻、邮件、时间管理、社交网络,这些用Web都够用了。试试SteveSouders的手机性能测试工具。另外,越来越多的框架结合WebGL,可以发挥OpenGL的优势了。比如ImpactJS,帮助开发JS游戏。

开发感受

正方:原生APP好写

原生APP使用强壮的程序语言(Java,ObjectiveC,C++)。适合写复杂程序,经过历史验证,API丰富。在桌面环境可以方便的用模拟器测试。而Web程序的runtimes和乱七八糟的各路浏览器让人头大。

反方:一般都是Web更简单,特别是需要兼容不同设备的时候。

Web最初的功能只限于文档展示,而不是程序应用,貌似最近俩星期才有了JS。但有了JS后,web的世界马上就不一样了。更何况web不只是静止的,HTML5,CSS3,EcmaScriptHarmony(谁知道这是什么?)都给开发者极大帮助。你是喜欢C++,java,JavaScript,那你的个人爱好,也是基于你已经攒下的代码。但是现在没人能否认JavaScript也和前者站在同一擂台上。

浏览器/runtime的互不兼容(碎片),反过来看做APP也是一样。用Java写了Androidapp,然后又要面对iOS的ObjectiveC。如果能写一个程序,马上能在Android和iOS上运行,多省事啊。这咱还没提WebOS,BlackBerry,WindowsMobile呢。当然,这是理论上的。要是想让程序在每个平台都跑得很漂亮,得做不少调试和妥协。这对很多原生APP也是一样的。不同OS版本,不同的设备。。

所谓的Web碎片化,一直都是如此。但好消息是现在已经有很多不错的解决办法。Modernizr库,用得好的话,可以帮你兼容一大批主流设备,不管是啥系统,哪个牌子的。看看我们2011年的GoogleIO演示。

用户体验

正方:原生APP更切合原有平台

操作感受的定义之一,就是用户希望在你的程序里,用与系统连贯统一的方式来操作。不同的平台,都有一些约定俗成的习惯。比如长按按钮会有啥反应。你不能指望用一套统一的HTML5App去满足所有用户。

此外,整个平台的操作感受都由用平台自有的软件库协调。直接调用平台工具包就能直接免费获得完整支持。

反方:我们Web有自己的传统,你要特想做原有平台那种感觉的web,也一样能做出来

前面说了,Web开发的方式,是先做一个大体适合所有平台的版本,然后再针对不同平台不断改进。当这些改进主要是针对功能时,你可以选择几个你最关心的平台做优化。类似于浏览器检测。技术论坛里的悲催技术员们,经常抱怨这事。太多不同的浏览器版本了。不过如果你优先关注两三种主流平台,是值得为他们多花点时间做做优化。

web本来就有自己的操作感受。我们也可以说,不同的默认浏览器以及运行环境造就了独特的”Web感受”。从更广的角度看,这本身就是一种用户公认的方式。此外,还有很多成功的案例并不遵循移动设备的原生操作习惯,人家也成功了。想想你最喜欢的手机游戏的界面?很多更传统的app也是一样,比如Twitter客户端。

传播途径

正方:原生应用更容易接触客户

象GooglePlay和AppleStore这样的app发布机制这几年势不可挡,推动了整个移动行业。每个程序员都能在市场里发布自己的应用。用户都挤在市场里浏览,搜索,接受推荐。不仅如此,只要你的程序够好,现有用户的打分会帮助你说服更多新的客户。

反方:其实web才容易接触到客户

。.05was chosen to indicate that a trend was significant.The frequency of each allele was also calculated as function of geographic regions of isolation following grouping non-redundant A/H1N1/09 HA protein sequences according to the regions where they were isolated(Asia,Europe,North America,South America,and Oceania)and tested using the v2test. Binding energy analysis

The MOE(molecular operating environment)program[27] was used to calculate the binding energy between A/H1N1/ 09HA and its cellular receptor based on a known binding structure(PDB ID:3GBN,X-ray diffraction)composed of the A/South Carolina/1/1918HA and a receptor analog. First,we partially minimized the complex by relaxing the ligand and the side chains within10nm from the ligand, while keeping all other atomsfixed.

Following calculation of energies,factor analysis(FA) and multiple regression analysis(MRA)were employed to generate an LRE-like equation[28,29]:

D G b FEB

ðÞ¼x1D G b vdWþx2D G b eleþx3D G b solvþx4D G b n

D G b¼D G complexþD G proteinþD G ligand

In this equation,D G(FEB)stands for the free energy of binding,D G vdW,D G ele,D G solv,and D G n stand for the van der Walls contribution,the electrostatic contribution,the polar solvation contribution,and the nonpolar solvation contribution to the binding process,respectively,where w1, w2,w3,and w4are weight factors,and D G b represents binding energy(i.e.,energy difference between ligand/ receptor complex and free protein and ligand).Results

Point mutation analysis

To specify the differences between the HA molecules of the A/H1N1/09virus and recently circulating seasonal IAVs,we generated a consensus sequence from704 A/H1N1/09HA amino acid sequences deposited in the GenBank(as of April21,2010)and a consensus sequence from the HA proteins of4seasonal H1N1IAV strains that had been recommended by WHO for production of influ-enza vaccines,and compared these two groups of HA sequences by aligning both consensus sequences and sev-eral individual HA sequences from each group(Fig.1).

As shown in Fig.1and Table1,while the overall dif-ference between the consensus sequences of seasonal H1N1and A/H1N1/09HA proteins was as high as19.61% (111/566),the cleavage sites of HA,at which the HA0 protein is recognized by specific protease(s)and enzy-matically cleaved into HA1and HA2,thereby becoming activated in mediating the entry of IAVs into host cells, remain identical(PSIQSR;GLFGAI)among the strains analyzed.Meanwhile,the Asp204,Asp239,Gln240,and Gly242residues at the RBS that are responsible for the viral attachment to the host cell receptor,a critical step for viral entry,were also identical between the two consensus sequences.It is of note that the four positions(i.e.,204, 239,240,and242)have been found previously to be involved in the specific binding capacity of HA to the host cell receptor[30–32].Whereas,amino acid variations were found at a number of positions at the HA RBS among different seasonal H1N1IAVs and the A/H1N1/09viruses, including positions150,152,206,207,210,211,and212. Whether variations at these sites could impact the infec-tivity of an influenza virus to a specific host needs to be further investigated biologically.

To address the differences in the antigenic properties between A/H1N1/09and human seasonal IAVs,previously proposed antigenic epitope regions were comparatively analyzed.In this context,two groups of epitope regions, namely,the highly conserved regions and the highly vari-able regions[33],were analyzed,respectively.As shown in Fig.1and Table2,four highly conserved epitope regions (1–4),located in HA2,involving residues345–354, 359–376,394–411,and436–453,were all identical between A/H1N1/09isolates and seasonal H1N1viruses. In contrast,the highly variable regions,which lie in the HA1globular head and include sites(residues86–91),Sa (residues141–142,170–174,and176–181),Sb(residues 201–212),Ca1(residues183–187,220–222,and252–254) and Ca2(residues154–159,and238–239)[34],showed dramatic changes in A/H1N1/09isolates when compared with those in seasonal IAVs(H1N1).Such changes might

Fig.1Alignment of HA amino acid sequences of human seasonal IAVs and their consensus sequence in comparison with the pandemic A/H1N1/09IAVs and their consensus sequence.Residues different from the consensus HA amino acid sequence(top line)of human seasonal H1N1IAVs are shown,and dots stand for identical residues. The sequences were numbered according to pandemic2009H1 numbering.Boxed residues indicate the Asn-X-Thr/Ser motifs of glycosylation sites

Table1Comparison between the new A/H1N1/09isolates and seasonal H1N1IAVs at HA0cleavage site and RBS

Functional sites Virus Residues

The cleavage site Human seasonal PSIQSR;GLFGAI

A/H1N1/09PSIQSR;GLFGAI

Difference:0

RBS(in HA1)Residue positions(pandemic2009H1numbering)

149–152204–212235–242

Human seasonal V S A S DQ RA LY HTE PKVRDQEG

A/H1N1/09V T A A DQ QS LY QNA PKVRDQEG

Difference:2/4Difference:5/9Difference:0

provide a molecular explanation for the observed lack of cross-protection from previous infection or vaccination of seasonal IAVs against the novel A/H1N1/09virus.

Since point mutations could cause emergence or loss of Asn-X-Thr/Ser motifs and thereby,attachment or loss of N-glycans,respectively,leading to alteration of the anti-genicity and receptor specificity of HA,we analyzed the glycosylation sites on H1HAs by further examining both consensus sequences of the seasonal H1N1and the novel pandemic H1N1,which revealedfive identical glycosyla-tion sites(28,40,104,304,498,pandemic2009H1 numbering)between the two consensus sequences,as shown in Fig.1.In marked contrast,the A/H1N1/09stains contained amino acid mutations predicted to lose three

Table2Comparison between A/H1N1/09isolates and seasonal H1N1IAVs at antigenic epitope regions Epitope region Virus Residues and positions b

Highly conserved regions(in HA2)1345–354in HA2

Human seasonal GLFGAIAGFI

A/H1N1/09GLFGAIAGFI

Difference:0

2359–376in HA2

Human seasonal TGMVDGWYGYHHQNEQGS

A/H1N1/09TGMVDGWYGYHHQNEQGS

Difference:0

3394–411in HA2

Human seasonal NKVNSVIEKMNTQFTAVG

A/H1N1/09NKVNSVIEKMNTQFTAVG

Difference:0

4436–453in HA2

Human seasonal WTYNAELLVLLENERTLD

A/H1N1/09WTYNAELLVLLENERTLD

Difference:0

Highly variable regions(in HA1)a Cb86–91in HA1

Human seasonal L ISK(R)E S

A/H1N1L STAS S

Difference:4/6

Sa141–142in HA1170–174in HA1176–181in HA1 Human seasonal PN G K NGL P N LSKS

A/H1N1/09PN K K GNS P K LSKS

Difference:0Difference:4/5Difference:1/6 Sb201–212in HA1

Human seasonal NIG D(N)Q R(K/M)A(T)LY HT(K)E

A/H1N1/09TSA DQ QS LY QNA

Difference:8/12

Ca1183–187in HA1220–222in HA1252–254in HA1 Human seasonal A(V)N N K E SS H EPG

A/H1N1/09I N D K G T S R EPG

Difference:3/5Difference:1/3Difference:0 Ca2154–159in HA1238–239in HA1

Human seasonal S H N G E(K)S RD

A/H1N1/09P H A G AK RD

Difference:4/6Difference:0

a Amino acid sequences for all highly variable epitope regions listed in this table are given using the consensus sequences generated for A/H1N1/09isolates and seasonal H1N1IAVs,respectively,as presented in Fig.1.Variations in individual sequences as compared with the consensus sequence are given in parenthesis

b Numbering according to pandemic2009H1numbering

glycosylation sites at position71,142,and177.It is of note that the two highly conserved glycosylation sites(i.e.,142 and177)have been found previously to be involved in the antigenic properties of H1N1IAVs,which was within or around the Sa site.Meanwhile,we found that the A/H1N1/ 09stains carried amino acid mutations predicted to acquire one potential glycosylation site at position293,raising the issues whether such glycosylation does occur at the site and alters the antigenicity and receptor specificity of2009 influenza A(H1N1)virus.

Point-to-point analysis of mutations in the HA protein was performed among704isolates of the novel A/H1N1/09 virus.As shown in Tables3and4,13out of566positions in the entire HA molecule displayed variations among704 non-redundant A/H1N1/09HA sequences(variation fre-quency was higher than2),including5positions at RBS, and11positions in the highly variable epitopes,and all other variations were shown in the supplement data Table S1.Specifically,the amino acid change at position342 (Gln342Leu)was at the cleavage site of one A/H1N1/09 isolate[A/Guangdong/03/2009(H1N1)].Whether such a change would influence the interaction of HA0with pro-teolytic enzyme(s)and consequently,the cleavage activity, remains unclear.It requires further investigation to clarify whether mutation Asp239Glu in two isolates[A/Paris/ 2591/2009(H1N1)and A/New Jersey/01/2009(H1N1)] was relevant to host adaptation,since Glu239had been previously found to be associated with acquisition of SA a-2,6Gal binding specificity[30,35].Most noteworthily is the high frequency(72.02%)of Ser220Thr mutation in the Ca1epitope,strongly indicating existence of a positive selection or,at a lesser likelihood(due to lack of other high-frequency mutated positions in the HA),more than one origin of the new A/H1N1/09viruses.

Structural modeling

To better characterize the variations in A/H1N1/09HA,we sought to map the altered amino acids to a predicted three-dimensional(3-D)HA structure.Wefirst BLAST searched the PDB database for deposited HA molecules with the

highest sequence homologies to A/H1N1/09HA.Three sequences,together with their previously X-ray determined 3-D structures,of similarities higher than80%were selected,which were1RUY[HA of A/swine/Iowa/15/30 (H1N1)],1RVT[HA of A/swine/Iowa/15/30(H1N1) complexed with receptor analog LSTC]and1RV0[HA of A/swine/Iowa/15/30(H1N1)complexed with receptor analog LSTA][32].Using the selected molecules as tem-plates,onto which the new A/H1N1/09HA consensus sequence was modeled for50times,a predicted3-D structure of the A/H1N1/09HA was obtained,visualized with Jmol,and demonstrated in Fig.2.

As shown in Fig.2,the structure of A/H1N1/09HA monomer was similar to those of other published HAs,as expected,with a globular head containing the RBS,a cleavage site,a trans-membrane domain,and an a-helical stalk.Variations found in various isolates of the A/H1N1/ 09HA,as well as differences between the HA molecules of human seasonal IAVs and the new A/H1N1/09virus, and the glycosylation attachment sites were mapped on the3-D structure as shown in distinct colors in Fig.2. These variations and different residues were mainly located in the HA1fragment,most of which lied in antigenic epitopes.

Table3Amino acid variations in the HA protein sequence among A/H1N1/09isolates

Positions a Primary

residue

Frequency

(%)

Variation(s)Variation

frequency(%) HA136V97.73I 2.13

L0.14 49L96.45I 3.41

X0.14 103D97.17E0.14

G 2.41

M0.28 114D95.74N 4.12

X0.14 145S95.32M 4.40

X0.28 220S26.99A0.14

T72.02

X0.85 222R96.17G0.14

K 3.13

S0.28

T0.14

X0.14 239D88.78E 4.69

G 3.55

N0.99

X 1.99 310Q95.31H 4.26

X0.43 314P97.30S 2.70

338V94.88I 4.69

S0.43

HA2391E88.78G0.71

K10.51 428V97.30I 2.56

X0.14

a Positions listed according to pandemic2009H1numbering

b‘‘X’’stands for any amino acid

Site-by-site analysis

To address the significance of the mutations identified in A/ H1N1/09HA,we conducted positive selection analysis on a site-by-site basis,as positive natural selection could drive the increase in prevalence of advantageous traits,which may lead to a new pandemic virus.In this study,the ratio between non-synonymous(dN)and synonymous(dS)substitutions were used to indicate selective pressure on each codon.According to Yang et al.[24],when the dN/dS value on a certain codon is greater than1and tested to be significant by the Bayes test,the site is considered to be under positive selection;and in con-trast,the site would be recognized as being under negative selection in the case if the dN/dS value is significantly smaller than1.Using the HyPhy program[23],our positive selection analysis revealed that34codon sites showed dN/dS[1with statistical significance(Bayes factor[1)(Fig.3).To mini-mize possible errors such as those caused by biased sampling, we chose to use a very conservative strategy in the identifi-cation of the site mutations under positive selection by selecting only those codon sites with the highest Bayes fac-tors,which accounted for the leading5%of all codon sites with a Bayes factor[1.Consequently,these procedures led to identification of two sites,namely,positions220and239 (pandemic2009H1numbering,equivalent to206and225, respectively,according to the H3numbering)in the HA1 protein.Notably,the residue220lied in the Ca1epitope region in the head of HA1,and residue239was found to be included in the RBS of HA1.It is of interest that previous studies have suggested that both regions are relevant to the determination of the severity and transmissibility of an IAV [35],raising the question whether the mutations at these sites could favor the prevalence of the novel virus.

Variation at position220

Particularly noteworthy was the relatively high frequencies of S220and T220present in the HA of A/H1N1/09

Table4Amino acid variations in functional regions of A/H1N1/09 isolates

Functional region Residue positions a Variations a

The cleavage site Between HA1and HA2None

RBS(in HA1)149–152151

204–212204

235–242238,239,240

Highly conserved epitopes(in HA2)345–354None 359–376None 394–411None 436–453None

Highly variable epitopes(in HA1)

Cb86–9190

Sa141–142None

170–174172

176–181179

Sb201–212202,203,204 Ca1183–187None

220–222220,222

252–254252

Ca2154–159None

238–239238,239

a Positions listed according to pandemic2009H1

numbering

Fig.2Mapping of mutations onto the3-D structure of HA monomer. Side views of the HA monomers are shown.Each has the highly conserved epitope regions in HA2colored violet and the highly variable epitope regions in HA1colored cyan.a the reference HA monomer,and b,c the HA monomers with mutations mapped.In b,regions colored blue stand for mutations detected among the A/H1N1/ 09viruses;in c,regions colored yellow demonstrate differences between the human seasonal influenza virus and this novel virus,and the amino acid variations at residues220and239and the glycosylation sites were numbered(Colorfigure online)

isolates,prompting us to ask whether they were a result of selection during the course of evolution.To address this issue,we further analyzed the frequency distribution of S220and T220in the HA among isolates of the novel A/H1N1/09virus as a function of isolation time and geo-graphic regions.When 704HA sequences derived from A/H1N1/09viruses isolated between the period of March 30,2009and April 21,2010,were grouped according to their isolation time,a total of 507isolates (72%)with T220were identified,and the percentage of S present at position 220was found to gradually decline,while the frequency of T220increased over time (Fig.4).To test the significance of the descending trend of S220,Kendall test and a linear model were used,and results revealed a Kendall test

P value of 2.503910-5and a descending rate of 0.06655with a P value of 9.84910-5in the linear model analysis.Both tests confirmed the significant descending trend of the residue S at position 220,with T220gradually becoming prevalent in the infected population.In contrast,no sig-nificant difference in the frequency distribution of S220versus T220among HA molecules derived from viruses isolated from Asia,Europe,North America,Oceania,and South America,with a v 2test P value of 0.2801,indicating that the changes in the frequencies of S220versus T220found in the above study probably were not geographic region-specific and might have been occurring worldwide.We next sought to investigate whether S220and T220were present in the HA molecules of previously isolated swine IAVs and seasonal human H1N1viruses.We down-loaded 272non-redundant HA protein sequences of North American classical swine flu virus (H1subtype)from the NCBI influenza virus sequence database and analyzed the frequency of amino acids present at position 220.Interest-ingly,251out of the 272sequences were found to have S220,as opposed to that only 20isolates carried T at the position 220.Moreover,all of the 20strains with T220were H1N1subtype swine influenza A viruses,among which 14were isolated from Tennessee in 1976,1977,and 1978,and the remaining six strains were isolated sporadically from several other US states.On the other hand,the frequency distribu-tion in 1767HA sequences derived from human-infected H1subtype IAVs isolated before the 2009pandemic showed an S220:T220ratio of 1760:7.Variation at position 239

Since our point mutation analysis found a notable vari-ability of position 239in HA,we analyzed the

frequencies

of different residues at the position in HA of704A/H1N1/09 isolates.Our analysis found that aspartic acid(D)was present at a frequency of88.78%(623/704),glycine(G)at 3.55%(25/704),and glutamic acid(E)at4.69%(33/704), with the remaining residues undetermined.Possible impact of mutations at this site on receptor binding was examined by calculating the minimal energy using the MOE(Molec-ular Operating Environment)program,and the result showed that the binding energy between receptor analog and wild type HA(D239)was-29.506kcal/mol,while for mutants G239and E239,it was-17.027and-16.445kcal/ mol,respectively,indicating possibly weakened receptor-binding capacities of the mutants.

Discussion

Our current study analyzed the site variations in the HA protein sequences among the novel A/H1N1/09viral iso-lates,as well as those between human seasonal influenza viruses and A/H1N1/09.3-D structure of the HA was also modeled,and identified point mutations were analyzed to predict their potential significance in molecular evolution and function of the pandemic virus.

In this study,the point mutation analysis showed that the A/H1N1/09strains carry amino acid mutations that might lead to acquisition of one glycosylation site(position293) and loss of three glycosylation sites(position71,142,and 177).It has been reported that variation in glycosylation is used by influenza viruses to interfere with surveillance by the host immune system.Acquisition of a glycosylation site masks the protein surface from antibody recognition because the glycans themselves are host-derived,and hence considered as‘‘self’’by the human immune system[36,37] Meanwhile,upon the addition of glycosylation to this region has been thought to slow down yearly antigenic drift,pre-sumably because glycosylation shielded this region from the antigenic pressure of antibodies[18,36].Recently,Wei et al.and Xu et al.have evaluated the cross-neutralization of pandemic1918and2009H1N1influenza viruses and found that they were both resistant to antisera directed to a rela-tively recent seasonal influenza virus of the same subtype [18,38].Interestingly,pandemic2009H1N1influenza virus (A/California/04/2009),like A/South Carolina/1/1918virus, does not have any glycosylation in or around the Sa site in HA and hence,the epitope is exposed for antibody recog-nition.They suggest that these N-glycans of the RBD and antigenic epitope regions may play roles in evading the human immune response and viral evolution in humans[18]. The probability of glycosylation of the predicted asparagine residues293above mentioned in our current study,and the role of the acquisition(position293)or loss of glycosylation (position71)in modulating immune recognition and its influence on viral evolution,is under investigation in our laboratory.

By mapping the variations in the HA protein sequence, we identified that the distinction between the HA antigenic specificities of the human seasonal influenza virus and the novel A/H1N1/09virus mainly lie in the HA1epitope regions,which might explain the lack of effective cross-protection by previous seasonal IAV vaccines or infections and the highly transmissible property of the A/H1N1/09 virus.

A key notion derived from this study is the possibility that two HA1sites in the novel A/H1N1/09virus,namely, residues220and239,might be positively selected during its evolution.Interestingly,residue239lies in a region possibly involved in both receptor binding and determi-nation of antigenic specificity[30–32,34].It has been previously reported that when residue239changes from Gly to Glu,the virus becomes more adaptive to human host [30].Recently,Chen et al.reported the D225G(H3num-bering,D239G according to2009pandemic H1number-ing)substitution in7(12.5%)of57patients with severe disease,and in0(0%)of60patients with mild disease,and the D225E mutation was identified in one patient with severe disease,by direct analysis of polymorphisms in126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N12009virus from117 clinical specimens in Hong Kong[39].Our current study revealed that the vast majority of pandemic A/H1N1/09 isolates carried D239(88.78%)and yet a small fraction of the A/H1N1/09isolates had glutamic acid(E,4.69%)and glycine(G,3.55%)at this position.Free binding energy analysis suggested that a D239E/G mutation tended to decrease the affinity of the H1subtype IAV to the sialic acid receptor.It remains to be determined,whether the identified positive selection of the D239E/G mutation is functionally significant during the spreading of the A/H1N1/09virus.

Residue220lies in the Ca1epitope region.In all A/H1N1/09isolates obtained as of August2009,T220was present at a frequency of72.02%,whereas the frequency of S220was26.99%.It is of note that both T220and S220 were present in H1subtype classical swine IAVs,from which the HA of the pandemic A/H1N1/09was originated, and that the frequency of T220was far lower than that of S220in these swine IAVs.Our analysis on the dynamic change of T220versus S220demonstrated an ascending trend of the percentage of T220and a descending curve of S220during the course of the pandemic spreading,sug-gesting that T220might have been positively selected over S220.Speculatively,T220was thenfixed through natural or other selection at the peak of this pandemic and favored the transmissibility of the new virus.Interestingly,com-pared with serine,amino acid threonine carries an extra

methyl group and therefore displays a different polarity than serine.Whether such a difference in the polarity could contribute to altering recognition and binding capacity between the antigenic epitope and specific antibodies and thereby should be taken into consideration when develop-ing more effective vaccines and therapeutic drugs remains to be further investigated.

Acknowledgments This study was supported by the National Natural Science Foundation of China-Guangdong Province joint grant (U0632002),a grant from the State Major Infectious Disease Research Program(China Central Government,2009ZX10004-213), a Key Science and Technique Research Project of Guangdong Province(2009A020101006,2009B020600001),National High-Tech R&D Program(863Program)(Ministry of Science and Technology, China,2006AA02A223,2007AA09Z448,2007AA09Z431),National Science and Technique Research Program for public welfare appli-cations(201005022),and a Key Project of Science&Technology Planning of Guangdong Province(2007A03260001). References

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。

普吉岛甲米蒂瓦娜广场酒店(DeevanaPlazaKrabi)

舒适度作为普吉岛甲米蒂瓦娜广场酒店所有的客房首要标准,一切设施都以此为目标,一定不会让您失望。酒店宽敞的客房,配有住客评分分数来自522条评语等设施,让您瞬间忘记旅途的疲倦。

中文名称普吉岛甲米蒂瓦娜广场酒店英文名称DeevanaPlazaKrabi酒店星级4星级房间数量213酒店地址186Moo3,AonangSoi8,Aonang,Muang,81180奥南海滩,泰国

【好巧网解读】4大卖点

1.很安全2.员工热情有礼貌,一部分客人带着两岁多的女儿,他们对小孩也很好3.酒店还设有池畔酒吧和24小时客房服务4.床也很舒服

酒店的图片

酒店房型房价介绍

每个客房都配有阳台,电话,DVD播放机,视频游戏,有线频道,平面电视,保险箱,空调,熨斗,书桌,熨衣设备,客厅角,瓷砖/大理石地板,衣柜/衣橱,淋浴,吹风机,浴袍,免费洗浴用品,卫生间,浴室,拖鞋,迷你吧,冰箱,电烧水壶,唤醒服务,希望能让客户在入住时更加愉快惬意。酒店的房型有多种选择,提供了豪华特大号床间-可使用游泳池(3位成人)、高级双床间(2位成年人、家庭间、豪华双床间-可直通泳池(3位成人)、高级双床间(3名成人)、豪华特大大床房(3人)、家庭间(3位成人)、豪华双床间(2位成人)、高级特大号床间(3位成人)、豪华双床间(3位成人)、豪华双床间-可使用游泳池(2位成人),房间布置都到位,服务员也很热情。简而言之,客人在普吉岛甲米蒂瓦娜广场酒店享受的服务与设施会有宾至如归的感觉。再讲究的客人也能在酒店得到满意的服务。

相关条款

入住时间从14:00时退房时间12:00时之前预订取消/预付政策不同类型的客房附带不同的取消预订和预先付费政策请输入您的入住日期并参阅您所需的客房的条款。儿童和加床允许客人携带儿童入住。允许1名12岁以下的儿童,使用现有床铺的收费是每人每晚THB200。允许1名2岁以下的儿童,加1张婴儿床,免费。允许1名年龄较大的儿童或者成人,一张加床收费:每人每晚THB1070。最多容纳:每间客房1张加床/婴儿床。所提出的任何加床或婴儿床的要求均需获得酒店的确认。附加费用不会自动计算在总价中,您需在入住时另行支付。宠物不允许携带宠物入住。团体如果预订客房数超过7间,住宿方将采用不同的政策和额外补充规定。酒店接受的银行卡类型将鼠标悬停在卡片标志上,即可查看更多信息。

好巧网酒店评价分

非常好,88分

酒店优缺点

游客提到的优点:安全适合带小孩,服务好餐厅很棒床铺好干净游客提到的缺点:浴缸没有那么好晚上有蚊子

备注:好巧网综合打分是基于100多个酒店预订网站的评分综合算出。以上是该酒店在几家酒店预订网站上的评分,供您参考。好巧网对酒店特色的分析数据,来自主流的酒店预订网站、旅游社区网站提到该酒店的评论,尽量展示客观中立的分析。查询更多用户评论信息,请访问http://www.haoqiao.cn/Phuket_c3/274779.html

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现因借款人__________身份证号:__________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。借款种类为现金,借款日期为_____年____月____日,还款日期为______年____月____日前, 特立此据为凭。

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因___________(写借款用途)需向贷款人________借款人民币_______________元整(小写_____元整)。借得款种类为现金,借得款日期为_____年____月____日,还款日期为____年____月____日前, 按时一次性偿还清¥:____________借款加利息。借款利息为: ___%(年利率),特立此据为凭。

借款人:__________(亲笔签名并按手印)

贷款人:__________ (亲笔签名并按手印)

见证人1:_________ (亲笔签名并按手印)

见证人2:__________ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。(注:因借款人超过还款日期不守信用赖账的,需在省级权威媒体向贷款人道歉,借款人按借得款两倍偿还给贷款人。)

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

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  中新网8月20日电 据美国中文网报道,美国康州、佛州和俄亥俄州当局表示,最近挫败了3起独立的大规模枪击图谋,3名白人男子被捕。

  据报道,公众线报帮助破获了这3起事件,三州警方在15和16日将3名嫌犯逮捕。当局称,3名嫌犯均是20出头的白人男性,他们或在网上发帖、或发短信进行大规模枪击威胁。近日,加州、得州和俄亥俄州相继发生大规模枪击事件,最近几周,多次误报和枪击威胁恶作剧让美国民众心神不宁,要求变更枪支立法的呼声迭起。

  康州

  诺沃克(Narwalk)市警部门称,22岁瓦格肖(Brandon Wagshol)被捕。瓦格肖在诺沃克警方和FBI的联合调查后被捕,FBI收到线报,据称他试图从外州购买大容量的步枪弹夹。

  警方表示,瓦格肖在网上购买了步枪零件,打算自己制造武器,并在“脸书”上展示出“他对进行大规模枪击的兴趣”。诺沃克警方说,在执行搜查令时,警方发现两把注册在瓦格肖父亲名下的枪,多发弹药,防弹衣和其他战术装备。

  佛州

  沃卢西亚郡(Volusia)治安官办公室称,25岁代托纳比奇(Daytona Beach)市居民威克斯(Scott Wix)16日被捕,被控威胁要进行大规模射击。警方接到多条线报,据称威克斯发送了多条短信,阐述了他进行大规模枪击的计划。警方没有说明威克斯向谁发送了这些短信。

  警长办公室描述威克斯的短信时称,“学校是个弱的目标……我更有可能向3英里外的一大群人开枪……我想打破连续杀人时间最长的世界纪录。”据称另一条短信写道:“但是要能杀死100人会很好。我已经有一个地点选择(笑出眼泪的emoji)很坏吗?”

  警方在一份声明中表示,威克斯称他并未拥有枪支,但“对大规模枪击事件着迷”。警方同时发布了威克斯被捕时的视频。

  俄亥俄州

  FBI称,警方接到一个线报,一名男子在网上发视频称,自己是犹太社区中心枪击事件的枪手。虽然该枪击事件并未发生,但警方将该男子逮捕,他是20岁的里尔顿(James P. Reardon)。FBI克利夫兰分部表示,里尔顿于16日被捕,涉嫌电信骚扰和加重恐吓。

  警方称,里尔顿在“图片墙”(Instagram)上发布的视频帖子,标记了扬斯敦犹太社区中心。警方收到该视频线报的当天,在里尔顿父母家执行了搜查令。他随后顺利被警方逮捕,警方还在现场发现了弹药,半自动武器和反犹信息。

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联邦快递明年1月起每周7天都提供快递服务

(综合三十日电)为了满足电子商务市场持续增加的送货需求,美国联邦快递公司(FedEx)将从明年开始,每周送货七天,而且不会收取额外的费用。联邦快递明年1月起每周七天都提供快递服务,以试图跟上网购的繁荣景气。该公司也将取回目前由邮局处理的每天近200万件包裹快递能量,并表示,将SmartPost包裹转移回自有快递网络将使送货司机能将更多的包裹紧密安排规画,提高送货效率。除了每周增加一天送货服务,联邦快递公司还计划配合陆运路线,将更多包裹送到客户的家门口,以降低成本。此外,该公司明年将会有大约200万个包裹,不再委托美国邮局运送。「消费者每周七天在线购物」,联邦快递总裁兼首席运营官苏伯拉曼尼(Raj Subramaniam)表示,因此,网购者以及电商业者对每周七天送货服务的需求不断增加。他强调,每周增加一天送货服务,不会提高收费,也不是针对特定电商业者,而是对小型托运者到大型零售商都一视同仁,提供全面送货服务。联邦快递公司估计,到了2026年,美国小包裹出货量将翻倍,该公司必须提前准备。联邦快递和其竞争对手联合包裹服务公司(UPS)近年来投入资金改善送货管理,并且将所谓的「最后一英里」(Last-Mile,即送到客户家门口)利润较低的送货,外包给美国邮局(USPS)或其它业者。然而,随著网购增加,包裹数量也与日俱增,全美每天国内包裹达到5,000万件。为了因应这个趋势,FedEx及UPS正在调整业务,以增加市场份额及处理周末的送货需求。与此同时,由于亚马逊及沃尔玛等零售商在全美增建仓库,以缩短送货距离,导致联邦快递在全美或全球范围内的空运快递业务量逐渐萎缩。此外,多家零售商建立自己的送货系统,销售订单管理软件公司OrderDynamics营销副总裁迪莫夫(Charles Dimov)说:「联邦快递如果不提供周日送货服务,很有可能被市场淘汰。」UPS目前是每周送货六天,发言人扎卡拉(Glenn Zaccara)表示,该公司「持续评估扩大服务的时机」。美国邮局正在考虑将包裹递送服务扩大到每周七天,目前邮局在周日为亚马逊提供送货服务,并在假日期间为其它业者提供送货服务。

。

猜对了吗

教学内容

大象版小学科学三年级(下)第一单元第2课。

教学目标

1、利用实验验证的方法,明白猜想只是一种可能的答案,它和事实并不总是一致。

2、学会使用酒精灯加热的技能,了解材料传热的性能。

3、培养重视实验和证明的科学态度。

教学重点

了解猜想、假设和事实的区别。

教学难点

培养学生重视实验和证明的科学态度。

实验器材

酒精灯、铁架台、纸杯、火柴、水槽、烧杯、镊子、铁棒、纸团、纸条等。教学过程

一、激趣导入

同学们,你们喜欢猜谜语吗?(喜欢)老师今天给大家带来了几个谜语,请大家猜一猜,看谁最聪明。(课件出示谜语)

1、上边毛,下边毛,中间一个黑葡萄。生:眼睛。

2、麻屋子,红帐子,里面住着白胖子。生:花生。

3、身体生来瘦又长,五彩衣裳黑心肠,嘴儿尖尖说黑话,只见短来不见长。生:铅笔。

4、小铁驴,真是好,又不踢,又不咬,屁股后面把烟冒,突突突叫着跑。生:摩托车。这几则谜语你猜对了吗?生:猜对了。

二、猜想和验证

1、大胆猜想

你们想挑战更难的吗?(想)请同学们来猜想。(课件出示猜想问题图)

用酒精灯烧空纸杯的底部,纸杯会烧着吗?

把空纸杯倒扣在酒精灯上,纸杯会烧着吗?

用酒精灯烧装水的纸杯底部,谁能烧开吗?

生把猜想填入表格,指明说说自己猜想的根据。

2、学习使用酒精灯

我们进行了大胆的猜想,猜对了吗?我们要设法验证

自己的猜想。要验证自己的猜想,就必须学会安全使用酒精灯。请同学们打开书第6页,阅读左下角的“安全使用酒精灯。”

生阅读。

请同学们在小组内互相说一说怎样安全使用酒精灯。生在小组内互说,指明说一说怎样安全使用酒精灯。

师示范安全使用酒精灯。

3、验证

小组讨论所用实验材料。小组组长到材料超市选取实验材料,分组实验,是巡视指导,生填写实验报告单。汇报实验结果。

4、我进步我成功

课件出示:在几次猜想中,我的猜想与实验结果:

总是不一样()嘿,没关系!

有时不一样()呵,再努力!

总是一样的()瞧,我真棒!

指名说,是鼓励。

5、你在猜想与验证活动中取得了哪些收获?

师:猜想只是一种可能的答案,它和事实并不总是一

样的,要想知道猜想是否正确,必须设法验证。

三、课外延伸

我们再来进行两个猜想。

杯子竖直扣入水底,塞在杯底的纸团会湿吗?

用一张普通的纸,裁成长条,以螺旋状紧绕在一根铁棒上,然后火柴去烧铁棒上的纸条,纸条会被烧着吗?

想知道你猜对了吗?(想)怎么办?

。

  原标题:印尼狮航空难一周年 美国对波音的调查怎么样了?

  参考消息网10月29日报道 外媒称,10月29日是印尼狮航737 MAX客机坠毁一周年纪念日,美国议员表示,在“99.9%的美国公众”和决策者确信客机是安全的之前,波音该机型客机不会复飞。

  新加坡《联合早报》网站10月28日援引路透社报道称,波音公司首席执行长米伦伯格将连续两天出席听证会。此前,有多份报告发现,波音在设计737 MAX客机时,没有充分考虑飞行员应该如何应对驾驶舱紧急情况。

  据报道,美国联邦航空管理局已经花了数月时间,对波音提交的关于关键安全系统软件的升级、培训以及系统变化等进行评估。但报道称,预计最早要到12月才可能让该机型飞机复飞。

  美国参院商业委员会主席、共和党参议员威克说,“除非99.9%的美国公众和政策制定者相信,它(737 MAX客机)是绝对安全的,否则这一型号飞机不会复飞。”

  威克称,他还计划在推进调查期间,加强波音与美国联邦航空管理局的沟通,并在听证会期间加强“监管机构与制造商之间的关系”。

。

  原标题:川普催促美国实行负利率 美联储官员不乐意

  中新网11月21日电 综合报道,尽管美国总统川普一再呼吁在美国实行负利率政策,但20日公布的一项会议记录显示,美联储的官员们并不乐意通过这种方式来刺激经济。

资料图:美联储主席鲍威尔中新社记者 陈孟统 摄资料图:美联储主席鲍威尔中新社记者 陈孟统 摄

  据报道,一直以来,川普声称欧洲与其他地区采取的负利率措施,为这些国家带来了竞争优势,于美国不利,因而呼吁美联储降息。

  然而,这份美联储10月份的会议记录明确显示,美联储不仅在当前美国经济仍在增长的情况下不愿采用负利率,也对经济陷入衰退时采用负利率手段刺激经济深表怀疑。

  根据会议记录,美联储的17位决策委员会成员都认同,将借款成本推低至低于零“在美国似乎并不是有吸引力的货币政策工具”。

  决策者们也不热衷于通过购买国债来控制长期利率,一些决策者认为这样做会被视为干涉财政部对国家债务的管理,其他决策者则担心这会使美联储资产负债表膨胀。

  美联储公开市场委员会(FOMC)利率决策委员在会中陈述,在一些已尝试执行负利率的国家,证据显示效果好坏并存。因此委员们认为,近期内进一步降息“并无必要”,除非迎来重大变化。

  据悉,自2019年7月以来,美联储已3次降息,但此前从2015年末开始连续9次加息。川普曾多次指责美联储没能更大幅度地降息,影响了他任期内的经济增长。

 

。.05was chosen to indicate that a trend was significant.The frequency of each allele was also calculated as function of geographic regions of isolation following grouping non-redundant A/H1N1/09 HA protein sequences according to the regions where they were isolated(Asia,Europe,North America,South America,and Oceania)and tested using the v2test. Binding energy analysis

The MOE(molecular operating environment)program[27] was used to calculate the binding energy between A/H1N1/ 09HA and its cellular receptor based on a known binding structure(PDB ID:3GBN,X-ray diffraction)composed of the A/South Carolina/1/1918HA and a receptor analog. First,we partially minimized the complex by relaxing the ligand and the side chains within10nm from the ligand, while keeping all other atomsfixed.

Following calculation of energies,factor analysis(FA) and multiple regression analysis(MRA)were employed to generate an LRE-like equation[28,29]:

D G b FEB

ðÞ¼x1D G b vdWþx2D G b eleþx3D G b solvþx4D G b n

D G b¼D G complexþD G proteinþD G ligand

In this equation,D G(FEB)stands for the free energy of binding,D G vdW,D G ele,D G solv,and D G n stand for the van der Walls contribution,the electrostatic contribution,the polar solvation contribution,and the nonpolar solvation contribution to the binding process,respectively,where w1, w2,w3,and w4are weight factors,and D G b represents binding energy(i.e.,energy difference between ligand/ receptor complex and free protein and ligand).Results

Point mutation analysis

To specify the differences between the HA molecules of the A/H1N1/09virus and recently circulating seasonal IAVs,we generated a consensus sequence from704 A/H1N1/09HA amino acid sequences deposited in the GenBank(as of April21,2010)and a consensus sequence from the HA proteins of4seasonal H1N1IAV strains that had been recommended by WHO for production of influ-enza vaccines,and compared these two groups of HA sequences by aligning both consensus sequences and sev-eral individual HA sequences from each group(Fig.1).

As shown in Fig.1and Table1,while the overall dif-ference between the consensus sequences of seasonal H1N1and A/H1N1/09HA proteins was as high as19.61% (111/566),the cleavage sites of HA,at which the HA0 protein is recognized by specific protease(s)and enzy-matically cleaved into HA1and HA2,thereby becoming activated in mediating the entry of IAVs into host cells, remain identical(PSIQSR;GLFGAI)among the strains analyzed.Meanwhile,the Asp204,Asp239,Gln240,and Gly242residues at the RBS that are responsible for the viral attachment to the host cell receptor,a critical step for viral entry,were also identical between the two consensus sequences.It is of note that the four positions(i.e.,204, 239,240,and242)have been found previously to be involved in the specific binding capacity of HA to the host cell receptor[30–32].Whereas,amino acid variations were found at a number of positions at the HA RBS among different seasonal H1N1IAVs and the A/H1N1/09viruses, including positions150,152,206,207,210,211,and212. Whether variations at these sites could impact the infec-tivity of an influenza virus to a specific host needs to be further investigated biologically.

To address the differences in the antigenic properties between A/H1N1/09and human seasonal IAVs,previously proposed antigenic epitope regions were comparatively analyzed.In this context,two groups of epitope regions, namely,the highly conserved regions and the highly vari-able regions[33],were analyzed,respectively.As shown in Fig.1and Table2,four highly conserved epitope regions (1–4),located in HA2,involving residues345–354, 359–376,394–411,and436–453,were all identical between A/H1N1/09isolates and seasonal H1N1viruses. In contrast,the highly variable regions,which lie in the HA1globular head and include sites(residues86–91),Sa (residues141–142,170–174,and176–181),Sb(residues 201–212),Ca1(residues183–187,220–222,and252–254) and Ca2(residues154–159,and238–239)[34],showed dramatic changes in A/H1N1/09isolates when compared with those in seasonal IAVs(H1N1).Such changes might

Fig.1Alignment of HA amino acid sequences of human seasonal IAVs and their consensus sequence in comparison with the pandemic A/H1N1/09IAVs and their consensus sequence.Residues different from the consensus HA amino acid sequence(top line)of human seasonal H1N1IAVs are shown,and dots stand for identical residues. The sequences were numbered according to pandemic2009H1 numbering.Boxed residues indicate the Asn-X-Thr/Ser motifs of glycosylation sites

Table1Comparison between the new A/H1N1/09isolates and seasonal H1N1IAVs at HA0cleavage site and RBS

Functional sites Virus Residues

The cleavage site Human seasonal PSIQSR;GLFGAI

A/H1N1/09PSIQSR;GLFGAI

Difference:0

RBS(in HA1)Residue positions(pandemic2009H1numbering)

149–152204–212235–242

Human seasonal V S A S DQ RA LY HTE PKVRDQEG

A/H1N1/09V T A A DQ QS LY QNA PKVRDQEG

Difference:2/4Difference:5/9Difference:0

provide a molecular explanation for the observed lack of cross-protection from previous infection or vaccination of seasonal IAVs against the novel A/H1N1/09virus.

Since point mutations could cause emergence or loss of Asn-X-Thr/Ser motifs and thereby,attachment or loss of N-glycans,respectively,leading to alteration of the anti-genicity and receptor specificity of HA,we analyzed the glycosylation sites on H1HAs by further examining both consensus sequences of the seasonal H1N1and the novel pandemic H1N1,which revealedfive identical glycosyla-tion sites(28,40,104,304,498,pandemic2009H1 numbering)between the two consensus sequences,as shown in Fig.1.In marked contrast,the A/H1N1/09stains contained amino acid mutations predicted to lose three

Table2Comparison between A/H1N1/09isolates and seasonal H1N1IAVs at antigenic epitope regions Epitope region Virus Residues and positions b

Highly conserved regions(in HA2)1345–354in HA2

Human seasonal GLFGAIAGFI

A/H1N1/09GLFGAIAGFI

Difference:0

2359–376in HA2

Human seasonal TGMVDGWYGYHHQNEQGS

A/H1N1/09TGMVDGWYGYHHQNEQGS

Difference:0

3394–411in HA2

Human seasonal NKVNSVIEKMNTQFTAVG

A/H1N1/09NKVNSVIEKMNTQFTAVG

Difference:0

4436–453in HA2

Human seasonal WTYNAELLVLLENERTLD

A/H1N1/09WTYNAELLVLLENERTLD

Difference:0

Highly variable regions(in HA1)a Cb86–91in HA1

Human seasonal L ISK(R)E S

A/H1N1L STAS S

Difference:4/6

Sa141–142in HA1170–174in HA1176–181in HA1 Human seasonal PN G K NGL P N LSKS

A/H1N1/09PN K K GNS P K LSKS

Difference:0Difference:4/5Difference:1/6 Sb201–212in HA1

Human seasonal NIG D(N)Q R(K/M)A(T)LY HT(K)E

A/H1N1/09TSA DQ QS LY QNA

Difference:8/12

Ca1183–187in HA1220–222in HA1252–254in HA1 Human seasonal A(V)N N K E SS H EPG

A/H1N1/09I N D K G T S R EPG

Difference:3/5Difference:1/3Difference:0 Ca2154–159in HA1238–239in HA1

Human seasonal S H N G E(K)S RD

A/H1N1/09P H A G AK RD

Difference:4/6Difference:0

a Amino acid sequences for all highly variable epitope regions listed in this table are given using the consensus sequences generated for A/H1N1/09isolates and seasonal H1N1IAVs,respectively,as presented in Fig.1.Variations in individual sequences as compared with the consensus sequence are given in parenthesis

b Numbering according to pandemic2009H1numbering

glycosylation sites at position71,142,and177.It is of note that the two highly conserved glycosylation sites(i.e.,142 and177)have been found previously to be involved in the antigenic properties of H1N1IAVs,which was within or around the Sa site.Meanwhile,we found that the A/H1N1/ 09stains carried amino acid mutations predicted to acquire one potential glycosylation site at position293,raising the issues whether such glycosylation does occur at the site and alters the antigenicity and receptor specificity of2009 influenza A(H1N1)virus.

Point-to-point analysis of mutations in the HA protein was performed among704isolates of the novel A/H1N1/09 virus.As shown in Tables3and4,13out of566positions in the entire HA molecule displayed variations among704 non-redundant A/H1N1/09HA sequences(variation fre-quency was higher than2),including5positions at RBS, and11positions in the highly variable epitopes,and all other variations were shown in the supplement data Table S1.Specifically,the amino acid change at position342 (Gln342Leu)was at the cleavage site of one A/H1N1/09 isolate[A/Guangdong/03/2009(H1N1)].Whether such a change would influence the interaction of HA0with pro-teolytic enzyme(s)and consequently,the cleavage activity, remains unclear.It requires further investigation to clarify whether mutation Asp239Glu in two isolates[A/Paris/ 2591/2009(H1N1)and A/New Jersey/01/2009(H1N1)] was relevant to host adaptation,since Glu239had been previously found to be associated with acquisition of SA a-2,6Gal binding specificity[30,35].Most noteworthily is the high frequency(72.02%)of Ser220Thr mutation in the Ca1epitope,strongly indicating existence of a positive selection or,at a lesser likelihood(due to lack of other high-frequency mutated positions in the HA),more than one origin of the new A/H1N1/09viruses.

Structural modeling

To better characterize the variations in A/H1N1/09HA,we sought to map the altered amino acids to a predicted three-dimensional(3-D)HA structure.Wefirst BLAST searched the PDB database for deposited HA molecules with the

highest sequence homologies to A/H1N1/09HA.Three sequences,together with their previously X-ray determined 3-D structures,of similarities higher than80%were selected,which were1RUY[HA of A/swine/Iowa/15/30 (H1N1)],1RVT[HA of A/swine/Iowa/15/30(H1N1) complexed with receptor analog LSTC]and1RV0[HA of A/swine/Iowa/15/30(H1N1)complexed with receptor analog LSTA][32].Using the selected molecules as tem-plates,onto which the new A/H1N1/09HA consensus sequence was modeled for50times,a predicted3-D structure of the A/H1N1/09HA was obtained,visualized with Jmol,and demonstrated in Fig.2.

As shown in Fig.2,the structure of A/H1N1/09HA monomer was similar to those of other published HAs,as expected,with a globular head containing the RBS,a cleavage site,a trans-membrane domain,and an a-helical stalk.Variations found in various isolates of the A/H1N1/ 09HA,as well as differences between the HA molecules of human seasonal IAVs and the new A/H1N1/09virus, and the glycosylation attachment sites were mapped on the3-D structure as shown in distinct colors in Fig.2. These variations and different residues were mainly located in the HA1fragment,most of which lied in antigenic epitopes.

Table3Amino acid variations in the HA protein sequence among A/H1N1/09isolates

Positions a Primary

residue

Frequency

(%)

Variation(s)Variation

frequency(%) HA136V97.73I 2.13

L0.14 49L96.45I 3.41

X0.14 103D97.17E0.14

G 2.41

M0.28 114D95.74N 4.12

X0.14 145S95.32M 4.40

X0.28 220S26.99A0.14

T72.02

X0.85 222R96.17G0.14

K 3.13

S0.28

T0.14

X0.14 239D88.78E 4.69

G 3.55

N0.99

X 1.99 310Q95.31H 4.26

X0.43 314P97.30S 2.70

338V94.88I 4.69

S0.43

HA2391E88.78G0.71

K10.51 428V97.30I 2.56

X0.14

a Positions listed according to pandemic2009H1numbering

b‘‘X’’stands for any amino acid

Site-by-site analysis

To address the significance of the mutations identified in A/ H1N1/09HA,we conducted positive selection analysis on a site-by-site basis,as positive natural selection could drive the increase in prevalence of advantageous traits,which may lead to a new pandemic virus.In this study,the ratio between non-synonymous(dN)and synonymous(dS)substitutions were used to indicate selective pressure on each codon.According to Yang et al.[24],when the dN/dS value on a certain codon is greater than1and tested to be significant by the Bayes test,the site is considered to be under positive selection;and in con-trast,the site would be recognized as being under negative selection in the case if the dN/dS value is significantly smaller than1.Using the HyPhy program[23],our positive selection analysis revealed that34codon sites showed dN/dS[1with statistical significance(Bayes factor[1)(Fig.3).To mini-mize possible errors such as those caused by biased sampling, we chose to use a very conservative strategy in the identifi-cation of the site mutations under positive selection by selecting only those codon sites with the highest Bayes fac-tors,which accounted for the leading5%of all codon sites with a Bayes factor[1.Consequently,these procedures led to identification of two sites,namely,positions220and239 (pandemic2009H1numbering,equivalent to206and225, respectively,according to the H3numbering)in the HA1 protein.Notably,the residue220lied in the Ca1epitope region in the head of HA1,and residue239was found to be included in the RBS of HA1.It is of interest that previous studies have suggested that both regions are relevant to the determination of the severity and transmissibility of an IAV [35],raising the question whether the mutations at these sites could favor the prevalence of the novel virus.

Variation at position220

Particularly noteworthy was the relatively high frequencies of S220and T220present in the HA of A/H1N1/09

Table4Amino acid variations in functional regions of A/H1N1/09 isolates

Functional region Residue positions a Variations a

The cleavage site Between HA1and HA2None

RBS(in HA1)149–152151

204–212204

235–242238,239,240

Highly conserved epitopes(in HA2)345–354None 359–376None 394–411None 436–453None

Highly variable epitopes(in HA1)

Cb86–9190

Sa141–142None

170–174172

176–181179

Sb201–212202,203,204 Ca1183–187None

220–222220,222

252–254252

Ca2154–159None

238–239238,239

a Positions listed according to pandemic2009H1

numbering

Fig.2Mapping of mutations onto the3-D structure of HA monomer. Side views of the HA monomers are shown.Each has the highly conserved epitope regions in HA2colored violet and the highly variable epitope regions in HA1colored cyan.a the reference HA monomer,and b,c the HA monomers with mutations mapped.In b,regions colored blue stand for mutations detected among the A/H1N1/ 09viruses;in c,regions colored yellow demonstrate differences between the human seasonal influenza virus and this novel virus,and the amino acid variations at residues220and239and the glycosylation sites were numbered(Colorfigure online)

isolates,prompting us to ask whether they were a result of selection during the course of evolution.To address this issue,we further analyzed the frequency distribution of S220and T220in the HA among isolates of the novel A/H1N1/09virus as a function of isolation time and geo-graphic regions.When 704HA sequences derived from A/H1N1/09viruses isolated between the period of March 30,2009and April 21,2010,were grouped according to their isolation time,a total of 507isolates (72%)with T220were identified,and the percentage of S present at position 220was found to gradually decline,while the frequency of T220increased over time (Fig.4).To test the significance of the descending trend of S220,Kendall test and a linear model were used,and results revealed a Kendall test

P value of 2.503910-5and a descending rate of 0.06655with a P value of 9.84910-5in the linear model analysis.Both tests confirmed the significant descending trend of the residue S at position 220,with T220gradually becoming prevalent in the infected population.In contrast,no sig-nificant difference in the frequency distribution of S220versus T220among HA molecules derived from viruses isolated from Asia,Europe,North America,Oceania,and South America,with a v 2test P value of 0.2801,indicating that the changes in the frequencies of S220versus T220found in the above study probably were not geographic region-specific and might have been occurring worldwide.We next sought to investigate whether S220and T220were present in the HA molecules of previously isolated swine IAVs and seasonal human H1N1viruses.We down-loaded 272non-redundant HA protein sequences of North American classical swine flu virus (H1subtype)from the NCBI influenza virus sequence database and analyzed the frequency of amino acids present at position 220.Interest-ingly,251out of the 272sequences were found to have S220,as opposed to that only 20isolates carried T at the position 220.Moreover,all of the 20strains with T220were H1N1subtype swine influenza A viruses,among which 14were isolated from Tennessee in 1976,1977,and 1978,and the remaining six strains were isolated sporadically from several other US states.On the other hand,the frequency distribu-tion in 1767HA sequences derived from human-infected H1subtype IAVs isolated before the 2009pandemic showed an S220:T220ratio of 1760:7.Variation at position 239

Since our point mutation analysis found a notable vari-ability of position 239in HA,we analyzed the

frequencies

of different residues at the position in HA of704A/H1N1/09 isolates.Our analysis found that aspartic acid(D)was present at a frequency of88.78%(623/704),glycine(G)at 3.55%(25/704),and glutamic acid(E)at4.69%(33/704), with the remaining residues undetermined.Possible impact of mutations at this site on receptor binding was examined by calculating the minimal energy using the MOE(Molec-ular Operating Environment)program,and the result showed that the binding energy between receptor analog and wild type HA(D239)was-29.506kcal/mol,while for mutants G239and E239,it was-17.027and-16.445kcal/ mol,respectively,indicating possibly weakened receptor-binding capacities of the mutants.

Discussion

Our current study analyzed the site variations in the HA protein sequences among the novel A/H1N1/09viral iso-lates,as well as those between human seasonal influenza viruses and A/H1N1/09.3-D structure of the HA was also modeled,and identified point mutations were analyzed to predict their potential significance in molecular evolution and function of the pandemic virus.

In this study,the point mutation analysis showed that the A/H1N1/09strains carry amino acid mutations that might lead to acquisition of one glycosylation site(position293) and loss of three glycosylation sites(position71,142,and 177).It has been reported that variation in glycosylation is used by influenza viruses to interfere with surveillance by the host immune system.Acquisition of a glycosylation site masks the protein surface from antibody recognition because the glycans themselves are host-derived,and hence considered as‘‘self’’by the human immune system[36,37] Meanwhile,upon the addition of glycosylation to this region has been thought to slow down yearly antigenic drift,pre-sumably because glycosylation shielded this region from the antigenic pressure of antibodies[18,36].Recently,Wei et al.and Xu et al.have evaluated the cross-neutralization of pandemic1918and2009H1N1influenza viruses and found that they were both resistant to antisera directed to a rela-tively recent seasonal influenza virus of the same subtype [18,38].Interestingly,pandemic2009H1N1influenza virus (A/California/04/2009),like A/South Carolina/1/1918virus, does not have any glycosylation in or around the Sa site in HA and hence,the epitope is exposed for antibody recog-nition.They suggest that these N-glycans of the RBD and antigenic epitope regions may play roles in evading the human immune response and viral evolution in humans[18]. The probability of glycosylation of the predicted asparagine residues293above mentioned in our current study,and the role of the acquisition(position293)or loss of glycosylation (position71)in modulating immune recognition and its influence on viral evolution,is under investigation in our laboratory.

By mapping the variations in the HA protein sequence, we identified that the distinction between the HA antigenic specificities of the human seasonal influenza virus and the novel A/H1N1/09virus mainly lie in the HA1epitope regions,which might explain the lack of effective cross-protection by previous seasonal IAV vaccines or infections and the highly transmissible property of the A/H1N1/09 virus.

A key notion derived from this study is the possibility that two HA1sites in the novel A/H1N1/09virus,namely, residues220and239,might be positively selected during its evolution.Interestingly,residue239lies in a region possibly involved in both receptor binding and determi-nation of antigenic specificity[30–32,34].It has been previously reported that when residue239changes from Gly to Glu,the virus becomes more adaptive to human host [30].Recently,Chen et al.reported the D225G(H3num-bering,D239G according to2009pandemic H1number-ing)substitution in7(12.5%)of57patients with severe disease,and in0(0%)of60patients with mild disease,and the D225E mutation was identified in one patient with severe disease,by direct analysis of polymorphisms in126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N12009virus from117 clinical specimens in Hong Kong[39].Our current study revealed that the vast majority of pandemic A/H1N1/09 isolates carried D239(88.78%)and yet a small fraction of the A/H1N1/09isolates had glutamic acid(E,4.69%)and glycine(G,3.55%)at this position.Free binding energy analysis suggested that a D239E/G mutation tended to decrease the affinity of the H1subtype IAV to the sialic acid receptor.It remains to be determined,whether the identified positive selection of the D239E/G mutation is functionally significant during the spreading of the A/H1N1/09virus.

Residue220lies in the Ca1epitope region.In all A/H1N1/09isolates obtained as of August2009,T220was present at a frequency of72.02%,whereas the frequency of S220was26.99%.It is of note that both T220and S220 were present in H1subtype classical swine IAVs,from which the HA of the pandemic A/H1N1/09was originated, and that the frequency of T220was far lower than that of S220in these swine IAVs.Our analysis on the dynamic change of T220versus S220demonstrated an ascending trend of the percentage of T220and a descending curve of S220during the course of the pandemic spreading,sug-gesting that T220might have been positively selected over S220.Speculatively,T220was thenfixed through natural or other selection at the peak of this pandemic and favored the transmissibility of the new virus.Interestingly,com-pared with serine,amino acid threonine carries an extra

methyl group and therefore displays a different polarity than serine.Whether such a difference in the polarity could contribute to altering recognition and binding capacity between the antigenic epitope and specific antibodies and thereby should be taken into consideration when develop-ing more effective vaccines and therapeutic drugs remains to be further investigated.

Acknowledgments This study was supported by the National Natural Science Foundation of China-Guangdong Province joint grant (U0632002),a grant from the State Major Infectious Disease Research Program(China Central Government,2009ZX10004-213), a Key Science and Technique Research Project of Guangdong Province(2009A020101006,2009B020600001),National High-Tech R&D Program(863Program)(Ministry of Science and Technology, China,2006AA02A223,2007AA09Z448,2007AA09Z431),National Science and Technique Research Program for public welfare appli-cations(201005022),and a Key Project of Science&Technology Planning of Guangdong Province(2007A03260001). References

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如对您有帮助,可购买打赏,谢谢

生活常识分享孕妇可以吃花生酥吗

导语:花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是

花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是一种高热量的饮食。生活中很多的孕妇对于花生酥也是情有独钟,但是考虑到孕妇身体的特殊性使得人们比较的重视,那么,孕妇可以吃花生酥吗?

花生酥香甜而不腻,具有极高的营养价值,可以与鸡蛋、鱼肉等相媲美,孕妇在生活中可以适当的吃一些花生酥,补充身体所需要的能量,但是不可以多吃,以免对身体不利。

孕妇可以吃花生酥吗?花生酥的营养价值比粮食高,可以与鸡蛋、牛奶、肉类等一些动物性食物媲美。它含有大量的蛋白质和脂肪,特别是不饱和脂肪酸的含量很高,很适宜制造各种营养食品。

孕妈妈常吃花生酥能够预防产后缺乳,而且花生衣中含有止血成分,可以对抗纤维蛋白溶解,增强骨髓制造血小板的功能,缩短出血时间,提高血小板量,改善血小板质,加强毛细血管的收缩功能.是孕妇防治再生障碍性贫血的最佳选择。此外,用新鲜花生叶煎水代茶饮,还能够有效防治妊娠高血压综合征。

以上的内容就是对于孕妇可以吃花生酥吗的介绍,希望能够给您带去一定的帮助。在一般的情况下人们都是可以食用花生酥的,但是对于孕妇来说不可过量的食用,花生酥中的糖分会引起妊娠高血糖等病变,对孕妇和胎儿的健康不利,我们需要避免。

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普吉岛甲米蒂瓦娜广场酒店(DeevanaPlazaKrabi)

舒适度作为普吉岛甲米蒂瓦娜广场酒店所有的客房首要标准,一切设施都以此为目标,一定不会让您失望。酒店宽敞的客房,配有住客评分分数来自522条评语等设施,让您瞬间忘记旅途的疲倦。

中文名称普吉岛甲米蒂瓦娜广场酒店英文名称DeevanaPlazaKrabi酒店星级4星级房间数量213酒店地址186Moo3,AonangSoi8,Aonang,Muang,81180奥南海滩,泰国

【好巧网解读】4大卖点

1.很安全2.员工热情有礼貌,一部分客人带着两岁多的女儿,他们对小孩也很好3.酒店还设有池畔酒吧和24小时客房服务4.床也很舒服

酒店的图片

酒店房型房价介绍

每个客房都配有阳台,电话,DVD播放机,视频游戏,有线频道,平面电视,保险箱,空调,熨斗,书桌,熨衣设备,客厅角,瓷砖/大理石地板,衣柜/衣橱,淋浴,吹风机,浴袍,免费洗浴用品,卫生间,浴室,拖鞋,迷你吧,冰箱,电烧水壶,唤醒服务,希望能让客户在入住时更加愉快惬意。酒店的房型有多种选择,提供了豪华特大号床间-可使用游泳池(3位成人)、高级双床间(2位成年人、家庭间、豪华双床间-可直通泳池(3位成人)、高级双床间(3名成人)、豪华特大大床房(3人)、家庭间(3位成人)、豪华双床间(2位成人)、高级特大号床间(3位成人)、豪华双床间(3位成人)、豪华双床间-可使用游泳池(2位成人),房间布置都到位,服务员也很热情。简而言之,客人在普吉岛甲米蒂瓦娜广场酒店享受的服务与设施会有宾至如归的感觉。再讲究的客人也能在酒店得到满意的服务。

相关条款

入住时间从14:00时退房时间12:00时之前预订取消/预付政策不同类型的客房附带不同的取消预订和预先付费政策请输入您的入住日期并参阅您所需的客房的条款。儿童和加床允许客人携带儿童入住。允许1名12岁以下的儿童,使用现有床铺的收费是每人每晚THB200。允许1名2岁以下的儿童,加1张婴儿床,免费。允许1名年龄较大的儿童或者成人,一张加床收费:每人每晚THB1070。最多容纳:每间客房1张加床/婴儿床。所提出的任何加床或婴儿床的要求均需获得酒店的确认。附加费用不会自动计算在总价中,您需在入住时另行支付。宠物不允许携带宠物入住。团体如果预订客房数超过7间,住宿方将采用不同的政策和额外补充规定。酒店接受的银行卡类型将鼠标悬停在卡片标志上,即可查看更多信息。

好巧网酒店评价分

非常好,88分

酒店优缺点

游客提到的优点:安全适合带小孩,服务好餐厅很棒床铺好干净游客提到的缺点:浴缸没有那么好晚上有蚊子

备注:好巧网综合打分是基于100多个酒店预订网站的评分综合算出。以上是该酒店在几家酒店预订网站上的评分,供您参考。好巧网对酒店特色的分析数据,来自主流的酒店预订网站、旅游社区网站提到该酒店的评论,尽量展示客观中立的分析。查询更多用户评论信息,请访问http://www.haoqiao.cn/Phuket_c3/274779.html

。

现因借款人__________身份证号:__________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。借款种类为现金,借款日期为_____年____月____日,还款日期为______年____月____日前, 特立此据为凭。

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因___________(写借款用途)需向贷款人________借款人民币_______________元整(小写_____元整)。借得款种类为现金,借得款日期为_____年____月____日,还款日期为____年____月____日前, 按时一次性偿还清¥:____________借款加利息。借款利息为: ___%(年利率),特立此据为凭。

借款人:__________(亲笔签名并按手印)

贷款人:__________ (亲笔签名并按手印)

见证人1:_________ (亲笔签名并按手印)

见证人2:__________ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

现因借款人_______身份证号:_____________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。(注:因借款人超过还款日期不守信用赖账的,需在省级权威媒体向贷款人道歉,借款人按借得款两倍偿还给贷款人。)

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

见证人1:______ (亲笔签名并按手印)

见证人2:______ (亲笔签名并按手印)

借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

年月日

。

  中新网8月20日电 据美国中文网报道,美国康州、佛州和俄亥俄州当局表示,最近挫败了3起独立的大规模枪击图谋,3名白人男子被捕。

  据报道,公众线报帮助破获了这3起事件,三州警方在15和16日将3名嫌犯逮捕。当局称,3名嫌犯均是20出头的白人男性,他们或在网上发帖、或发短信进行大规模枪击威胁。近日,加州、得州和俄亥俄州相继发生大规模枪击事件,最近几周,多次误报和枪击威胁恶作剧让美国民众心神不宁,要求变更枪支立法的呼声迭起。

  康州

  诺沃克(Narwalk)市警部门称,22岁瓦格肖(Brandon Wagshol)被捕。瓦格肖在诺沃克警方和FBI的联合调查后被捕,FBI收到线报,据称他试图从外州购买大容量的步枪弹夹。

  警方表示,瓦格肖在网上购买了步枪零件,打算自己制造武器,并在“脸书”上展示出“他对进行大规模枪击的兴趣”。诺沃克警方说,在执行搜查令时,警方发现两把注册在瓦格肖父亲名下的枪,多发弹药,防弹衣和其他战术装备。

  佛州

  沃卢西亚郡(Volusia)治安官办公室称,25岁代托纳比奇(Daytona Beach)市居民威克斯(Scott Wix)16日被捕,被控威胁要进行大规模射击。警方接到多条线报,据称威克斯发送了多条短信,阐述了他进行大规模枪击的计划。警方没有说明威克斯向谁发送了这些短信。

  警长办公室描述威克斯的短信时称,“学校是个弱的目标……我更有可能向3英里外的一大群人开枪……我想打破连续杀人时间最长的世界纪录。”据称另一条短信写道:“但是要能杀死100人会很好。我已经有一个地点选择(笑出眼泪的emoji)很坏吗?”

  警方在一份声明中表示,威克斯称他并未拥有枪支,但“对大规模枪击事件着迷”。警方同时发布了威克斯被捕时的视频。

  俄亥俄州

  FBI称,警方接到一个线报,一名男子在网上发视频称,自己是犹太社区中心枪击事件的枪手。虽然该枪击事件并未发生,但警方将该男子逮捕,他是20岁的里尔顿(James P. Reardon)。FBI克利夫兰分部表示,里尔顿于16日被捕,涉嫌电信骚扰和加重恐吓。

  警方称,里尔顿在“图片墙”(Instagram)上发布的视频帖子,标记了扬斯敦犹太社区中心。警方收到该视频线报的当天,在里尔顿父母家执行了搜查令。他随后顺利被警方逮捕,警方还在现场发现了弹药,半自动武器和反犹信息。

。

 

联邦快递明年1月起每周7天都提供快递服务

(综合三十日电)为了满足电子商务市场持续增加的送货需求,美国联邦快递公司(FedEx)将从明年开始,每周送货七天,而且不会收取额外的费用。联邦快递明年1月起每周七天都提供快递服务,以试图跟上网购的繁荣景气。该公司也将取回目前由邮局处理的每天近200万件包裹快递能量,并表示,将SmartPost包裹转移回自有快递网络将使送货司机能将更多的包裹紧密安排规画,提高送货效率。除了每周增加一天送货服务,联邦快递公司还计划配合陆运路线,将更多包裹送到客户的家门口,以降低成本。此外,该公司明年将会有大约200万个包裹,不再委托美国邮局运送。「消费者每周七天在线购物」,联邦快递总裁兼首席运营官苏伯拉曼尼(Raj Subramaniam)表示,因此,网购者以及电商业者对每周七天送货服务的需求不断增加。他强调,每周增加一天送货服务,不会提高收费,也不是针对特定电商业者,而是对小型托运者到大型零售商都一视同仁,提供全面送货服务。联邦快递公司估计,到了2026年,美国小包裹出货量将翻倍,该公司必须提前准备。联邦快递和其竞争对手联合包裹服务公司(UPS)近年来投入资金改善送货管理,并且将所谓的「最后一英里」(Last-Mile,即送到客户家门口)利润较低的送货,外包给美国邮局(USPS)或其它业者。然而,随著网购增加,包裹数量也与日俱增,全美每天国内包裹达到5,000万件。为了因应这个趋势,FedEx及UPS正在调整业务,以增加市场份额及处理周末的送货需求。与此同时,由于亚马逊及沃尔玛等零售商在全美增建仓库,以缩短送货距离,导致联邦快递在全美或全球范围内的空运快递业务量逐渐萎缩。此外,多家零售商建立自己的送货系统,销售订单管理软件公司OrderDynamics营销副总裁迪莫夫(Charles Dimov)说:「联邦快递如果不提供周日送货服务,很有可能被市场淘汰。」UPS目前是每周送货六天,发言人扎卡拉(Glenn Zaccara)表示,该公司「持续评估扩大服务的时机」。美国邮局正在考虑将包裹递送服务扩大到每周七天,目前邮局在周日为亚马逊提供送货服务,并在假日期间为其它业者提供送货服务。

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猜对了吗

教学内容

大象版小学科学三年级(下)第一单元第2课。

教学目标

1、利用实验验证的方法,明白猜想只是一种可能的答案,它和事实并不总是一致。

2、学会使用酒精灯加热的技能,了解材料传热的性能。

3、培养重视实验和证明的科学态度。

教学重点

了解猜想、假设和事实的区别。

教学难点

培养学生重视实验和证明的科学态度。

实验器材

酒精灯、铁架台、纸杯、火柴、水槽、烧杯、镊子、铁棒、纸团、纸条等。教学过程

一、激趣导入

同学们,你们喜欢猜谜语吗?(喜欢)老师今天给大家带来了几个谜语,请大家猜一猜,看谁最聪明。(课件出示谜语)

1、上边毛,下边毛,中间一个黑葡萄。生:眼睛。

2、麻屋子,红帐子,里面住着白胖子。生:花生。

3、身体生来瘦又长,五彩衣裳黑心肠,嘴儿尖尖说黑话,只见短来不见长。生:铅笔。

4、小铁驴,真是好,又不踢,又不咬,屁股后面把烟冒,突突突叫着跑。生:摩托车。这几则谜语你猜对了吗?生:猜对了。

二、猜想和验证

1、大胆猜想

你们想挑战更难的吗?(想)请同学们来猜想。(课件出示猜想问题图)

用酒精灯烧空纸杯的底部,纸杯会烧着吗?

把空纸杯倒扣在酒精灯上,纸杯会烧着吗?

用酒精灯烧装水的纸杯底部,谁能烧开吗?

生把猜想填入表格,指明说说自己猜想的根据。

2、学习使用酒精灯

我们进行了大胆的猜想,猜对了吗?我们要设法验证

自己的猜想。要验证自己的猜想,就必须学会安全使用酒精灯。请同学们打开书第6页,阅读左下角的“安全使用酒精灯。”

生阅读。

请同学们在小组内互相说一说怎样安全使用酒精灯。生在小组内互说,指明说一说怎样安全使用酒精灯。

师示范安全使用酒精灯。

3、验证

小组讨论所用实验材料。小组组长到材料超市选取实验材料,分组实验,是巡视指导,生填写实验报告单。汇报实验结果。

4、我进步我成功

课件出示:在几次猜想中,我的猜想与实验结果:

总是不一样()嘿,没关系!

有时不一样()呵,再努力!

总是一样的()瞧,我真棒!

指名说,是鼓励。

5、你在猜想与验证活动中取得了哪些收获?

师:猜想只是一种可能的答案,它和事实并不总是一

样的,要想知道猜想是否正确,必须设法验证。

三、课外延伸

我们再来进行两个猜想。

杯子竖直扣入水底,塞在杯底的纸团会湿吗?

用一张普通的纸,裁成长条,以螺旋状紧绕在一根铁棒上,然后火柴去烧铁棒上的纸条,纸条会被烧着吗?

想知道你猜对了吗?(想)怎么办?

。

  原标题:印尼狮航空难一周年 美国对波音的调查怎么样了?

  参考消息网10月29日报道 外媒称,10月29日是印尼狮航737 MAX客机坠毁一周年纪念日,美国议员表示,在“99.9%的美国公众”和决策者确信客机是安全的之前,波音该机型客机不会复飞。

  新加坡《联合早报》网站10月28日援引路透社报道称,波音公司首席执行长米伦伯格将连续两天出席听证会。此前,有多份报告发现,波音在设计737 MAX客机时,没有充分考虑飞行员应该如何应对驾驶舱紧急情况。

  据报道,美国联邦航空管理局已经花了数月时间,对波音提交的关于关键安全系统软件的升级、培训以及系统变化等进行评估。但报道称,预计最早要到12月才可能让该机型飞机复飞。

  美国参院商业委员会主席、共和党参议员威克说,“除非99.9%的美国公众和政策制定者相信,它(737 MAX客机)是绝对安全的,否则这一型号飞机不会复飞。”

  威克称,他还计划在推进调查期间,加强波音与美国联邦航空管理局的沟通,并在听证会期间加强“监管机构与制造商之间的关系”。

。.05was chosen to indicate that a trend was significant.The frequency of each allele was also calculated as function of geographic regions of isolation following grouping non-redundant A/H1N1/09 HA protein sequences according to the regions where they were isolated(Asia,Europe,North America,South America,and Oceania)and tested using the v2test. Binding energy analysis

The MOE(molecular operating environment)program[27] was used to calculate the binding energy between A/H1N1/ 09HA and its cellular receptor based on a known binding structure(PDB ID:3GBN,X-ray diffraction)composed of the A/South Carolina/1/1918HA and a receptor analog. First,we partially minimized the complex by relaxing the ligand and the side chains within10nm from the ligand, while keeping all other atomsfixed.

Following calculation of energies,factor analysis(FA) and multiple regression analysis(MRA)were employed to generate an LRE-like equation[28,29]:

D G b FEB

ðÞ¼x1D G b vdWþx2D G b eleþx3D G b solvþx4D G b n

D G b¼D G complexþD G proteinþD G ligand

In this equation,D G(FEB)stands for the free energy of binding,D G vdW,D G ele,D G solv,and D G n stand for the van der Walls contribution,the electrostatic contribution,the polar solvation contribution,and the nonpolar solvation contribution to the binding process,respectively,where w1, w2,w3,and w4are weight factors,and D G b represents binding energy(i.e.,energy difference between ligand/ receptor complex and free protein and ligand).Results

Point mutation analysis

To specify the differences between the HA molecules of the A/H1N1/09virus and recently circulating seasonal IAVs,we generated a consensus sequence from704 A/H1N1/09HA amino acid sequences deposited in the GenBank(as of April21,2010)and a consensus sequence from the HA proteins of4seasonal H1N1IAV strains that had been recommended by WHO for production of influ-enza vaccines,and compared these two groups of HA sequences by aligning both consensus sequences and sev-eral individual HA sequences from each group(Fig.1).

As shown in Fig.1and Table1,while the overall dif-ference between the consensus sequences of seasonal H1N1and A/H1N1/09HA proteins was as high as19.61% (111/566),the cleavage sites of HA,at which the HA0 protein is recognized by specific protease(s)and enzy-matically cleaved into HA1and HA2,thereby becoming activated in mediating the entry of IAVs into host cells, remain identical(PSIQSR;GLFGAI)among the strains analyzed.Meanwhile,the Asp204,Asp239,Gln240,and Gly242residues at the RBS that are responsible for the viral attachment to the host cell receptor,a critical step for viral entry,were also identical between the two consensus sequences.It is of note that the four positions(i.e.,204, 239,240,and242)have been found previously to be involved in the specific binding capacity of HA to the host cell receptor[30–32].Whereas,amino acid variations were found at a number of positions at the HA RBS among different seasonal H1N1IAVs and the A/H1N1/09viruses, including positions150,152,206,207,210,211,and212. Whether variations at these sites could impact the infec-tivity of an influenza virus to a specific host needs to be further investigated biologically.

To address the differences in the antigenic properties between A/H1N1/09and human seasonal IAVs,previously proposed antigenic epitope regions were comparatively analyzed.In this context,two groups of epitope regions, namely,the highly conserved regions and the highly vari-able regions[33],were analyzed,respectively.As shown in Fig.1and Table2,four highly conserved epitope regions (1–4),located in HA2,involving residues345–354, 359–376,394–411,and436–453,were all identical between A/H1N1/09isolates and seasonal H1N1viruses. In contrast,the highly variable regions,which lie in the HA1globular head and include sites(residues86–91),Sa (residues141–142,170–174,and176–181),Sb(residues 201–212),Ca1(residues183–187,220–222,and252–254) and Ca2(residues154–159,and238–239)[34],showed dramatic changes in A/H1N1/09isolates when compared with those in seasonal IAVs(H1N1).Such changes might

Fig.1Alignment of HA amino acid sequences of human seasonal IAVs and their consensus sequence in comparison with the pandemic A/H1N1/09IAVs and their consensus sequence.Residues different from the consensus HA amino acid sequence(top line)of human seasonal H1N1IAVs are shown,and dots stand for identical residues. The sequences were numbered according to pandemic2009H1 numbering.Boxed residues indicate the Asn-X-Thr/Ser motifs of glycosylation sites

Table1Comparison between the new A/H1N1/09isolates and seasonal H1N1IAVs at HA0cleavage site and RBS

Functional sites Virus Residues

The cleavage site Human seasonal PSIQSR;GLFGAI

A/H1N1/09PSIQSR;GLFGAI

Difference:0

RBS(in HA1)Residue positions(pandemic2009H1numbering)

149–152204–212235–242

Human seasonal V S A S DQ RA LY HTE PKVRDQEG

A/H1N1/09V T A A DQ QS LY QNA PKVRDQEG

Difference:2/4Difference:5/9Difference:0

provide a molecular explanation for the observed lack of cross-protection from previous infection or vaccination of seasonal IAVs against the novel A/H1N1/09virus.

Since point mutations could cause emergence or loss of Asn-X-Thr/Ser motifs and thereby,attachment or loss of N-glycans,respectively,leading to alteration of the anti-genicity and receptor specificity of HA,we analyzed the glycosylation sites on H1HAs by further examining both consensus sequences of the seasonal H1N1and the novel pandemic H1N1,which revealedfive identical glycosyla-tion sites(28,40,104,304,498,pandemic2009H1 numbering)between the two consensus sequences,as shown in Fig.1.In marked contrast,the A/H1N1/09stains contained amino acid mutations predicted to lose three

Table2Comparison between A/H1N1/09isolates and seasonal H1N1IAVs at antigenic epitope regions Epitope region Virus Residues and positions b

Highly conserved regions(in HA2)1345–354in HA2

Human seasonal GLFGAIAGFI

A/H1N1/09GLFGAIAGFI

Difference:0

2359–376in HA2

Human seasonal TGMVDGWYGYHHQNEQGS

A/H1N1/09TGMVDGWYGYHHQNEQGS

Difference:0

3394–411in HA2

Human seasonal NKVNSVIEKMNTQFTAVG

A/H1N1/09NKVNSVIEKMNTQFTAVG

Difference:0

4436–453in HA2

Human seasonal WTYNAELLVLLENERTLD

A/H1N1/09WTYNAELLVLLENERTLD

Difference:0

Highly variable regions(in HA1)a Cb86–91in HA1

Human seasonal L ISK(R)E S

A/H1N1L STAS S

Difference:4/6

Sa141–142in HA1170–174in HA1176–181in HA1 Human seasonal PN G K NGL P N LSKS

A/H1N1/09PN K K GNS P K LSKS

Difference:0Difference:4/5Difference:1/6 Sb201–212in HA1

Human seasonal NIG D(N)Q R(K/M)A(T)LY HT(K)E

A/H1N1/09TSA DQ QS LY QNA

Difference:8/12

Ca1183–187in HA1220–222in HA1252–254in HA1 Human seasonal A(V)N N K E SS H EPG

A/H1N1/09I N D K G T S R EPG

Difference:3/5Difference:1/3Difference:0 Ca2154–159in HA1238–239in HA1

Human seasonal S H N G E(K)S RD

A/H1N1/09P H A G AK RD

Difference:4/6Difference:0

a Amino acid sequences for all highly variable epitope regions listed in this table are given using the consensus sequences generated for A/H1N1/09isolates and seasonal H1N1IAVs,respectively,as presented in Fig.1.Variations in individual sequences as compared with the consensus sequence are given in parenthesis

b Numbering according to pandemic2009H1numbering

glycosylation sites at position71,142,and177.It is of note that the two highly conserved glycosylation sites(i.e.,142 and177)have been found previously to be involved in the antigenic properties of H1N1IAVs,which was within or around the Sa site.Meanwhile,we found that the A/H1N1/ 09stains carried amino acid mutations predicted to acquire one potential glycosylation site at position293,raising the issues whether such glycosylation does occur at the site and alters the antigenicity and receptor specificity of2009 influenza A(H1N1)virus.

Point-to-point analysis of mutations in the HA protein was performed among704isolates of the novel A/H1N1/09 virus.As shown in Tables3and4,13out of566positions in the entire HA molecule displayed variations among704 non-redundant A/H1N1/09HA sequences(variation fre-quency was higher than2),including5positions at RBS, and11positions in the highly variable epitopes,and all other variations were shown in the supplement data Table S1.Specifically,the amino acid change at position342 (Gln342Leu)was at the cleavage site of one A/H1N1/09 isolate[A/Guangdong/03/2009(H1N1)].Whether such a change would influence the interaction of HA0with pro-teolytic enzyme(s)and consequently,the cleavage activity, remains unclear.It requires further investigation to clarify whether mutation Asp239Glu in two isolates[A/Paris/ 2591/2009(H1N1)and A/New Jersey/01/2009(H1N1)] was relevant to host adaptation,since Glu239had been previously found to be associated with acquisition of SA a-2,6Gal binding specificity[30,35].Most noteworthily is the high frequency(72.02%)of Ser220Thr mutation in the Ca1epitope,strongly indicating existence of a positive selection or,at a lesser likelihood(due to lack of other high-frequency mutated positions in the HA),more than one origin of the new A/H1N1/09viruses.

Structural modeling

To better characterize the variations in A/H1N1/09HA,we sought to map the altered amino acids to a predicted three-dimensional(3-D)HA structure.Wefirst BLAST searched the PDB database for deposited HA molecules with the

highest sequence homologies to A/H1N1/09HA.Three sequences,together with their previously X-ray determined 3-D structures,of similarities higher than80%were selected,which were1RUY[HA of A/swine/Iowa/15/30 (H1N1)],1RVT[HA of A/swine/Iowa/15/30(H1N1) complexed with receptor analog LSTC]and1RV0[HA of A/swine/Iowa/15/30(H1N1)complexed with receptor analog LSTA][32].Using the selected molecules as tem-plates,onto which the new A/H1N1/09HA consensus sequence was modeled for50times,a predicted3-D structure of the A/H1N1/09HA was obtained,visualized with Jmol,and demonstrated in Fig.2.

As shown in Fig.2,the structure of A/H1N1/09HA monomer was similar to those of other published HAs,as expected,with a globular head containing the RBS,a cleavage site,a trans-membrane domain,and an a-helical stalk.Variations found in various isolates of the A/H1N1/ 09HA,as well as differences between the HA molecules of human seasonal IAVs and the new A/H1N1/09virus, and the glycosylation attachment sites were mapped on the3-D structure as shown in distinct colors in Fig.2. These variations and different residues were mainly located in the HA1fragment,most of which lied in antigenic epitopes.

Table3Amino acid variations in the HA protein sequence among A/H1N1/09isolates

Positions a Primary

residue

Frequency

(%)

Variation(s)Variation

frequency(%) HA136V97.73I 2.13

L0.14 49L96.45I 3.41

X0.14 103D97.17E0.14

G 2.41

M0.28 114D95.74N 4.12

X0.14 145S95.32M 4.40

X0.28 220S26.99A0.14

T72.02

X0.85 222R96.17G0.14

K 3.13

S0.28

T0.14

X0.14 239D88.78E 4.69

G 3.55

N0.99

X 1.99 310Q95.31H 4.26

X0.43 314P97.30S 2.70

338V94.88I 4.69

S0.43

HA2391E88.78G0.71

K10.51 428V97.30I 2.56

X0.14

a Positions listed according to pandemic2009H1numbering

b‘‘X’’stands for any amino acid

Site-by-site analysis

To address the significance of the mutations identified in A/ H1N1/09HA,we conducted positive selection analysis on a site-by-site basis,as positive natural selection could drive the increase in prevalence of advantageous traits,which may lead to a new pandemic virus.In this study,the ratio between non-synonymous(dN)and synonymous(dS)substitutions were used to indicate selective pressure on each codon.According to Yang et al.[24],when the dN/dS value on a certain codon is greater than1and tested to be significant by the Bayes test,the site is considered to be under positive selection;and in con-trast,the site would be recognized as being under negative selection in the case if the dN/dS value is significantly smaller than1.Using the HyPhy program[23],our positive selection analysis revealed that34codon sites showed dN/dS[1with statistical significance(Bayes factor[1)(Fig.3).To mini-mize possible errors such as those caused by biased sampling, we chose to use a very conservative strategy in the identifi-cation of the site mutations under positive selection by selecting only those codon sites with the highest Bayes fac-tors,which accounted for the leading5%of all codon sites with a Bayes factor[1.Consequently,these procedures led to identification of two sites,namely,positions220and239 (pandemic2009H1numbering,equivalent to206and225, respectively,according to the H3numbering)in the HA1 protein.Notably,the residue220lied in the Ca1epitope region in the head of HA1,and residue239was found to be included in the RBS of HA1.It is of interest that previous studies have suggested that both regions are relevant to the determination of the severity and transmissibility of an IAV [35],raising the question whether the mutations at these sites could favor the prevalence of the novel virus.

Variation at position220

Particularly noteworthy was the relatively high frequencies of S220and T220present in the HA of A/H1N1/09

Table4Amino acid variations in functional regions of A/H1N1/09 isolates

Functional region Residue positions a Variations a

The cleavage site Between HA1and HA2None

RBS(in HA1)149–152151

204–212204

235–242238,239,240

Highly conserved epitopes(in HA2)345–354None 359–376None 394–411None 436–453None

Highly variable epitopes(in HA1)

Cb86–9190

Sa141–142None

170–174172

176–181179

Sb201–212202,203,204 Ca1183–187None

220–222220,222

252–254252

Ca2154–159None

238–239238,239

a Positions listed according to pandemic2009H1

numbering

Fig.2Mapping of mutations onto the3-D structure of HA monomer. Side views of the HA monomers are shown.Each has the highly conserved epitope regions in HA2colored violet and the highly variable epitope regions in HA1colored cyan.a the reference HA monomer,and b,c the HA monomers with mutations mapped.In b,regions colored blue stand for mutations detected among the A/H1N1/ 09viruses;in c,regions colored yellow demonstrate differences between the human seasonal influenza virus and this novel virus,and the amino acid variations at residues220and239and the glycosylation sites were numbered(Colorfigure online)

isolates,prompting us to ask whether they were a result of selection during the course of evolution.To address this issue,we further analyzed the frequency distribution of S220and T220in the HA among isolates of the novel A/H1N1/09virus as a function of isolation time and geo-graphic regions.When 704HA sequences derived from A/H1N1/09viruses isolated between the period of March 30,2009and April 21,2010,were grouped according to their isolation time,a total of 507isolates (72%)with T220were identified,and the percentage of S present at position 220was found to gradually decline,while the frequency of T220increased over time (Fig.4).To test the significance of the descending trend of S220,Kendall test and a linear model were used,and results revealed a Kendall test

P value of 2.503910-5and a descending rate of 0.06655with a P value of 9.84910-5in the linear model analysis.Both tests confirmed the significant descending trend of the residue S at position 220,with T220gradually becoming prevalent in the infected population.In contrast,no sig-nificant difference in the frequency distribution of S220versus T220among HA molecules derived from viruses isolated from Asia,Europe,North America,Oceania,and South America,with a v 2test P value of 0.2801,indicating that the changes in the frequencies of S220versus T220found in the above study probably were not geographic region-specific and might have been occurring worldwide.We next sought to investigate whether S220and T220were present in the HA molecules of previously isolated swine IAVs and seasonal human H1N1viruses.We down-loaded 272non-redundant HA protein sequences of North American classical swine flu virus (H1subtype)from the NCBI influenza virus sequence database and analyzed the frequency of amino acids present at position 220.Interest-ingly,251out of the 272sequences were found to have S220,as opposed to that only 20isolates carried T at the position 220.Moreover,all of the 20strains with T220were H1N1subtype swine influenza A viruses,among which 14were isolated from Tennessee in 1976,1977,and 1978,and the remaining six strains were isolated sporadically from several other US states.On the other hand,the frequency distribu-tion in 1767HA sequences derived from human-infected H1subtype IAVs isolated before the 2009pandemic showed an S220:T220ratio of 1760:7.Variation at position 239

Since our point mutation analysis found a notable vari-ability of position 239in HA,we analyzed the

frequencies

of different residues at the position in HA of704A/H1N1/09 isolates.Our analysis found that aspartic acid(D)was present at a frequency of88.78%(623/704),glycine(G)at 3.55%(25/704),and glutamic acid(E)at4.69%(33/704), with the remaining residues undetermined.Possible impact of mutations at this site on receptor binding was examined by calculating the minimal energy using the MOE(Molec-ular Operating Environment)program,and the result showed that the binding energy between receptor analog and wild type HA(D239)was-29.506kcal/mol,while for mutants G239and E239,it was-17.027and-16.445kcal/ mol,respectively,indicating possibly weakened receptor-binding capacities of the mutants.

Discussion

Our current study analyzed the site variations in the HA protein sequences among the novel A/H1N1/09viral iso-lates,as well as those between human seasonal influenza viruses and A/H1N1/09.3-D structure of the HA was also modeled,and identified point mutations were analyzed to predict their potential significance in molecular evolution and function of the pandemic virus.

In this study,the point mutation analysis showed that the A/H1N1/09strains carry amino acid mutations that might lead to acquisition of one glycosylation site(position293) and loss of three glycosylation sites(position71,142,and 177).It has been reported that variation in glycosylation is used by influenza viruses to interfere with surveillance by the host immune system.Acquisition of a glycosylation site masks the protein surface from antibody recognition because the glycans themselves are host-derived,and hence considered as‘‘self’’by the human immune system[36,37] Meanwhile,upon the addition of glycosylation to this region has been thought to slow down yearly antigenic drift,pre-sumably because glycosylation shielded this region from the antigenic pressure of antibodies[18,36].Recently,Wei et al.and Xu et al.have evaluated the cross-neutralization of pandemic1918and2009H1N1influenza viruses and found that they were both resistant to antisera directed to a rela-tively recent seasonal influenza virus of the same subtype [18,38].Interestingly,pandemic2009H1N1influenza virus (A/California/04/2009),like A/South Carolina/1/1918virus, does not have any glycosylation in or around the Sa site in HA and hence,the epitope is exposed for antibody recog-nition.They suggest that these N-glycans of the RBD and antigenic epitope regions may play roles in evading the human immune response and viral evolution in humans[18]. The probability of glycosylation of the predicted asparagine residues293above mentioned in our current study,and the role of the acquisition(position293)or loss of glycosylation (position71)in modulating immune recognition and its influence on viral evolution,is under investigation in our laboratory.

By mapping the variations in the HA protein sequence, we identified that the distinction between the HA antigenic specificities of the human seasonal influenza virus and the novel A/H1N1/09virus mainly lie in the HA1epitope regions,which might explain the lack of effective cross-protection by previous seasonal IAV vaccines or infections and the highly transmissible property of the A/H1N1/09 virus.

A key notion derived from this study is the possibility that two HA1sites in the novel A/H1N1/09virus,namely, residues220and239,might be positively selected during its evolution.Interestingly,residue239lies in a region possibly involved in both receptor binding and determi-nation of antigenic specificity[30–32,34].It has been previously reported that when residue239changes from Gly to Glu,the virus becomes more adaptive to human host [30].Recently,Chen et al.reported the D225G(H3num-bering,D239G according to2009pandemic H1number-ing)substitution in7(12.5%)of57patients with severe disease,and in0(0%)of60patients with mild disease,and the D225E mutation was identified in one patient with severe disease,by direct analysis of polymorphisms in126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N12009virus from117 clinical specimens in Hong Kong[39].Our current study revealed that the vast majority of pandemic A/H1N1/09 isolates carried D239(88.78%)and yet a small fraction of the A/H1N1/09isolates had glutamic acid(E,4.69%)and glycine(G,3.55%)at this position.Free binding energy analysis suggested that a D239E/G mutation tended to decrease the affinity of the H1subtype IAV to the sialic acid receptor.It remains to be determined,whether the identified positive selection of the D239E/G mutation is functionally significant during the spreading of the A/H1N1/09virus.

Residue220lies in the Ca1epitope region.In all A/H1N1/09isolates obtained as of August2009,T220was present at a frequency of72.02%,whereas the frequency of S220was26.99%.It is of note that both T220and S220 were present in H1subtype classical swine IAVs,from which the HA of the pandemic A/H1N1/09was originated, and that the frequency of T220was far lower than that of S220in these swine IAVs.Our analysis on the dynamic change of T220versus S220demonstrated an ascending trend of the percentage of T220and a descending curve of S220during the course of the pandemic spreading,sug-gesting that T220might have been positively selected over S220.Speculatively,T220was thenfixed through natural or other selection at the peak of this pandemic and favored the transmissibility of the new virus.Interestingly,com-pared with serine,amino acid threonine carries an extra

methyl group and therefore displays a different polarity than serine.Whether such a difference in the polarity could contribute to altering recognition and binding capacity between the antigenic epitope and specific antibodies and thereby should be taken into consideration when develop-ing more effective vaccines and therapeutic drugs remains to be further investigated.

Acknowledgments This study was supported by the National Natural Science Foundation of China-Guangdong Province joint grant (U0632002),a grant from the State Major Infectious Disease Research Program(China Central Government,2009ZX10004-213), a Key Science and Technique Research Project of Guangdong Province(2009A020101006,2009B020600001),National High-Tech R&D Program(863Program)(Ministry of Science and Technology, China,2006AA02A223,2007AA09Z448,2007AA09Z431),National Science and Technique Research Program for public welfare appli-cations(201005022),and a Key Project of Science&Technology Planning of Guangdong Province(2007A03260001). References

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如对您有帮助,可购买打赏,谢谢

生活常识分享孕妇可以吃花生酥吗

导语:花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是

花生酥是一种北京的美食,因为口感酥脆受到了人们的喜爱。花生酥富含丰富的热量、蛋白质、钙,磷等营养成分,能够为人体提供充足的能量,是一种高热量的饮食。生活中很多的孕妇对于花生酥也是情有独钟,但是考虑到孕妇身体的特殊性使得人们比较的重视,那么,孕妇可以吃花生酥吗?

花生酥香甜而不腻,具有极高的营养价值,可以与鸡蛋、鱼肉等相媲美,孕妇在生活中可以适当的吃一些花生酥,补充身体所需要的能量,但是不可以多吃,以免对身体不利。

孕妇可以吃花生酥吗?花生酥的营养价值比粮食高,可以与鸡蛋、牛奶、肉类等一些动物性食物媲美。它含有大量的蛋白质和脂肪,特别是不饱和脂肪酸的含量很高,很适宜制造各种营养食品。

孕妈妈常吃花生酥能够预防产后缺乳,而且花生衣中含有止血成分,可以对抗纤维蛋白溶解,增强骨髓制造血小板的功能,缩短出血时间,提高血小板量,改善血小板质,加强毛细血管的收缩功能.是孕妇防治再生障碍性贫血的最佳选择。此外,用新鲜花生叶煎水代茶饮,还能够有效防治妊娠高血压综合征。

以上的内容就是对于孕妇可以吃花生酥吗的介绍,希望能够给您带去一定的帮助。在一般的情况下人们都是可以食用花生酥的,但是对于孕妇来说不可过量的食用,花生酥中的糖分会引起妊娠高血糖等病变,对孕妇和胎儿的健康不利,我们需要避免。

。

普吉岛甲米蒂瓦娜广场酒店(DeevanaPlazaKrabi)

舒适度作为普吉岛甲米蒂瓦娜广场酒店所有的客房首要标准,一切设施都以此为目标,一定不会让您失望。酒店宽敞的客房,配有住客评分分数来自522条评语等设施,让您瞬间忘记旅途的疲倦。

中文名称普吉岛甲米蒂瓦娜广场酒店英文名称DeevanaPlazaKrabi酒店星级4星级房间数量213酒店地址186Moo3,AonangSoi8,Aonang,Muang,81180奥南海滩,泰国

【好巧网解读】4大卖点

1.很安全2.员工热情有礼貌,一部分客人带着两岁多的女儿,他们对小孩也很好3.酒店还设有池畔酒吧和24小时客房服务4.床也很舒服

酒店的图片

酒店房型房价介绍

每个客房都配有阳台,电话,DVD播放机,视频游戏,有线频道,平面电视,保险箱,空调,熨斗,书桌,熨衣设备,客厅角,瓷砖/大理石地板,衣柜/衣橱,淋浴,吹风机,浴袍,免费洗浴用品,卫生间,浴室,拖鞋,迷你吧,冰箱,电烧水壶,唤醒服务,希望能让客户在入住时更加愉快惬意。酒店的房型有多种选择,提供了豪华特大号床间-可使用游泳池(3位成人)、高级双床间(2位成年人、家庭间、豪华双床间-可直通泳池(3位成人)、高级双床间(3名成人)、豪华特大大床房(3人)、家庭间(3位成人)、豪华双床间(2位成人)、高级特大号床间(3位成人)、豪华双床间(3位成人)、豪华双床间-可使用游泳池(2位成人),房间布置都到位,服务员也很热情。简而言之,客人在普吉岛甲米蒂瓦娜广场酒店享受的服务与设施会有宾至如归的感觉。再讲究的客人也能在酒店得到满意的服务。

相关条款

入住时间从14:00时退房时间12:00时之前预订取消/预付政策不同类型的客房附带不同的取消预订和预先付费政策请输入您的入住日期并参阅您所需的客房的条款。儿童和加床允许客人携带儿童入住。允许1名12岁以下的儿童,使用现有床铺的收费是每人每晚THB200。允许1名2岁以下的儿童,加1张婴儿床,免费。允许1名年龄较大的儿童或者成人,一张加床收费:每人每晚THB1070。最多容纳:每间客房1张加床/婴儿床。所提出的任何加床或婴儿床的要求均需获得酒店的确认。附加费用不会自动计算在总价中,您需在入住时另行支付。宠物不允许携带宠物入住。团体如果预订客房数超过7间,住宿方将采用不同的政策和额外补充规定。酒店接受的银行卡类型将鼠标悬停在卡片标志上,即可查看更多信息。

好巧网酒店评价分

非常好,88分

酒店优缺点

游客提到的优点:安全适合带小孩,服务好餐厅很棒床铺好干净游客提到的缺点:浴缸没有那么好晚上有蚊子

备注:好巧网综合打分是基于100多个酒店预订网站的评分综合算出。以上是该酒店在几家酒店预订网站上的评分,供您参考。好巧网对酒店特色的分析数据,来自主流的酒店预订网站、旅游社区网站提到该酒店的评论,尽量展示客观中立的分析。查询更多用户评论信息,请访问http://www.haoqiao.cn/Phuket_c3/274779.html

。

现因借款人__________身份证号:__________________ 因________________(写借款用途)需向贷款人_________借款得到人民币______________元整(小写_____元整)。借款种类为现金,借款日期为_____年____月____日,还款日期为______年____月____日前, 特立此据为凭。

借款人:_______ (亲笔签名并按手印)

贷款人:_______ (亲笔签名并按手印)

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借款人身份证复印件粘贴处:

借款人:向拥有资金的一方借出款项的人;

贷款人:向需要资金的一方发放贷款的人。

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现因借款人_______身份证号:_____________________ 因___________(写借款用途)需向贷款人________借款人民币_______________元整(小写_____元整)。借得款种类为现金,借得款日期为_____年____月____日,还款日期为____年____月____日前, 按时一次性偿还清¥:____________借款加利息。借款利息为: ___%(年利率),特立此据为凭。