2008～2009年珠海市H3N2亚型流感病毒HA1基因特性分析

为了解2008~2009年珠海市H3N2亚型流感病毒HA1基因变异情况,选择珠海市2008~2009年期间不同时间点的经狗肾传代细胞(MDCK)培养分离的H3N2亚型流感毒株20株,提取病毒RNA,通过RT-PCR扩增HA1基因片段,将产物纯化并测序,推导氨基酸序列,进行基因进化特性分析。与同时期的疫苗株比较,2008年珠海市流行的H3N2亚型流感毒株HA1区抗原决定簇的氨基酸位点变异数少于4个;2009年珠海市流行的H3N2亚型流感毒株除09-0056外,HA1区存在5个位于抗原决定簇内的变异氨基酸位点。2008年H3N2亚型流感毒株的HA1区的糖基化位点与疫苗株一致;2009年H3N2亚型流感毒株HA1区丢失第144位糖基化位点。2008~2009年H3N2亚型流感毒株RBS氨基酸序列未见明显变异。与2008年H3N2亚型流感毒株比较,2009年H3N2亚型流感毒株HA1区抗原决定簇内存在多个位点的氨基酸替换。这些说明2008年珠海市流行的H3N2亚型流感病毒不是新变种;2009年流行的H3N2亚型流感病毒为新的变异株,这可能是H3N2亚型流感病毒在2009年6-9月为珠海地区季节性流感流行优势株的原因。     

新发甲型H1N1流感病毒HA分子的变异分析

目的:从分子进化水平上分析流感的起源及发展问题,研究目前爆发的H1N1病毒的HA分子的变异行为.方法:以GenBank公布的甲型流感H1N1病毒血凝素(hemagglutinin,HA)核酸序列和我国及世界范围内近几年来报告的H1N1流感病毒HA的核酸及氨基酸序列为研究对象,利用CLUSTAL 1.83和NetNGlyc 1.0等生物信息学软件对HA核酸和氨基酸序列进行了比对分析;将其糖基化位点、氨基酸序列和抗原决定簇与以往流感病毒进行了比较.同时,还将人源和猪源甲型H1N1流感病毒的HA氨基酸序列进行了序列比对和系统发育分析.结果:最新爆发的甲型H1N1流感病毒的HA除了在60,259,453,512位点高度保守区域与之前爆发的流感病毒一致外,在249位点新出现1个"-NTT-"的糖基化位点.发现所有的甲型病毒的氨基酸序列在8个氨基酸位点均发生改变,而8个氨基酸位点位于6个抗原抗原决定簇上.结论:糖基化位点的增加,氨基酸位点的改变导致抗原决定簇的改变,即抗原性漂移现象,都成为引起其传染性改变的重要原因.     

2009年中国流行的甲型流感病毒（H1N1）血凝素特征分析

目的研究甲型流感病毒（H1N1）暴发流行以来中国各地甲型流感病毒血凝素（HA）的特征。方法搜索甲型流感病毒（H1N1）暴发流行以来中国各地报道的血凝素（HA）的氨基酸序列,比较当年不同时期血凝素（HA）的氨基酸序列的变化,并比较2009年报道的血凝素（HA）的氨基酸序列和2008年、2007年报道的血凝素（HA）的氨基酸序列作比较,以分析和前2年血凝素（HA）氨基酸序列相比所发生的变化。结果2009年中国各地甲型流感病毒（H1N1）的血凝素（HA）的氨基酸序列（人源）的同源性为99%-100%,但和2008年以及2007年的同源性非常低,分别为70%-77%和71%-90%。结论2009年暴发流行的甲型流感病毒（H1N1）的血凝素氨基酸序列较往年发生了很大程度的变异,这可能是今年甲型流感病毒（H1N1）暴发流行的主要原因。     

2010?2011年全球季节性H3N2流感病毒表面蛋白基因的分子遗传特性分析

[目的]分析2010年1月至2011年9月间全球季节性H3N2流感病毒血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)基因的演变和分子特征,为流感病毒的防制提供分子信息依据.[方法]搜集期间季节性H3N2流感病毒HA和NA基因的完整核苷酸序列,分别绘制两基因编码序列的进化树;推导出相应的氨基酸序列,统计不同毒株间氨基酸位点差异并分析重要功能位点的变化.[结果]在136条完整的片段4和131条片段6中,2条HA和l条NA序列源自猪群流感病毒,剩余的序列根据进化特征可被分为两群.相比疫苗毒株,发生在HA和NA蛋白抗原位点的平均差异数分别为5.33和2.01个,3个毒株分别在HA宿主受体结合位点和二硫键及NA耐药位点出现突变,多数毒株的糖基化位点增多.江苏毒株和广东毒株分别属于群l和群2,且两省毒株间在HA蛋白抗原位点的差异数从7到13个不等.[结论]2010年1月至2011年9月间的全球季节性H3N2病毒主要呈现两种基因进化特征.因抗原性差异对疫苗开发具有指导作用,而多数毒株的抗原性检测信息仍然未知,但从抗原位点和糖基化位点的变异情况来看,多数毒株的抗原性可能已经变化,为判断是否形成新的流行株,应开展进一步的抗原性检测;并且各地区卫生行政部门应根据耐药位点的变化,制定相应的抗病毒治疗措施.     

A(H3N2)亚型流感病毒血凝素基因特性及蛋白结构分析,以南宁市2009～2012年为研究案例

利用生物信息学软件和数据库研究南宁市2009~2012年度A(H3N2)亚型流感病HA基因的遗传变异规律及蛋白结构变化。通过RT-PCR扩增H3N2病毒HA基因并测序,同源比对统计毒株氨基酸位点的差异、构建系统进化树分析进化规律和同源建模分析蛋白结构的变化。系统进化树表明53株HA基因序列被分为4个类群,呈多侧支流行;所有毒株的二硫键和受体结合位点(RBS)高度保守;部分毒株HA在第45位点增加一个糖基化位点,在第144位点丢失一个糖基化位点;HA抗原决定簇氨基酸累计有30个位点发生变异,涉及5个抗原决定簇;HA晶体结构分析抗原决定簇突变位点主要发生在无规则卷曲处,大多数替换的氨基酸种类和性质相同或相似。南宁市A(H3N2)亚型流感病毒株变异活跃,2012年A(H3N2)亚型流感病毒与当年WHO推荐的疫苗株具有较远的进化距离,多数毒株具备了形成新变种的条件,是否形成新的流行株,有待深入研究。     

甲型H1N1流感监测及其HA1基因特性分析,以2013~2014年新余市为例

因HA基因的头部重链区(HA1)是流感病毒的抗原位点和受体结合位点,所以了解流感病毒的HA1基因特性可以评估当前WHO推荐的疫苗株的预防效果。本文选取新余市2013~2014年通过狗肾传代细胞(MDCK)培养分离到的H1N1流感毒株12株,提取病毒RNA,反转录-聚合酶链反应(RT-PCR)扩增HA1基因片段。对序列分析发现2013~2014年新余市甲型H1N1流感病毒HA1基因序列与疫苗推荐株有较高的同源性,核苷酸同源性97.9%~98.5%。2013年的6株毒株HA1区与疫苗株比较,氨基酸变异位点7~11个,其中3株有2个变异位点在不同的抗原决定簇区。而2014年毒株单独集中在进化树的一支。以上提示,目前流感疫苗对2014年的流感人群仍有保护作用,对2013年的部分流行的H1N1预防效果有限;甲型H1N1爆发流行的可能性很大,应加强防控。     

山东省甲型H1N1流感病毒病原学及基因特性研究

在2009~2010年监测年度开展甲型H1N1流感病毒学监测并进行病原学分离鉴定,以及对血凝素基因(HA)和神经氨酸酶基因(NA)特性分析,研究其基因变异情况。采集了17 126份发热患者的鼻、咽拭子标本,采用逆转录实时荧光定量RT-PCR(Real-Time RT-PCR)进行核酸检测,其中甲型H1N1流感病毒核酸检测阳性4004份,总阳性率为23.38%。对阳性标本开展病毒分离,并对分离的甲型H1N1流感病毒的HA、NA基因序列进行测序。利用DNAStar软件对序列进行同源性分析发现与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株HA、NA基因同源性分别为96.9%~99.3%和99.1%~99.6%;利用Mega 4.0软件进行基因进化分析和氨基酸进化分析发现,与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株有21个血凝素基因的氨基酸发生替换,其中11个氨基酸位点位于抗原决定簇区,一个糖基化位点发生改变;有16个神经氨酸酶基因的氨基酸发生了替换,一个糖基化位点发生改变;未发生神经氨酸酶蛋白275位H→Y的替换。结果显示山东省甲型H1N1流感暴发流行株HA基因和NA基因均具有高度同源性,HA蛋白和NA蛋白均存在不同程度的氨基酸替换,部分流行株抗原决定簇和糖基化位点发生改变,所有病毒均对达菲类药物敏感。     

一株2011年出现的季节性甲型H1N1流行性感冒病毒血凝素基因特性的研究

本文通过比较2011年分离培养的1株季节性甲型H1N1流行性感冒(简称流感)病毒(A/Shanghai/1167/2011(H1N1))与历年季节性甲型H1N1流感病毒的血凝素(HA)基因,追溯该病毒的基因变异与来源,探讨该毒株的出现对流感防控工作的意义.采用反转录-聚合酶链反应(RT-PCR)方法扩增病毒的HA和神经氨酸酶(NA)片段,并进行测序;应用分子生物学软件对获得的序列进行分析,绘制基因进化树;同时,通过血凝抑制试验检测2011年下半年健康人群中该流感病毒的抗体水平.结果显示,A/Shanghai/1167/2011(H1N1)的HA基因序列与世界卫生组织(WHO)2007～2008年季节性甲型H1N1流感病毒疫苗株A/Brisbane/59/2007(H1N1)最接近,同源性达99.2%,与新型甲型H1N1流感病毒A/California/07/2009疫苗株同源性仅为72.4%.其HA基因裂解位点为PSIQSR↓GLF,尚未出现高致病性的分子特征.HA片段共编码557个氨基酸,有9个潜在的糖基化位点,序列与2009年前WHO疫苗株A/NewCaledonia/20/1999(H1N1)、A/SolomonIslands/3/2006(H1N1)和/Brisbane/59/2007(H1N1)相比,分别有15、12和4处不同,这些差异分布在Sa、Sb、Ca1、Ca2、Cb 5个抗原决定簇的氨基酸差异分别有5、5和2处.该毒株在健康人群血清的抗体阳性率为34.33%,几何平均效价(GMT)为10.38.A/Shanghai/1167/2011(H1N1)是2011年出现在上海地区的一个季节性甲型H1N1流感病毒毒株,其抗原变异与既往季节性甲型H1N1流感病毒相比不大,但在以A(H1N1)pdm09为主要流行株的年份检测到散在发生的既往季节性甲型H1N1流感病毒毒株应当引起重视,其在人群中的抗体水平较低,易引起流行,需要提高对类流感人群中此种毒株的持续监测.     

H1N1亚型猪流感病毒广东分离株全基因克隆及其遗传演化分析

为了研究H1N1亚型SIV遗传演化与变异的特性,采用RT-PCR技术分别扩增A/Swine/Guangdong/LM/2004(H1N1)的8个基因片段,分别将其克隆到pMD18-T载体,进行全基因组序列测定.核苷酸序列测定结果显示:LM株SIV各基因片段均未发现核苷酸插入或缺失现象.HA切割位点处的氨基酸序列序列为IPSIQSR↓G,与高致病性SIV的H1N1亚型毒株的分子特征不符合.HA基因含有6个潜在的N-糖基化位点,4个在HAl的第11、23、87、和276位,增加2个分别在HA2的154和213位点;NA基因不仅在58、63、68、88和146位含有高度保守的N-糖基化位点,而且在44和235位增加2个潜在的N-糖基化位点,这可能是近期H1N1亚型SIV的一个分子特征.核苷酸同源性结果:HA基因与类人谱系的流感病毒分离株有很高的同源性(99%),而其他基因均与古典猪谱系的流感病毒分离株同源性较高(87%～98%).从绘制的各个片段进化树和核苷酸同源性分析结果,可以推测该毒株HA基因可能来源于类人谱系的流感病毒;而其他基因来源于古典猪谱系的流感病毒.     

Molecular evolution of influenza A (H3N2) viruses circulated in Fujian Province, China during the 1996–2004 period

We studied the genetic and epidemic characteristics of influenza A (H3N2) viruses circulated in human in Fujian Province, south of China from 1996 to 2004. Phylogenetic analysis was carried out for genes encoding hemagglutinin1 (HA1) of influenza A virus (14 new and 11 previously reported reference se-quences). Our studies revealed that in the 8 flu seasons, the mutations of HA1 genes occurred from time to time, which were responsible for about four times of antigenic drift of influenza H3N2 viruses in Fujian, China. The data demonstrated that amino acid changes were limited to some key codons at or near antibody binding sites A through E on the HA1 molecule. The changes at the antibody binding site B or A or sialic acid receptor binding site 226 were critical for antigenic drift. But the antigenic sites might change and the key codons for antigenic drift might change as influenza viruses evolve. It seems important to monitor new H3 isolates for mutations in the positively selected codons of HA1 gene in south of Asia.     

辽宁省2016‒2017年H3N2亚型流感病毒 HA1基因特征分析

摘要：目的 了解2016?2017年辽宁省H3N2亚型流感病毒基因变异情况及流行株与疫苗株的匹配情况。方法 采用逆转录聚合酶链反应（RT-PCR）对分离得到的H3N2亚型流感毒株的HA1基因进行扩增,扩增片段经测序与近年来WHO推荐的北半球疫苗株进行比对和基因特征分析。结果 进化分析表明,2016?2017年H3N2亚型流感病毒与近三年的疫苗株均不在同一分支上;基因特性分析中,所有病毒均在A、B抗原决定簇上发生了两处以上的变异;19株病毒的受体结合位点131位氨基酸发生了新的变异;20株病毒中有1株突变产生了新的半胱氨酸,提示可能有新的二硫键产生;糖基化位点并未检测到新的突变。结论 2016?2017年辽宁省H3N2亚型流感病毒的抗原性及基因特性均发生了一定的变化,但变异程度不大,应密切关注疫苗株对流感病毒的免疫效果及流感毒株的变异情况。     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

2017年河北省蛋鸡养殖场H9N2亚型禽流感病毒的遗传演化和抗原性分析

【背景】H9N2亚型禽流感病毒在鸡群中广泛流行,引起巨大损失。【目的】了解河北省蛋鸡养殖场H9N2亚型禽流感病毒(avian influenza virus,AIV)的基因序列和抗原性的变异情况,为该病原的科学防控提供理论依据。【方法】于2017年从河北省部分蛋鸡养殖场分离鉴定出7株H9N2亚型AIV,对其HA基因进行序列测定,并进行遗传演化、关键氨基酸位点及抗原性分析。【结果】7株分离毒株HA基因同源性在95.5%?97.2%之间;与2016年前的流行毒株相比,分离病毒HA裂解位点均为典型低致病性AIV特征,在受体结合区域出现变异,潜在糖基化位点无明显差异;抗原分析结果显示分离毒株与早期分离株相比抗原性发生了变异,形成了新的抗原群;抗原性相关位点分析显示,分离毒株在9个位点发生了较为明显的突变,可能是导致抗原性变异的分子基础。【结论】河北省蛋鸡养殖场H9N2亚型AIV中的流行毒株在关键功能区发生基因突变,并且抗原性发生变异,提示应持续监测H9N2亚型AIV的遗传变异情况,并及时更换疫苗株。     

Predicting the Antigenic Structure of the Pandemic (H1N1) 2009 Influenza Virus Hemagglutinin

The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.     

Computational analysis of antigenic epitopes of avian influenza A (H7N9) viruses

Influenza virus can rapidly change its antigenicity, via mutation in the hemagglutinin (HA) protein, to evade host immunity. The emergence of the novel human-infecting avian H7N9 virus in China has caused widespread concern. However, evolution of the antigenicity of this virus is not well understood. Here, we inferred the antigenic epitopes of the HA protein from all H7 viruses, based on the five well-characterized HA epitopes of the human H3N2 virus. By comparing the two major H7 phylogenetic lineages, i.e., the Eurasian lineage and the North American lineage, we found that epitopes A and B are more frequently mutated in the Eurasian lineage, while epitopes B and C are more frequently mutated in the North American lineage. Furthermore, we found that the novel H7N9 virus (derived from the Eurasian lineage) isolated in China in the year 2013, contains six frequently mutated sites on epitopes that include site 135, which is located in the receptor binding domain. This indicates that the novel H7N9 virus that infects human may already have been subjected to gradual immune pressure and receptor-binding variation. Our results not only provide insights into the antigenic evolution of the H7 virus but may also help in the selection of suitable vaccine strains.     

Immunodominance of Antigenic Site B over Site A of Hemagglutinin of Recent H3N2 Influenza Viruses

H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A-E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006-07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008-09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). "Native ELISA" analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006-07 and/or 2008-09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.     

1981～2005年中国H1N1甲型流感病毒血凝素基因的HA1演变特征

为了解1981~2005年我国H1N1甲型流感病毒血凝素基因的HA1演变特征,选取H1N1甲型流感病毒370株,提取病毒RNA,经逆转录和聚合酶链反应扩增HA1并测序,测定的序列用生物信息软件分析,与GenBank中相关序列比较,并对推导的编码氨基酸序列进行基因特性分析。结果表明:HA1氨基酸的变异表现为抗原决定簇4个区均有变异,Sb区和Ca区变化较大;HA1受体结合位点(RBS)的前壁130环的第134位赖氨酸从1991年起在部分毒株HA1序列上开始缺失,以后缺失株逐步增多,自2000年起测定的所有毒株上该氨基酸全部缺失,同时这些缺失株的第137位氨基酸也全部由苏氨酸替换为丝氨酸;糖基化位点从增多到减少,最后稳定在7个;1981~2004年我国H1N1甲型流感病毒血凝素HA1编码的氨基酸在种系发育树上同年代基本呈现集中分布,与时间和地域无关,2005年毒株分成两个分支在时间上有明显差异。     

Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies

We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) ΔHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants’ HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.