季节性流感病毒 H1N1 BALB / c 鼠肺适应株的建立及其分子机制简

目的 建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究.方法 以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株.结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变.结论 野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用.     

季节性流感病毒H1N1 BALB/c鼠肺适应株的建立及其分子机制

目的建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究。方法以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株。结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变。结论野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用。     

甲型H1N1／2009流感病毒的种属跨越机制

在世界范围流行的甲型H1N1／2009流感病毒具有下述3个重要特征：可寄生于人体,易感人群很多,患者年龄偏低．本研究确定了病毒蛋白中的一块关键区域．该区域对病毒所寄生的物种的种属范围起决定性作用,并且是全球性流感病毒的一个标志性区域．正是该区域氨基酸的特性导致了上述3个特点．具体来说,对宿主的免疫系统而言,病毒蛋白质结构的变化会形成新的标靶结构,并且可以进一步导致宿主范围的变化．基于多肽链发生致病性结构转换的概率,本研究确定了甲型流感病毒中对控制宿主范围起决定性作用的氨基酸的位置．研究发现甲型H1N1／2009流感病毒中处于这些位点的多肽链在本质上可以在寄生于人的毒株中表达,而之前仅在宿主为禽、猪的毒株中被发现．其与另一氨基酸短串的协同构象改变对于甲型H1N1／2009流感病毒的种属跨越具有重要作用．人体对这些关键位点的免疫缺陷导致了甲型H1N1／2009流感病毒宿主人群多和青年人易致病的特点．     

重组人禽流感H7N9疫苗株在Vero细胞上的适应性和传代稳定性研究

本文主要研究重组人感染高致病性禽流感H7N9株作为主工作种子批在Vero细胞连续传代中的高产特性和传代稳定性,为疫苗开发提供前期研究。将重组获得的H7N9流感病毒Vero细胞适应株(A/Anhui/1/2013Va)按照病毒感染复数(MOI) 0. 01的病毒量接种于Vero细胞,并连续传15代。血凝试验检测每代病毒血凝效价,并绘制病毒在Vero细胞上的生长曲线;每代病毒用MDCK细胞检测病毒的感染性滴度;通过单向免疫扩散试验对重组病毒H7N9的第1及第16代进行型别鉴定;取第1、5及16代病毒,用鸡胚半数感染量及致死量检测病毒在传代过程中毒力变化;分别提取第1和16代H7N9流感病毒的RNA,反转录及克隆后测序,对比病毒在连续传代过程中血凝素(HA)和神经氨酸酶(NA)基因的变化。试验结果表明:在连续传代过程中,病毒效价越来越趋于稳定,血凝效价稳定维持在384～448;病毒感染性滴度维持在7 Log10TCID50/mL左右;病毒在传代过程中毒力未发生显著变化; HA和NA在连续传代过程中并未出现因基因变异导致的编码氨基酸改变的状况。H7N9安徽株病毒在Vero细胞传代过程中不仅产量稳定,其毒力和基因也很稳定,可以将其作为H7N9流感疫苗候选株。     

PR8F/NS1不同位点突变株对小鼠致病性的比较研究

为了探索NS1不同位点突变对流感病毒毒力的影响以及NS1毒力相关位点的作用机制。分析流感病毒NS1蛋白的毒力相关关键基因区段和关键位点,以实验室保存的H1N1亚型流感病毒PR8F为模板,对其NS1基因的第42、81、149位氨基酸分别进行定点突变。利用反向遗传操作技术,成功拯救出突变毒株PR8F-42、PR8F-81、PR8F-149。将获救病毒接种SPF鸡胚,连续传代5代,至病毒能够稳定扩增后,通过血凝效价分析病毒复制效率。并将获救病毒与PR8F均以106 TCID50/100μL感染BALB/c小鼠,观察各组小鼠临床表现、体重变化及存活率。剖检攻毒后死亡小鼠,观察病理特征,制作肺组织病理切片。并提取小鼠肺脏RNA,通过qPCR检测各组小鼠肺脏的病毒载量。结果表明,PR8F的NS1蛋白第42位氨基酸由丝氨酸(Ser)改变为脯氨酸(Pro)对小鼠的致病性无明显改变。第81位、149位氨基酸突变则都能减弱突变株对小鼠的致病性,进而为研究NS1毒力相关位点的作用机制提供平台。     

甲型流感病毒X31(H3N2)小鼠适应后毒力增强与HA及PB2突变相关

流感小鼠适应毒株及感染模型是研究流感病毒致病机理、评价抗流感病毒药物和疫苗效果的重要工具。为建立甲型流感病毒X31(H3N2)的鼠肺适应毒株,并探讨其在小鼠适应过程中毒力增强的分子机制,本研究将X31在BALB/c小鼠肺组织中连续传代,通过观察病毒滴度测定、感染小鼠症状变化、体重改变和致死力等指标,发现X31经过鼠肺连续传代10次以后,病毒毒力逐渐增强,与原代病毒相比,鼠肺中病毒滴度提高10倍,半数致死量(50%lethal dose,LD_(50))提高大于1 000倍。确定获得高致病力的鼠肺适应毒株(Mouse adapted X31,X31_(MA));对X31_(MA)毒株全基因组测序发现,血凝素(Hemagglutinin,HA)基因出现3个有义突变(P221H、V309I、K411T),聚合酶碱性蛋白2(Basic protein 2,PB2)基因出现1个有义突变(M483T)。结果表明,通过在小鼠肺组织中连续传代X31(H3N2)致病力增强,其HA和PB2蛋白中四个适应性突变位点可能与流感病毒X31_(MA)毒力增加相关。本研究为流感疫苗评价及致病机制研究提供了重要技术支持。     

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.     

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.     

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。     

深圳市新甲型H1N1流感病毒感染病例的血清学及分子特点分析

本研究通过对深圳市4名甲流重症患者的血清抗体及其所感染的新甲型H1N1流感病毒的抗原性和分子特点的分析,发现这些患者在感染后短期内产生的血清中和抗体滴度均不超过1:20,不能起到有效的保护作用;交叉血凝抑制实验的结果显示新H1N1病毒与季节性H1N1和H3N2流感病毒无任何交叉反应,抗原性差异很大,而患者所感染的病毒与标准株的抗原性则没有太大差异;分子特点的分析表明新H1N1病毒进入人群后依然属于经典的猪流感亚系,4名重症患者感染的病毒不具备高致病性流感病毒的遗传特点,几个氨基酸位点的变异没有影响病毒的毒力和致病性,只有一株毒株的NA蛋白发生了His275Tyr的突变,产生了对达菲等神经氨酸酶抑制剂的耐药性。     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

甲型H1N1流感病毒HA蛋白结构和功能研究进展

自2009年3月,甲型H1N1流感疫情相继在包括我国在内的许多国家暴发,对人体健康和社会经济发展造成了严重危害。血凝素（HA）蛋白是重要的病毒表面糖蛋白,主要有3种功能：①与宿主细胞表面受体结合;②引起病毒包膜与靶细胞间的膜融合;③刺激机体产生中和性抗体。本文综合了近年来的研究成果,对甲型H1N1流感病毒HA蛋白结构、主要功能、进化、抗原性的研究进展进行了综述。     

Analysis of cellular proteome alterations in porcine alveolar macrophage cells infected with 2009 (H1N1) and classical swine H1N1 influenza viruses

The H1N1/2009 influenza virus has the potential to cause a human pandemic, and sporadic cases of human-to-pig transmission have been reported. In this study, two influenza viruses were isolated from pigs. A phylogenetic analysis showed that the A/swine/NanChang/F9/2010(H1N1) (F9/10) strain shared a high degree of homology with the pandemic H1N1/2009 virus, and A/swine/GuangDong/34/2006 (H1N1) (34/06) strains was a classical swine influenza virus. A proteomic analysis was performed to investigate possible alterations of protein expression in porcine alveolar macrophage (PAM) cells infected by the F9/10 and 34/06 viruses over different time courses. Using 2-DE in association with MALDI-TOF MS/MS, we identified 13 up-regulated and 21 down-regulated protein spots, including cytoskeleton proteins, cellular signal transduction proteins, molecular biosynthesis proteins and heat shock proteins. The most significant changes in the infected cells were associated with molecular biosynthesis proteins and heat shock proteins. We analysed the biological characteristics of the F9/10 and 34/06 viruses in vivo and in vitro. The F9/10 virus showed greater pathogenicity than the 34/06 virus in PAM cells and mice. This study provides insights into the biologic characteristics, potential virulence alteration and cross-species transmission mechanisms of the pandemic H1N1/2009.     

Absence of 2009 Pandemic H1N1 Influenza A Virus in Fresh Pork

The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.     

Epidemiological update on swine influenza (H1N1) in pigs

The 2009 H1N1 pandemic has slowed down its spread after initial speed of transmission. The conventional swine influenza H1N1 virus (SIV) in pig populations worldwide needs to be differentiated from pandemic H1N1 influenza virus, however it is also essential to know about the exact role of pigs in the spread and mutations taking place in pig-to-pig transmission. The present paper reviews epidemiological features of classical SIV and its differentiation with pandemic influenza.     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.     

Cytokine and chemokine responses in pediatric patients with severe pneumonia associated with pandemic A/H1N1/2009 influenza virus

Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty‐seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)‐10 (P = 0.027) and IL‐5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon‐γ (P = 0.009), tumor necrosis factor‐α (P = 0.01), IL‐4 (P = 0.024), and IL‐2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL‐8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.