H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用

为建立H1亚型猪流感病毒抗体检测方法,扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分,构建原核表达载体pET30a-HA1,并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原,建立间接ELISA检测方法。利用该检测方法检测了2008?2009年采集的猪血清785份,阳性率为15.54％,不同省份的阳性率存在差异 (8％~47％)。以IDEXX相关试剂盒检测结果作为参照,该方法的诊断特异性达到91％,诊断敏感性达到95％。     

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.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

中国“人-猪-禽”基因重排H1N2亚型猪流感病毒全基因克隆及遗传进化的研究

[目的]为了研究2006年从广西病猪肺组织中分离的H1N2亚型猪流感病毒(SIV)A/Swine/Guangxi/13/2006(H1N2)(Sw/Gx/13/06)的遗传学特性和8个基因的来源.[方法]运用RT PCR方法对其全基因进行了克隆并运用分子生物学软件对其基因序列进行了遗传进化分析.[结果]血凝素(HA)、核蛋白(NP)、基质蛋白(M)和非结构蛋白(NS)基因来源于猪古典H1N1亚型流感病毒;神经氨酸酶(NA)和聚合酶蛋白(PB1)基因来源于人的H3N2亚型流感病毒;聚合酶蛋白(PA)和聚合酶蛋白(PB2)基因来自于禽流感病毒.[结论]可见Sw/GX/13/06是一株"人-猪-禽"三源基因重排H1N2亚型SIV且与美国(1999-2001年)和韩国(2002年)分离到该型病毒的有明显的亲缘关系.据我们所知,这是中国首次报道含有禽流感病毒基因片段的重排H1N2 SIV,该病毒是否对养猪业和人类公共卫生健康具有潜在的威胁,有待于进一步研究.     

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009

In 2013, three reassortant swine influenza viruses (SIVs)—two H1N2 and one H3N2—were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human‐like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human‐like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human‐lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human‐lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk.     

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems. Chinese Nature Science Foundation Key Project (Grant No. 30599433), Chinese Basic Science Research Program (973)Key Project (Grant No. 2005CB523006)     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-X_L expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-X_L) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

一株人感染高致病性禽流感(H5N1)病毒基因组分子特征分析

【背景】H5N1禽流感病毒可以感染人类导致重症呼吸道感染,致死率高。【目的】研究我中心确认的一例人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015的可能起源及基因组分子特征。【方法】对病人痰液样本中的H5N1病毒进行全基因组测序,使用CLC Genomics Workbench 9.0对序列进行拼接,使用BLAST和MEGA 5.22软件进行同源性比对和各片段分子特征分析。【结果】该株禽流感病毒属于H5亚型的2.3.2.1c家系,其8个片段均与江浙地区禽类中分离的病毒高度同源,未发现有明显的重配。分子特征显示,该病毒血凝素(Hemagglutinin,HA)蛋白裂解位点为PQRERRRR/G,受体结合位点呈现禽类受体特点,但出现D94N、S133A和T188I氨基酸置换增强了病毒对人类受体的亲和性。神经氨酸酶(Neuraminidase,NA)蛋白颈部在49-68位缺失20个氨基酸,非结构蛋白1 (Non-structure protein,NS1)存在P42S置换和80-84位氨基酸的缺失。其他蛋白中也存在多个增强病毒致病力和对人类细胞亲和力的氨基酸突变。对耐药位点分析发现存在对奥司他韦的耐药突变H_274Y,病毒对金刚烷胺仍旧敏感。【结论】人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015属于2.3.2.1c家系,禽类来源,关键位点较保守,但仍出现了多个氨基酸的进化与变异使其更利于感染人类。H5N1禽流感病毒进化活跃,持续动态监测不能放松。     

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.     

H9N2亚型猪流感病毒感染BALB/c小鼠动物模型的建立

目的建立H9N2亚型猪流感病毒感染BALB/c小鼠动物模型,为研究病毒致病机制提供模型动物。方法通过滴鼻的方法将H9N2亚型猪流感病毒感染BALB/c小鼠,观察小鼠的症状和组织病理变化。结果 BALB/c小鼠的临床症状明显,病理变化典型。结论 H9N2亚型猪流感病毒感染BALB/c小鼠的疾病模型成功建立。     

A/swine/H1N1流感病毒中国分离株血凝素进化分析

2009年3月以来,甲型H1N1病毒已在全球包括中国造成了巨大的危害,所以利用生物信息技术对其进行研究显得十分必要。从NCBI数据库下载中国大陆境内A/swine/H1N1病毒HA基因核酸序列及其编码蛋白序列,利用MEGA4.0软件对核苷酸编码序列构建系统进化树,利用BioEdit软件对蛋白序列进行比对,分析重要抗原位点变异情况,结果显示2010年在广东流行的病毒,从其他地方传播到广东的,而非早期广东流行的的病毒变异而来。2008年福建,山东,北京等地区的病毒传播比较迅速.这些分析结果阐明了中国大陆境内A/swine/H1N1病毒血凝素(HA)基因的进化关系和变异趋向,对于研究A/swine/H1N1病毒具有重要的参考价值。     

21世纪的首次大流行：2009甲型（H1N1）流感

2009年4月初,在墨西哥和美国出现一种新型甲型（H1N1）流感病毒。该病毒通过人-人传播迅速在全球范围蔓延。该病毒拥有来自人流感病毒、禽流感病毒和猪流感病毒的基因片段,其HA基因与引发1918年大流行的流感病毒株的HA基因同源性很高。该病毒倾向于感染儿童、青少年、孕妇,以及具有心肺疾病的人。据观察,它在人群中的传播能力高于季节性流感。部分感染患者具有在季节性流感中罕见的呕吐和腹泻症状。先前的流感病毒大流行和2009年爆发的甲型H1N1流感病毒大流行表明,由于流感病毒变异速度快、容易发生基因重排,新产生的变异毒株很可能造成新的大流行,威胁人类健康。由于禽流感病毒和人流感病毒都能感染猪,猪被认为是通过基因重排生成新的大流行病毒的＂混合容器＂。     

一株H1N1亚型猪流感病毒的分离鉴定及全基因序列分析

2006年从辽宁省某猪场采集具有流感症状猪鼻拭子共30份,经9~11日龄SPF鸡胚分离,对分离株进行了血凝试验、血凝抑制试验、RT-PCR亚型鉴定,全基因序列比对和接种试验动物试验。结果表明:该毒株具有凝集0.5%鸡红细胞的活性,且与抗猪流感H1标准血清发生血凝抑制反应,RT-PCR分别扩增得到全基因8个片段,利用DNAStar生物学软件进行序列分析,HA基因与GenBank登录的H1~H16中的H1基因序列同源性最高,NA基因与N1~N9中的N1基因序列同源性最高,故该LN株分离毒为猪流感H1N1亚型病毒。全基因序列中,除了M基因不是猪源的,其它基因片段均与国内H1N1亚型猪流感参照株高度同源,推测LN株可能是由类人和类禽谱系的流感病毒与古典猪谱系的流感病毒重排形成的;用该病毒接种试验动物可成功复制出猪流感典型症状。该病毒的分离鉴定及全基因组序列分析为我国进一步调查猪流感的流行规律提供了基础数据。     

A/H1N1流感—世界关注的焦点

2009年4月,A/H1N1流感在墨西哥和美国暴发。随后,疫情迅速蔓延到美洲、欧洲、亚洲多个国家。A/H1N1流感病毒是一种以前在人或动物身上从未观测到的新病毒。遗传进化和抗原特性分析表明该病毒和猪流感病毒密切相关,与人类的季节性流感病毒有明显区别。但是流行病学信息表明A/H1N1流感病毒只攻击人类,并在人与人之间传播,尚未发现动物向人类传播的情况。本文从A/H1N1流感病毒的生物学特性、临床特征、公共卫生意义等方面全面阐述了A/H1N1流感的最新研究进展,为正确认识和科学防控A/H1N1流感提供参考。     

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil

The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

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.     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.