2004—2005年中国A(H1N亚型流感病毒抗原性及基因特性研究

目的阐明2004—2005年中国流行的A(H1N1)亚型流感病毒血凝素抗原性及其基因变异情况。方法对2004—2005年分离的A(H1N1)亚型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的A(H1N1)亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析。结果单向血凝抑制实验结果表明,2004年A(H1N1)亚型病毒株对鉴定血清的血凝抑制效价与A/Shanghai/1/1999(H1N1)毒株没有4倍差异;2005年分离的A(H1H1)亚型毒株中有62株(占6·2%)病毒与A/Shanghai/1/1999(H1N1)毒株本身的血凝抑制效价相比有4倍差异。HA1区核苷酸序列和氨基酸序列分析表明,我国2005年分离到的A(H1N1)亚型流感病毒株有以下位点发生变异,54K>R、90T>K、101Y>H、149R>K、169V>A、190D>N、212R>K、219K>R、245W>R、246Y>F、258T>N、318V>A,其中54、190位氨基酸位于抗原决定簇。结论我国2005年分离的A(H1N1)亚型流感毒株基因特性和抗原性已开始发生变异。     

2004年中国甲3亚型流感病毒(H3N2)抗原性及基因特性研究

目的阐明2004年中国流行的甲3亚型流感病毒血凝素抗原性及其基因变异情况。方法对2004年分离的甲3亚型毒株先进行单向血凝抑制试验及交叉血凝抑制试验;在此基础上选取不同时间、地点的甲3亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析。结果单向血凝抑制实验结果表明,2004年共有52.3%毒株与A/Fujian/411/2002(H3N2)(20042005毒株)有4倍或以上的血凝抑制滴度差异,交叉血凝抑制实验结果表明,它们间的抗原比为4。HA1区核苷酸序列和氨基酸序列分析表明,我国从2004年2月分离的甲3亚型毒株开始出现了与A/Fujian/411/2002(H3N2)和A/Wellington/1/2004(H3N2)(2005年国际代表株)相比较,在其HA1蛋白分子上存在有4个氨基酸位点(159位Y>F,189位S>N,145位K>N,226位V>I)发生了替换。此类毒株首发于我国南方,然后到我国北方。结论我国2004年2月份以后所分离的甲3亚型流感毒株已经发生抗原性及基因特性的改变。     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

2004-2005年中国B型流感病毒抗原性及基因特性研究

目的 阐明2004-2005年中国流行的B型流感病毒血凝素抗原性及其基因变异情况.方法 对2004-2005年分离的B型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的B型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析.结果 2004-2005年我国人群中同时流行着B型Yamagata系和Victoria系毒株.Yamagata系毒株与B/Shanghai/361/02比较,2004年有3.7%病毒单向血凝抑制效价有4倍以上差异,2005年有4.5%病毒单向血凝抑制效价有4倍以上差异,并且在血凝素基因HA1区发生9个氨基酸替换,在196为增加一个糖基化位点.Victoria系毒株与B/Hong kong/330/01比较,2004年有8.5%病毒单向血凝抑制效价有4倍以上差异,2005年有20.6%病毒单向血凝抑制效价有4倍以上差异,并且在HA1区发生9个氨基酸替换,在197位增加一个糖基化位点.结论 2004-2005年我国人群中流行的B型流感病毒的抗原性与B/Shanghai/361/02、B/Hong kong/330/01相比抗原性已经发生了变化.     

2004-2008年我国流行的A型流感病毒抗原性及HA1基因特性的研究

目的 了解我国2004-2008年A(H1N1、H3N2)型流感病毒流行情况、抗原性和基因特性变异关系,了解疫苗株与我国流行株之间抗原性变化情况.方法 选择2004年以来我国分离的A(H1N1、H3N2)型流感病毒进行抗原性及HA1区基因序列,通过比对HA1蛋白位点变异情况,分析我国流感病毒抗原性及基因特性变化情况.结果 A(H1N1)亚型流感毒株抗原性2004-2007年分离的A(H1N1)亚型流感病毒的抗原性与疫苗株A/New Caledonia/20/1999(H1N1)类似;2008年我国流行的A(H1N1)亚型毒株的抗原性与2008-2009年北半球的流感疫苗株A/Brisben/59/2007(H1N1)类似.2004-2005年分离的A(H3N2)亚型流感病毒的抗原性与疫苗株A/Fujian/411/12002(H3N2)比较发生了变异;2006-2007年我国流行的H3N2毒株与A/Wiscansin/67/2006(H3N2)类似,2008年我国流行的H3N2毒株与疫苗株A/Brisben/10/2006(H3N2)类似.结论 2004-2008年我国流行的A(H1N1、H3N2)亚型流感病毒的抗原性和基因特性发生了改变.     

不同抗原性甲3 (H3N2) 亚型流感病毒的鉴定及分子生物学研究

目的：对不能用当年标准血清进行鉴定的甲3（H3N2）亚型流感病毒进行抗原性分析及分子生物学研究。方法：用交互血凝抑制试验对毒株进行抗原性分析,提取病毒RNA,用一步法逆转录聚合酶链反应（RT－PCR）扩增HA1（血凝素重链区）基因片段,产物纯化后测序并分析结果。结果：发现两株从鸡胚分离到的甲3（H3N2）亚型“D”相毒株与2株从MDCK细胞分离到的“0”相毒株抗原性已明显不同;其中1999年分离的“D”相与“0”相2毒株的抗原比大于256,氨基酸序列同源性为93％,抗原决定簇A区和B区氨基酸位点相差分别为50％及35％;受体结合部位（RBS）有6个氨基酸位点发生变异（左侧壁1个、前壁3个、右壁2个）。结论：人群中存在没有发生“0”相变异的甲（H3N2）亚型“D”相毒株,其抗原性与当前流行的“O”相变异株已明显不同,提示今后在鉴定甲3（H3N2）亚型毒株时,需根据毒株的来源和相特性选择适合的标准血清进行鉴定。     

2010年广州市甲型H1N1流感病毒分离株HA基因变异分析

目的比较2010年广州市分离到的甲型H1N1流感病毒血凝素（HA）基因和2009年中国大陆甲型H1N1流感病毒HA基因的变异情况,为甲型H1N1流感的监测和防控提供理论依据。方法收集2010年广州市有发热和呼吸道症状病人的咽拭子标本,用H1N1流感特异性引物进行PCR检测,扩增分离到的H1N1病毒HA片段,测序后与2009年的H1N1毒株进行比对和分析,并用生物信息学方法对抗原位点和糖基化位点进行分析。结果共收集到426份标本,甲型流感阳性211份,其中H1N1流感4株,与2009年分离的甲型H1N1流感相比,有12个氨基酸碱基位点发生了有意义突变,其中6个位点位于抗原位点上;4株毒株HA基因145位氨基酸都发生了变异;其中2株毒株在第180位氨基酸位点的抗原位点发生了变异。进化分析表明4株毒株与2009年中国大陆分离的8株毒株进化关系较远。结论 2010年广州市甲型H1N1毒株与2009年相比发生了较大变异。HA基因145位和180位氨基酸位点变异对H1N1毒株抗原变异有重要意义。本文分离的A/Guangdong/ZS03/2010（H1N1）和A/Guangdong/ZS01/2010（H1N1）毒株可能已经发生了抗原性漂移。     

甲1型流感病毒新分离株HA基因的序列分析

目的 研究新分离的H1N1亚型流感病毒株的HA1基因序列。方法 甲型流感病毒通过鸡胚增殖后提取RNA、逆转录合成cDNA,经PCR扩增和产物纯化构建重组质粒,用双脱氧链终止法进行核苷酸序列测定;并进行基因特性分析。结果 新分离到的3株流感病毒株（H1N1）HA1区基因长度为981bp,编码327个氨基酸;与A/桂防/10/94和A/Bayern/07/95（H1N1）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点;新分离的3株甲型流感病毒（H1地）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点,新分离的3株甲型流感病毒株（H1N1）HA1区氨基酸同源性高达98%;A/桂防/10/94和A/Bayern/07/95(H1N1)毒株HN1氨基酸的同源性高达96%。结论 新分离到的3株H1N1毒株HA编码氨基酸不同于A/Baydrn/07/95(H1N1)和A/桂防/10/94（H1N1）标准株,它们可能为新的甲型流感病毒变异性。     

2000～2002年我国流行的甲3(H3N2)亚型流感病毒抗原性及基因特性的研究

目的 了解2000～2002年我国流行的甲3流感病毒HA基因突变及其抗原变异情况。方法 鸡胚传代流感病毒，收获尿囊液作为抗原性分析抗原并提取病毒的RNA，进行逆转录—聚合酶链反应(RT-PCR)，扩增产物用纯化试剂盒纯化后测序，用MegAlign软件进行基因种系发生树分析。结果 与A/武汉/359/1995(H3N2)、A／Sydney／5／1997(H3N2)相比，2000～2002年我国分离到的甲3亚型流感病毒的血凝素重链区氨基酸序列存在差异。2001～2002年分离到的甲3毒株与2000年分离出的毒株的血凝素蛋白重链区(HA2)氨基酸序列有4个位点差异，它们分别位于83、186、202和222位，其中83和186分别位于抗原决定簇E和B区，其余均位于受体结合位点(RBS)的左臂。结论 2000～2002年分离到的甲3亚型流感病毒的基因特性发生突变并导致其抗原性发生漂移。     

2008年深圳市甲3亚型流感病毒基因特异性分析

目的了解2008年深圳市H3N2亚型流感流行及病毒变异情况。方法用狗肾传代细胞（MDCK）和鸡胚进行流感病毒分离,收获病毒后提取病毒RNA,进行逆转录-聚合酶链式反应（RT-PCR）,扩增产物经琼脂糖凝胶电泳鉴定后送大连宝生物公司纯化及测序,测序结果用SIMMONIC软件和MEGA3.1软件进行序列分析。结果深圳2008年流行的H3N2亚型流感病毒血凝素与深圳2007年流行的差异不大,而与同期疫苗毒株A/Wisconsin/67/2005比较有一定的差异,B抗原决定簇上有1个位点发生替换,158位R替换了K;1个糖基化位点增加,在122位氨基酸由D变成了N;与世界卫生组织推荐的2009年流感疫苗毒株比较差异不大。结论深圳市2008年流行的H3N2亚型流感病毒并未形成新的变种,其漂移趋势与世界H3N2亚型流感漂移趋势一致。     

Genetic Conservation of Hemagglutinin Gene of H9 Influenza Virus in Chicken Population in Mainland China

The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995–2002 were genetically analyzed. All the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage (except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.     

Molecular and serological investigations of the Influenza A(H1N1) 2009 pandemic virus in Turkey

Intense research has been conducted on influenza A(H1N1)pdm09 virus to determine the virulence markers. Limited information on characteristics of pandemic virus has become available in Turkey since the pandemic. In this first report from Turkey, we investigated the molecular markers that have been associated with increased virulence and oseltamivir resistance. We also conducted serological studies in people after infection, vaccination, exposure, and no-exposure controls to determine the level of protection against the pandemic H1N1 influenza virus. Thirteen rRT-PCR positive samples were analyzed for presence of mutations that have been associated with host range, virulence, and antiviral resistance: substitution D222G in the HA, E627K in the PB2, and H275Y in the neuraminidase (NA). In addition, 135 serum samples from vaccinated, recovered, asymptomatic contacts, and control individuals were tested using hemagglutination inhibition (HI) assay. D222G was detected in nasal samples from two severe cases. No specified mutations in the PB2 and NA were identified. Additional substitutions, I216V, V321I, E374K, S203T in HA, V655I in PB2, and I163V in NA, were detected. HI testing from vaccinated individuals, recovered patients, asymptomatic contacts, and control individuals showed that 97.9, 99.7, 88.2, and 44.2 % had HI titers ≥40, respectively. Molecular markers promoting influenza A(H1N1)pdm09 to become a pandemic virus are still under investigation. Serological results confirm that younger, un-exposed individuals are at increased risk of pandemic virus infections. Influenza A(H1N1)pdm09 viruses are still in circulation around the globe. Therefore, these viruses need to be monitored closely for development of new markers including antiviral resistance mutations.     

Generation of the influenza B viruses with improved growth phenotype by substitution of specific amino acids of hemagglutinin

Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.     

Two residues in the hemagglutinin of A/Fujian/411/02-like influenza viruses are responsible for antigenic drift from A/Panama/2007/99

The H3N2 vaccine strain (A/Panama/2007/99) for the 2003-2004 influenza season did not antigenically match the circulating A/Fujian/411/02-like H3N2 viruses and had reduced effectiveness against influenza outbreaks. A/Wyoming/03/2003, an A/Fujian-like virus, was recommended as the vaccine strain for the 2004-2005 season. A/Wyoming differed from A/Panama by 16 amino acids in the HA1 molecule. Reverse genetics was used to determine the minimal amino acid changes that were responsible for the antigenic drift from A/Panama to A/Wyoming. After substitutions of 2 of the 16 amino acids in the HA (H155T, Q156H), the A/Panama HA variant was antigenically equivalent to A/Wyoming as determined by hemagglutination inhibition and microneutralization assays using ferret postinfection antisera. Conversely, A/Wyoming containing the His-155 and Gln-156 residues from A/Panama was antigenically equivalent to A/Panama. These results indicated that only these two HA residues specified the antigenic drift from A/Panama to A/Wyoming; other amino acid differences between these two H3N2 viruses had minimal impact on virus antigenicity but impacted virus replication efficiency in eggs.     

Identification of the binding sites to monoclonal antibodies on A/USSR/90/77 (H1N1) hemagglutinin and their involvement in antigenic drift in H1N1 influenza viruses

We have determined nucleotide sequences of the HA1 portion of the hemagglutinin (HA) gene of the parental A/USSR/90/70 (H1N1) virus and its eight variants selected in vitro with six monoclonal antibodies to study antigenic determinants. The HA1 gene of one of the variants (B-1-23) was cloned in bacteria and its nucleotide sequence was determined by the Maxam-Gilbert method. The nucleotide sequence of the variant was confirmed by the dideoxy chain termination method. The gene sequences of the other viruses were determined by the latter method. Three variants with reduced reactivity in HI test only with the selecting antibodies possessed one amino acid substitution. On the other hand, most other variants which had the changed reactivity to multiple antibodies in HI test possessed more than one substitution. Comparison of the amino acid sequences of the HA1 molecule, deduced from the nucleotide sequences, suggested that the monoclonal antibodies W18 and 264 reacted with epitopes located on the area involving amino acid residues 125C and 189-190, respectively, whereas, the antibodies 22 and 70 reacted with epitopes involving amino acid residues 129, 132, and 157. The epitope recognized by antibody 110 overlapped with that of W18, and the epitope recognized by antibody 385 was located on the area involving at least amino acid residues 129, 159, and 189, which overlapped with some of the above epitopes. The sequence analysis with B-1-23 variant selected with antibody 264 clearly showed that in A/USSR/77 viruses, a single substitution at amino acid residue 190 effectively changes the epitope and caused a significant antigenic variation detectable by postinfection ferret sera.