4株甲型流感病毒的分离鉴定

从24份(人)上感患儿咽拭中,分离到4株甲_3型(H_3N_2)流感病毒,按新命名法定为A/桂医/1/83(H_3N_2)、A/桂医/2/83(H_3N_2)、A/桂医/3/83(H_3N_2)和A/桂医/4/83(H_3N_2)株.经电镜及免疫电镜检查,形态与标准株基本一致;血抑试验鉴定能被甲_3型A/京科/2/79(H_3N_2)株的免疫血清抑制,血抑效价最高5120,最低1280,而不被甲_1型A/津防/78/77(H_1N_1)株及乙型B/湘防/2/74株的免疫血清抑制.证明新分离毒株是甲_3型(H_3N_2)流感病毒.用交叉血抑试验,比较各毒株间抗原性关系,以抗原比表示,A/桂医/2/83(H_3N_2)株与A/京科/2/79(H_3N_2)株无明显差异,而其他各株间则有大小不等的差异,表明不同亚型病毒株抗原性差异大,同一亚型毒株间抗原性差异小.     

2009年新型甲型H1N1流感病毒基质蛋白及核蛋白基因进化分析

目的:探讨2009年新型甲型流感病毒(A/H1N1)基质蛋白(M)及核蛋白(NP)基因的进化规律。方法:从NCBI数据库下载147条甲型H1N1流感病毒M基因及NP基因序列,采用Molecular Evolutionary Genetics Analysis version 4.0(MEGA4.0)软件对M基因和NP基因序列进行比对,并用NJ法构建进化树,同时采用Epi Info软件分析1918~2009年人H1N1病毒的M基因和NP基因序列进化距离的线性趋势。采用MEGA4.0软件对M2蛋白氨基酸序列进行比对。结果:不同地区的2009年新型甲型H1N1流感病毒M基因、NP基因同源性高,但与历史上流行的H1N1流感病毒M基因、NP基因差异较大,且M基因进化距离随分离年限变化的趋势性检验结果有统计学意义(Ptrend=0.001)。2009年新型甲型A/H1N1流感病毒M2蛋白与1918~2008年人A/H1N1病毒M2蛋白氨基酸序列进行比对,结果显示在第11、43、54、57、77、78氨基酸位点发生了改变;与猪、禽A/H1N1的M2蛋白氨基酸序列进行比对,结果显示仅在第43、77位氨基酸位点发生改变。结论:2009年新型甲型A/H1N1流感病毒NP基因片段较以往流行的人H1N1流感病毒NP基因发生了改变;M2蛋白位于胞外编码区的第11位氨基酸、位于TM结构域的第43位氨基酸突变可能导致了新型甲型A/H1N1流感病毒对金刚烷胺类特异性抗病毒药物产生耐药。     

用于儿童的A型流感冷适应减毒重组疫苗的反应原性、抗原活性及遗传稳定性分析

用流感野毒株A/列宁格勒/322/79(H_1N_1)和A/曼谷/1/79(H_3N_2),杂交与冷适应减毒供体A/列宁格勒/134/47/57获得重组株47/25/1(H_1N_1)和47/7/2(H_3N_2)制备成含10~(7·5)/mlE1D_(50)重组病毒的疫苗。免疫组使用每人每侧鼻腔喷雾0.25ml,间隔     

流感病毒变异规律的一些新发现及其在流感监测和生态学研究中的应用

通过近10年的研究,在流感病毒生态学研究方面取得了一些重要的研究成果。填补了我国在这方面的空白。在国际上首先从猪中分离到丙型流感病毒,并通过基因分析,证实了丙型流感病毒在猪群中也能发生抗原性漂移甚至基因重组;并证实在猪群中有乙型流感病毒抗体的存在且至今猪群中仍含有甲_2型(H_2N_2)流感病毒抗体。在国内,首次从猪中分离到人甲1_型(H_1N_1)流感病毒,发现人与     

人和几种动物血清中人类流感抗体分布

本文报告用血凝抑制试验检查人和猪、鸭、鸡、兔血清中的人类流感病毒(A/Hsw_1N_1,A/H_1N_1,A/H_2N_2和A/H_3N_2)抗体,并用血抑抗体滴度较高的动物血清作单扩溶血试验复核,两种试验的结果悬殊很大,最后对这些结果的意义加以讨论。     

A型流感病毒株核蛋白基因的克隆与重组质粒的构建

目的克隆A型流感病毒株Swine/Henan/703/2001(H3N2)的核蛋白(NP)基因序列并构建重组质粒。方法采用逆转录聚合酶链式反应技术从病毒株基因组中扩增出编码核蛋白的基因序列,克隆至pGEM-TEasy载体,经蓝白筛选、菌落PCR及酶切鉴定挑取阳性克隆并测序。结果利用RT-PCR扩增到目的基因片段,并将其克隆至T载体。结论成功构建重组质粒pGEM-TEasy-NP,为NP相关功能及流感疫苗的进一步研究奠定实验基础。     

RNA干扰对流感病毒NP和PA基因表达及病毒增殖的抑制

目的:应用RNA干扰技术(RNAi)研究针对流感病毒NP或/和PA基因的siRNA抑制NP或/和PA基因的表达及抑制流感病毒在MDCK细胞中的增殖. 方法: 分别构建针对NP,PA及同时干扰NP和PA的三种siRNA表达质粒pEGFP/NP,pGenesil/PA和pEGFP/NP PA,转染MDCK后,H5N1亚型流感病毒感染细胞,荧光显微镜判断转染效率,蛋白质印迹和半定量RT-PCR检测NP及PA蛋白表达及基因转录水平的变化,并在不同时间点检测细胞培养上清中的血凝值(HA),观察siRNA抑制流感病毒增殖的能力. 结果: 荧光显微镜结果表明,重组质粒转染效率达65.0%. 蛋白质印迹结果表明,重组质粒pEGFP/NP和pEGFP/NP PA均能抑制NP蛋白在MDCK细胞内的表达,两者的抑制率分别为64.5%和69.6%. 半定量RT-PCR检测到pEGFP/NP质粒在MDCK细胞内对NP基因的转录抑制率为66.0%;pGenesil/PA质粒对PA基因的转录抑制率为63.0%;pEGFP/NP PA质粒对NP和PA基因的转录同时抑制率,分别为71.4%和69.3%. 而对照质粒pEGFP/HK对NP或/和PA基因的蛋白表达及转录均没有抑制作用. HA结果表明,三种质粒均能抑制流感病毒在MDCK细胞中的增殖,以pEGFP6/NP PA的作用最为显著,它们抑制流感病毒增殖的能力分别为87.0%,75.0%和96.9%. 结论: NP或/和PA基因的siRNA质粒可明显抑制NP或/和PA基因的转录和表达,并有效地抑制流感病毒在MDCK细胞中的增殖.     

连翘苷对甲型流感病毒核蛋白基因表达的影响研究

目的探讨连翘苷对甲型流感病毒核蛋白(NP)基因转染后表达的影响。方法将甲型流感病毒NP基因转染Hela细胞,用甲型流感病毒胶体金法检测连翘苷对转染后细胞内和上清核蛋白表达情况,用实时定量反转录聚合酶链反应(RT-PCR)检测Hela细胞内NP基因的拷贝数。结果 NP重组质粒组甲型流感病毒核蛋白含量高。空质粒组、脂质体组、Hela细胞组基本不含甲型流感病毒核蛋白。连翘苷组上清无或可能含有微量核蛋白。连翘苷组胞内甲型流感病毒含量不高。RT-PCR校正曲线相关性为0.998,效率为97.4%。重复4次转染后48 h连翘苷组NP基因表达量为(2.1±0.3)×105拷贝数/μl,NP重组质粒组NP基因表达量为(61.5±15.0)×105拷贝数/μl,连翘苷组NP基因表达量低于NP重组质粒组,差异有统计学意义(t=7.672,P<0.05)。结论连翘苷可以抑制甲型流感病毒NP基因转染后表达。     

甲型流感病毒H_1和H_2之间的抗原关系(摘要)

一般认为,甲型流感病毒在H_(1)N_(1)和H_(2)N_(2)之间没有血清交叉反应。而梁荣根等报道了末期H_(1)N_(1)毒株(Dutch/56组)和H_(2)N_(2)病毒之间存在着明确的血清学关系。 但有人对梁氏资料持有异议,认为两者的交叉反应可能是所用病毒交叉污染等因所致。为澄清这个问题,作者进行下列试验。     

海南省2019—2020监测年度A (H1N1) pdm09流感病毒基质蛋白基因特性分析

目的 分析2019—2020监测年度（2019年4月1日—2020年3月29日）海南省A (H1N1) pdm09亚型流感病毒基质蛋白基因（Matrix, M）进化规律和M2蛋白氨基酸位点变异情况。方法 选取14株2019—2020年海南省分离的A (H1N1) pdm09亚型流感病毒进行基因序列测定,采用Neighbor-Joining方法进行种系进化分析,通过系统进化树比较海南流行株与疫苗株的差异。测序结果用MEGA 10.1.8和DNASTAR7.0.1软件进行基因特性分析及基因同源性分析。结果 2019—2020监测年度,海南省A (H1N1) pdm09亚型流感病毒分离株占分离株的4.9%。序列分析显示,海南省A (H1N1) pdm09亚型流感病毒与国际疫苗株A/Brisbane/02/2018的M基因在核苷酸系统进化树6B.1分支上,核苷酸与氨基酸同源性范围为98.7%~100.0%、98.8%~100.0%。12株分离株胞外区编码区S23N氨基酸发生变异;跨膜区14株分离株均发生S31N位点突变,突变率为100.0%;1株I39V位点氨基酸变异;9株分离株在胞浆区E70D位氨基酸位点发生变异。结论 2019—2020监测年度海南省A (H1N1) pdm09亚型流感病毒活动水平较低,与疫苗株相匹配,对金刚烷胺类药物耐药,应特别关注M2蛋白氨基酸变异情况,为临床抗流感病毒药物的使用提供科学依据。     

A Novel Reassortant H2N3 Influenza Virus Lsolated from China

Objective To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market （LPM） in 2009 in Guangdong province of China. Methods PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed. Results The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus. Conclusion market. Its A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry potential impacts and evolution should be closely monitored.     

A Novel Reassortant H2N3 Influenza Virus Isolated from China

Objective To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.Methods PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced,and phylogenetic trees were constructed and analyzed.Results The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant withthe HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.Conclusion A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.     

2009年新型甲型H1N1流感病毒全基因组序列重组分析

目的:分析2009年流行的新型甲型流感病毒（A/H1N1）全序列的基因重组现象。方法:从NCBI基因数据库下载2009年新型甲型流感病毒（A/H1N1）全基因组序列,采用Molecular Evolutionary Genetics Analysis version 4.0（MEGA 4.0）软件对8条基因序列进行拼接和比对,分析2009年爆发株与历史流行株序列间的同源性;同时采用Simplot 3.5.1软件分析新型流感病毒A/H1N1基因重组现象。结果:2009年3月以来爆发的新型A/H1N1病毒株聚合酶B1(polymerase B1,PB1)基因来自于人H3N2,其同源性为93.7%;聚合酶B2 (polymerase B2,PB2)和聚合酶A (polymerase A,PA)与禽H5N1同源性较高,同源性分别为89.0%、89.9%;血凝素(hemagglutinin,HA)、核蛋白(nucleoprotein,NP)和非结构蛋白(non-structural protein,NS)与北美地区猪H1N1同源性较高,同源性分别为91.7%、93.1%和93.1%;神经氨酸酶(neuraminidase,NA)和基质蛋白(matrix protein,MP)与欧洲地区猪H1N1同源性较高,同源性分别为90.5%、95.5%。全基因序列同源性分析发现2009年新型A/H1N1病毒与北美地区猪H1N1病毒同源性最高,为83.9%。结论:2009年新型甲型H1N1流感病毒可能是人H3N2、北美地区猪H1N1、欧洲地区猪H1N1、禽H5N1的基因重排病毒。     

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate

Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics, Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 （H3N2） in a background of internal genes derived from the master donor viruses （MDV）, cold-adapted （ca）, temperature sensitive （ts）, live attenuated influenza virus strain A/Ann Arbor/6/60 （MDV-A）. Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1：1024. A fluorescent focus assay （FFU） was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition （HI） test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.     

Response of ferrets and monkeys to intranasal infection with human, equine and avian influenza viruses

Rhesus monkeys and ferrets were exposed to intranasal inoculation of several strains of egg-adapted avian, equine and human influenza viruses and to strains of mouse-adapted equine influenza viruses. Local replication of virus and seroconversion were observed in the majority of these animals. However, clinical infection was observed only in ferrets.     

2009年新型甲型H1N1流感病毒进化过程中猪作为宿主及“混合器”的作用

不同种属流感病毒通过基因重排产生变异病毒导致了多次周期性全球流感大流行。2009年爆发流行的新型甲型H1N1流感病毒是猪H1N1流感病毒、禽H5N1流感病毒和人H1N1流感病毒的基因重排病毒,其8条基因片段均有自己的进化特点。禽类流感病毒是导致人类流感流行的流感病毒的起源,其常在猪体内进行基因重排进化为人类流感病毒。猪是流感病毒的中间宿主,也是不同种属流感病毒基因重排的“混合器”,在2009年新型甲型H1N1流感病毒进化过程中起重要作用,是未来流感防制的重要环节。     

2009年新型甲型H1N1流感病毒神经氨酸酶基因进化分析

目的:探讨2009年新型甲型H1N1流感病毒神经氨酸酶(NA)基因的进化及NA基因编码蛋白抗原性、酶活性位点、糖基化位点变异情况。方法:从NCBI基因库检索获得43株不同年代不同地域甲型流感病毒NA基因序列,用Molecular Evolutionary Genetics Analysis version 4.0（MEGA 4.0）软件进行基因进化分析和氨基酸序列分析。结果:2009年新型甲型H1N1流感病毒与禽H5N1流感病毒NA基因的同源性达到85％,潜在抗原位点氨基酸分布相同;所有毒株的酶活性中心位点高度保守,但糖基化位点有变异。结论:2009年新型甲型H1N1流感病毒的NA基因可能来源于禽H5N1流感病毒;神经氨酸酶抑制剂治疗有效。     

Use of temperature-sensitive mutants of influenza A virus as live virus vaccine strains. Evaluation in laboratory animals, adults and children

Temperature-sensitive (ts) recombinants of influenza A virus were evaluated for use in a live virus vaccine. Evidence from several sources suggested that the ts lesions were responsible for attenuation of these mutants. Specification of attenuation by defined genetic lesions which can be assayed for in the laboratory offers an advantage to the use of ts viruses for vaccination. This means that ts recombinants can be assessed for genetic stability during vaccine development, production and later during usage in man. One ts virus, influenza A/Hong Kong/68-ts-1[E], with a 38°C shut-off temperature, had the following properties desirable for a live virus vaccine: (1) satisfactory infectivity for seronegative (serum HI antibody titre ≤ 1:8) adults; (2) satisfactory attenuation for adults; (3) capacity to stimulate local and serum anti-haemagglutinin and anti-neuraminidase antibodies in seronegative volunteers; (4) stimulation of resistance to virulent, wild type virus; (5) relative genetic stability in vivo; (6) lack of communicability in man; (7) replication to high titre in avian leucosis virus-free eggs; and (8) localization of ts lesions to genes that do not code for the haemagglutinin and neuraminidase. The ts lesions of influenza A/Hong Kong/68-ts-1[E] virus were transferred to more current viruses within the H₃N₂ subtype (influenza A/Udorn/307/72 and influenza A/Georgia/101/74). These recombinant Udorn/72 and Georgia/74 ts viruses, which possessed the same shut-off temperature and the same ts lesions as the influenza A/Hong Kong/68-ts-1[E] parent virus, exhibited a pattern of infection and attenuation in hamsters and man similar to their ts parent. These data suggest that ts mutants which are sufficiently attenuated for man, could serve as donors of ts lesions for the rapid production of an attenuated vaccine when new antigenic variants arise.     

2009年新型甲型H1N1流感病毒非结构蛋白基因进化分析

目的:探讨2009年新型甲型H1N1流感病毒的非结构蛋白(NS)基因进化规律。方法:从NCBI下载2009年新型甲型H1N1流感病毒以及北美、欧洲、亚洲地区以往流行的甲型H1N1流感病毒NS基因序列,利用Molecular Evolutionary Genetics Analysis version 4.0(MEGA 4.0)软件对所选序列进行基因进化分析,并用NJ法构建进化树;对2009年新型甲型H1N1流感病毒NS基因核苷酸序列同源性及编码蛋白氨基酸序列进行分析。结果:2009年新型甲型H1N1流感病毒NS基因来源于猪A/H1N1流感病毒,与2005~2007年猪A/H1N1流感病毒具有较高的同源性(97.5％~97.6％),与1930~2007年猪A/H1N1流感病毒具有明显的时间进化关系;其重要抗原及拮抗宿主抗病毒能力的氨基酸位点基本没有变异。结论:2009年新型甲型H1N1流感病毒NS基因来源于猪A/H1N1流感病毒,NS基因编码蛋白拮抗宿主抗病毒能力并没有改变。