浙江省人感染H7N9禽流感病毒的基因组序列分析

目的分析浙江省2013年4月初一例人感染甲型H7N9禽流感患者的病原学和基因组序列特征.方法提取患者标本的病毒RNA并用荧光定量RT-PCR方法检测,一步法RT-PCR扩增H7N9禽流感病毒基因组的8个片段,测序并拼接出基因组序列.下载目前已经公布的H7N9禽流感病毒和其他H7、N9亚型的病毒的HA和NA序列,采用Mega 5.1软件对其进行序列比对并构建进化树.根据测序的结果分析该例H7N9禽流感病毒的序列变异情况.结果该患者标本甲型流感、H7亚型和N9亚型均为阳性,通过扩增基因组各片段并进行测序.对血凝素和神经氨酸酶基因进行比对和进化树构建,结果显示该H7N9病毒HA基因与A/duck/Zhejiang/12/2011(H7N3)的亲缘关系最近,NA基因与A/wild bird/Korea/A14/2011 (H7N9)的亲缘关系最近.编码内部蛋白的6个基因均与中国大陆近两年的H9N2毒株最为相似.该标本的病毒HA蛋白发生了Q226L突变,而与已经公布的人感染H7N9禽流感毒株均不一致的是PB2未发生E627K突变.结论从该份临床标本完成了检测和基因组各片段的扩增与测序.该病毒与已报道的人感染H7N9禽流感病毒同源性高,存在Q226L等重要位点的突变,但PB2未发生E627K突变.     

甘肃省人感染和外环境来源的H9N2禽流感病毒基因特征分析

目的 分析甘肃省发现的人感染H9N2禽流感病毒分离株全基因组特征。方法 对甘肃省2016年流感样病例中发现并确诊的1例人感染H9N2禽流感病毒病例进行病原学分析,并使用MEGA 7.0等软件解析该毒株全基因组特征。结果 该毒株HA、NA、MP、NP、NS、PA、PB1和PB2各个基因片段与甘肃省2014-2019年外环境中分离获得的H9N2禽流感病毒各基因片段高度相似,且均>90%;其HA基因属于BJ/94-like支系,PB2和MP属于G1/97-like支系,PB1、PA、NS和NP基因属于F/98-like支系,MP和PB2与H7N9、H10N8和H5N6亲缘关系较近;氨基酸序列比对发现HA裂解位点呈PSRSSR↓GLF排列,发生H183N和Q226L突变,有7个HA糖基化位点;NA茎部62～64位均缺失ITE 3个氨基酸;M2-31N、NS1-42S、PA-356R和PA-409N突变。结论 人感染H9N2禽流感病毒为偶发感染,但甘肃省外环境中分离的H9N2禽流感病毒具有一系列哺乳动物适应性分子标记,提示人群感染风险较高,需多部门加强监测,共同应对流感大流行。     

长沙市1例人感染H3N8禽流感病例的流行病学调查和病原学分析

目的 了解人感染H3N8禽流感病例的流行病学特征和临床特征及其病原的分子生物学特征，为人感染H3N8禽流感防控提供科学依据。方法 采用流行病学调查方法对长沙市2022年5月1例人感染H3N8禽流感病例的可疑暴露史、感染途径、发病及就诊经过，以及密切接触者、可疑暴露者等涉疫重点人员进行现场调查，并进行描述性分析；采用核酸检测和基因测序技术，进行病原学检测和基因特征分析。结果 病例发病前6 d有活禽交易市场暴露史，在活禽交易市场、活禽来源批发市场均检测出禽流感H3、N8亚型阳性。深度基因测序显示，病毒对哺乳动物发生适应性突变，对烷胺类药物敏感性降低，未检测到神经氨酸酶抑制剂类药物和聚合酶抑制剂类药物相关的耐药性突变。结论 病例暴露于被H3N8禽流感病毒污染的活禽交易市场环境而感染，未发现人际间传播。     

安徽省2株人感染H9N2流感病毒基因特征

目的 分析2015年安徽省2株人感染H9N2禽流感病毒分离株全基因组特征。方法 对安徽省2015年4月和9月流感监测网络实验室先后发现并确诊的2例H9N2禽流感病毒感染病例及其病毒分离株全基因组序列进行分析,使用Mega 6.0等软件解析2株H9N2毒株全基因组特征。结果 安徽省首次报道人感染H9N2禽流感病毒病例,对病毒分离株分析显示,其HA和NA基因与中国2013年禽间流行的H9N2病毒高度同源,属于A/Chicken/Shanghai/F/98(H9N2)支系;而PB2和MP基因属于A/quail/Hong Kong/G1/97支系,与H7N9、H10N8和H6N2具有较高的相似度。氨基酸序列比对发现该2株病毒出现多个人易感倾向位点分子特征:HA蛋白发生Q226L、H183N和E190T突变,且HA裂解位点为PSRSSR\GL排列,NA蛋白茎部发生63~65位氨基酸缺失,M2蛋白S31N突变,以及PA-100A、PA-356R和PA-409N。结论 安徽省首次报道的人感染禽源H9N2流感病毒为H9N2系间重配病毒,存在多个人易感位点。     

广州市2014-2019年H5亚型禽流感病毒流行与基因遗传特征分析

目的 分析广州市H5亚型禽流感病毒流行特点及基因进化和变异特征,为禽流感的防控提供依据。方法 对2014-2019年广州市禽类市场外环境标本进行禽流感病毒核酸检测并分析H5亚型流行情况,随机选取48份（46份来源于环境和2份来源于病例）H5阳性标本进行HA和NA基因测序,应用生物信息学软件分析分子遗传特征。结果 2014-2019年监测的52 284份环境标本中,检出H5亚型阳性标本1 094份,阳性率为2.09%。遗传进化分析显示,HA基因属于Clade 2.3.4.4.C分支,NA基因主要归属于欧亚谱系H6N6进化分支,HA和NA基因的进化以时间聚类为特点,相近年份流行株聚成一个进化分支。分子特征显示,48份毒株裂解位点呈现高致病性分子特点,受体结合位点仍为禽源受体,但普遍发生S123P、S133A和T156A倾向结合人源受体的位点突变。抗原位点变异主要出现在B、E区,并表现时间分布的特点,同一年份流行株常发生与上一年份流行株不同的抗原位点变异。2017年开始所有毒株出现由氨基酸缺失导致的糖基化位点的增加（140-NHT）。结论 广州市禽类市场外环境H5亚型禽流感病毒长期流行,且阳性率占比逐渐升高。测序分析提示,广州市H5亚型禽流感病毒为Clade 2.3.4.4.C分支H5N6高致病性病毒,并且持续进化和变异,特别是普遍出现与人源受体结合能力增强的突变,需加强监测。     

云南边境地区禽流感H5N1亚型病毒NS基因进化分析

目的 阐明云南边境地区禽流感H5N1亚型病毒NSl、NS2基因变异特征及遗传进化关系。方法 在云南边境地区采集境外家禽和野生鸟类棉拭子样品,经H5N1亚型特异性多重RT-PCR检测,阳性样品对病毒NS基因进行扩增,克隆Z至pMDl8一T载体测序,获得NSl、NS2基因序列,并与已知参考毒株序列进行序列比对及系统发育分析。结果 自1240份样品中检出H5N]亚型阳性样品71份,阳性率为5．72％;30份代表性阳性样品病毒NS基因测序获得17种序列,存在3个不同进化(亚)分支(I一1、I一2、Ⅱ);NSl／NS2基因与病毒血凝素(HA)基因呈现不同进化关系;NSl蛋白涉及核定位信号区、RNA结合区、效应区及其他致病性相关的关键性氨基酸位点存在替代或突变。结论 云南边境地区H5N1亚型病毒NSl／NS2基因具有遗传差异,2010年以来NS基因进化分支I一2、Ⅱ已成为当地流行的优势毒株。     

云南省边境地区禽流感H5N1亚型病毒遗传多样性分析

目的 了解云南省边境地区禽流感H5N1亚型病毒遗传多样性.方法 2009-2011年7月在云南省边境地区采集境外家禽和野生鸟类棉拭子样品,经H5/Nl亚型特异性多重RT-PCR检测,阳性样品进行病毒HA基因扩增,克隆至pMD 18-T载体测序,并与已知参考毒株进行序列比对及系统发育分析.结果 36份阳性样品病毒HA基因测序获得15种HA序列,存在2个不同进化分支(2.3.2、2.3.4),2.3.2进一步可划分为3个进化小分支(Ⅱ-1～Ⅱ-3),2.3.4进一步可划分为2个进化小分支(Ⅰ-1和Ⅰ-2).2.3.2Ⅱ-1、Ⅱ-2毒株是新出现的H5N1亚型病毒变异株.结论 2009- 2011年7月云南省边境地区H5N1亚型病毒具有遗传多样性,病毒经历了多分支(2.3.2、2.3.4)至单一支(2.3.2)的进化过程.     

一起无防护禽肉加工引起人感染H7N9禽流感病毒的流行病学调查

目的 分析1例人感染高致病性H7N9禽流感病例的感染模式及病原变异情况,为禽流感防控提供依据。方法 采用流行病学方法调查病例可疑暴露史及感染途径,追踪调查病例病情进展;使用核酸检测、病毒分离、基因测序及进化分析等技术对采集的相关标本展开病原学分析。结果 病例无活禽接触史,发病前一周在狭小通风不畅厨房内不带手套加工烹饪光鸡;病例下呼吸道提取物、病家剩余冷冻光鸡表面涂抹标本、活禽来源市场环境标本均检出高度同源的H7N9禽流感病毒,且均在HA基因的裂解位点出现多个碱性氨基酸（PEVPKRKRTAR/GL）插入的突变。结论 无防护禽肉操作是"禽-人"传播模式下的重要感染方式之一,现行的活禽限售区防控措施效果有限,应尽快推进规模化标准养殖,实现活禽全城限售、集中屠宰、冰鲜上市。     

贵州省2014-2017年H7N9禽流感病毒HA和NA基因特征分析

目的 分析2014－2017年贵州省H7N9禽流感病毒HA和NA基因的分子特征和感染风险。方法 对2014－2017年贵州省获取的18例确诊人感染H7N9病例和6份环境样本分离的H7N9病毒株的HA和NA基因进行扩增,运用生物信息学软件解析其变异和遗传进化。结果 2014－2015年贵州省2株毒株与WHO推荐的A/Shanghai/2/2013和A/Anhui/1/2013疫苗株HA和NA基因的同源性最高,分别为98.8%～99.2%和99.2%;2016年2株和2017年14株与A/Hunan/02650/2016疫苗株同源性最高,分别为98.2%～99.3%和97.6%～98.8%;2017年其余6株与A/Guangdong/17SF003/2016疫苗株同源性最高,为99.1%～99.4%和98.9%～99.3%。所有毒株均属于长江三角洲分支,但主要聚集为3个次分支。2017年贵州省西部有6株毒株（含2例人感染病例毒株）裂解位点存在插入突变为PEVPKRKRTAR↓GLF,具备高致病性禽流感病毒的分子特征;受体结合关键位点存在A134V、G186V和Q226L突变,NA蛋白颈区“QISNT”均缺失,毒株A/Guizhou-Danzhai/18980/2017发生耐药突变R294K,9株毒株NA蛋白糖基化位点发生NCS42NCT突变。结论 2014－2017年贵州省H7N9禽流感病毒HA和NA基因存在遗传差异,关键位点的变异增强了病毒对人体的易感性和毒力,部分毒株发生耐药突变,其感染与致病风险增加。     

一株H5N1亚型禽流感病毒全基因组克隆及HA基因分子进化分析

目的对禽流感H5N1亚型病毒株A/Chicken/Hubei/489/2004的全基因组进行克隆和测序,并分析血凝素基因HA的遗传突变特点及其与1996年以来其他病毒株的亲缘关系。方法通过RT-PCR扩增病毒株A/Chicken/Hubei/489/2004的8个基因,并将其克隆到测序载体;在对病毒株全基因组序列测定基础上,利用生物信息学软件对HA基因进行遗传进化分析。结果病毒株A/Chicken/Hubei/489/2004的全基因组克隆到PMD18-T;遗传进化分析显示该毒株HA蛋白具有与致病性有关的切割位点"PQRERRRKKR",并且与2000～2006年在香港从人和禽体内分离的H5N1亲缘关系相近,也与2003～2004年在东南亚从人和禽体内分离的H5N1极其相关。结论 A/Chicken/Hubei/489/2004病毒分离株具有与A/Chicken/HongKong/YU777/2002更相似的遗传特性,极可能是2002年禽流感病毒家系的再次出现。     

Influenza activity in China: 1998-1999

During 1989-1999, influenza A H3N2 and H1N1 subtypes and B type viruses were still co-circulating in human population in China, while influenza A (H3N2) virus was predominant strain. The two antigenically and genetically distinguishable strains of influenza B virus were also still co-circulating in men in southern China. The antigenic analysis indicated that most of the H3N2 viruses were A/Panama/2007/99 (H3N2)-like strain, the most of the H1N1 viruses were antigenically similar to A/Beijing/262/95 (H1N1) virus. However, most of the influenza B viruses were B/Beijing/184/93-like strain, but few of them were antigenically similar to B/Shandong/7/97 virus. In the summer of 1998, the influenza outbreaks caused by H3N2 subtype of influenza A virus occurred widely in southern China. Afterwards, during 1998-1999 influenza season, a severe influenza epidemic caused by H3N2 virus emerged in northern China. The morbidity was reached as high as 10% in Beijing area. It was interesting that during influenza, surveillance from 1998 to 1999, five strains of avian influenza A (H9N2) virus were isolated from outpatients with influenza-like illness in July-August of 1998, and another one was repeatedly isolated from a child suffering from influenza-like disease in November of 1999 in Guangdong province. The genetic analysis revealed that the five strains isolated in 1998 were genetically closely related to H9N2 viruses being isolated from chickens (G9 lineage virus), whereas, A/Guangzhou/333/99 (H9N2) virus was a reassortant derived from reassortment between G9 and G1 lineage of avian influenza A (H9N2) viruses due to its genes encoding the HA, NA, NP and NS proteins, closely related to G9 lineage virus, the rest of the genes encoding the M and three polymerase (PB2, PB1 and PA) were closely related to G1 lineage strain of H9N2 virus. However, no avian influenza A (H5N1) virus has so far been isolated neither from in or outpatients with influenza-like disease in mainland China. Unfortunately, where did the reassortment occur and how did the reassortant transmit to men? These questions are still unknown.     

Clinical,epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus

A human infection with novel avian influenza A H5N6 virus emerged in Changsha city, China in February, 2014. This is the first detected human case among all human cases identified from 2014 to early 2016. We obtained and summarized clinical, epidemiological, and virological data from this patient. Complete genome of the virus was determined and compared to other avian influenza viruses via the construction of phylogenetic trees using the neighbor-joining approach. A girl aged five and half years developed fever and mild respiratory symptoms on Feb. 16, 2014 and visited hospital on Feb. 17. Throat swab specimens were obtained from the patient and a novel reassortant avian influenza A H5N6 virus was detected. All eight viral gene segments were of avian origin. The hemagglutinin (HA) and neuraminidase (NA) gene segments were closely related to A/duck/Sichuan/NCXN11/2014(H5N1) and A/chicken/Jiangxi/12782/2014(H10N6) viruses, respectively. The six internal genes were homologous to avian influenza A (H5N2) viruses isolated in duck from Jiangxi in China. This H5N6 virus has not gained genetic mutations necessary for human infection and was suggested to be sensitive to neuraminidase inhibitors, but resistant to adamantanes. Epidemiological investigation of the exposure history of the patient found that a live poultry market could be the source place of infection and the incubation period was 2–5 days. This novel reassortant Avian influenza A(H5N6) virus could be low pathogenic in humans. The prevalence and genetic evolution of this virus should be closely monitored.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

甲型H1N1流感病毒基因组序列分析及其特性研究

目的 分析甲型H1N1流感病毒的基因组序列特征,阐明该毒株的遗传变异及分子特性.方法 GenBank中获取流感病毒全序列,对各段基因与已知序列进行分析比较,绘制进化树,并分析和预测甲型毒株的致病性、药物敏感性和现有疫苗的预防保护作用.结果 甲型H1N1病毒的HA、PB2、PB1、PA、NP、NS基因与美国本土的猪流感病毒序列具有高度同源性,NA和M基因具有典型的欧亚株系猪流感病毒特征.该病毒具有人传人的分子基础,HA上HA1和HA2裂解位点序列为PSIQSR↓+GLFGAI,尚不具备高致病性流感病毒的特征.病毒对金刚烷胺类药物耐药,而对达菲和扎那米韦敏感.HA片段5个抗原决定区氨基酸序列与人用流感疫苗具有较大差异,推测现有疫苗对预防本次疫情基本无效.结论 甲型H1N1是一种北美和欧亚两种猪流感病毒的混合体,开发针对本病毒的流感疫苗有助于进一步控制疫情蔓延.     

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.     

Development of DIVA (differentiation of infected from vaccinated animals) vaccines utilizing heterologous NA and NS1 protein strategies for the control of triple reassortant H3N2 influenza in turkeys

Since 2003, triple reassortant (TR) swine H3N2 influenza viruses containing gene segments from human, avian, and swine origins have been detected in the U.S. turkey populations. The initial outbreak that occurred involved birds that were vaccinated with the currently available H3 swine- and avian-origin influenza vaccines. Antigenically, all turkey swine-lineage TR H3N2 isolates are closely related to each other but show little or no antigenic cross-reactivity with the avian origin or swine origin influenza vaccine strains that are currently being used in turkey operations. These results call for re-evaluation of currently available influenza vaccines being used in turkey flocks and development of more effective DIVA (differentiation of infected from vaccinated animals) vaccines. In this study, we selected one TR H3N2 strain, A/turkey/OH/313053/04 (H3N2) that showed broad cross reactivity with other recent TR turkey H3N2 isolates, and created NA- and NS-based DIVA vaccines using traditional reassortment as well as reverse genetics methods. Protective efficacy of those vaccines was determined in 2-week-old and 80-week-old breeder turkeys. The reassortant DIVA vaccines significantly reduced the presence of challenge virus in the oviduct of breeder turkeys as well as trachea and cloaca shedding of both young and old breeder turkeys, suggesting that proper vaccination could effectively prevent egg production drop and potential viral contamination of eggs in infected turkeys. Our results demonstrate that the heterologous NA and NS1 DIVA vaccines together with their corresponding serological tests could be useful for the control of TR H3N2 influenza in turkeys.     

金华市首例甲型H1N1流感死亡病例病毒分离及HA基因序列分析

目的分离甲型H1N1流感病毒,分析金华市首例甲型H1N1流感死亡病例病毒分离株HA基因序列特征,为研究病毒进化、致病性、流行规律提供科学依据。方法采用MDCK细胞和real-time PCR法进行病毒分离、鉴定;提取病毒RNA,通过RT-PCR扩增其HA基因片段,测定核苷酸序列;利用GeneBank中相关序列对病毒分离株A/Jinhua/01/2009（H1N1）进行基因进化树分析。结果从3份咽拭子样本中分离出1株甲型H1N1流感病毒。HA基因序列分析证明：该毒株与2009年大流行株高度同源,与古典型猪流感亲缘性较近,与欧亚地区分离的A/H1N1猪流感病毒及A/H1N1禽流感病毒进化关系较远。结论金华市首例甲型H1N1流感死亡病例分离病毒株与北美流行株高度同源,可能是由古典型猪流感进化而来,与WHO选定的甲型H1N1疫苗候选株同源性较高,对于人季节性流感A/H1N1疫苗可能不敏感。