A/鹅/广东/2/96(H5N1)流感毒株7和8 RNA节段核苷酸序列测定

目的　弄清A鹅广东296(H5N1)毒株RNA7和8核苷酸全序列及它们与AHK15697(H5N1)毒株RNA7和8之间的内在关系,并为今后流感病毒M和NS基因研究打下基础。方法　病毒粒RNA经逆转录合成cDNA,经聚合酶链反应(PCR)扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　A鹅广东296(H5N1)毒株RNA7长度为1027个核苷酸,编码M1(含252个氨基酸)和M2(含97个氨基酸)的蛋白。其RNA8长度为890个核苷酸,编码NS1(含230个氨基酸)和NS2(含121个氨基酸)非结构蛋白。其M1,M2,NS1和NS2蛋白分子上氨基酸序列与AHK15697(H5N1)毒株间同源性分别为976%,928%,657%和769%。结论　A鹅广东296(H5N1)毒株RNA7和8长度分别为1027和890个核苷酸,此两节段RNA均属禽类毒株。AHK15697(H5N1)毒株的RNA7和8不是来自A鹅广东296(H5N1)毒株。     

鹅流感病毒2／96—H5N1亚型毒株RNA1—3及RNA5节段核？…

目的 明确广东鹅流感病毒２／９６－Ｈ５Ｎ１亚型毒株ＲＮＡ１－３和ＲＮＡ５节段核芏酸全序列及其所编码蛋白的氨基酸序列,以及这些基因节段与香港禽流感病毒１５６／９７－Ｈ５Ｎ１亚型毒株相应节段间的关系。方法 病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果 广东鹅流感病毒２／９６－Ｈ５Ｎ１亚型毒株ＲＮＡ１－３和ＲＮＡ５节段长度分别为     

鹅流感病毒2/96-H5N1亚型毒株RNA1-3及RNA5节段核苷酸全序测定

目的　明确广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段核苷酸全序列及其所编码蛋白的氨基酸序列,以及这些基因节段与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应节段间的关系。方法　病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ） 扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２３４１,２ ３４１ ,２ ２３３ 和１５６５ 个核苷酸。它们分别编码ＰＢ２（ 含７５９ 个氨基酸）,ＰＢ１（ 含７５７个氨基酸） ,ＰＡ（ 含７１６ 个氨基酸） 和ＮＰ蛋白（ 含４９８ 个核苷酸） 。这些蛋白与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应蛋白氨基酸序列的同源性分别为９６－４％ ,９７－２％ ,９７－３ ％ 和９７－０％ 。结论　本毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２ ３４１,２ ３４１,２ ２３３ 和１ ５６５ 个核苷酸,它们与香港１５６９７Ｈ５ Ｎ１ 亚型毒株间存在着差异     

从人群中分离到1株A／PR／8／34（H1N1）类病毒

１９９１年５月从１名上呼吸道感染的儿童患者中分离出１株流感病毒。经血清学鉴定和分析，分离物为Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）类似毒株。病毒ＨＡ１区基因经核苷酸序列分析，分离物与Ａ／ＰＲ／８／３４毒株间有１２个位点不同，导致了ＨＡ１区蛋白分子上４个氨基酸的替换。在病毒基因组寡核苷酸指纹图分析中发现，分离物与Ａ／ＰＲ／８／３４毒株相比较丢失了２个点，而插入了９个点。因此，Ａ／广东／６／９１（Ｈ１Ｎ１）毒株不是由Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）通过实验室污染而来。     

甲型流感病毒（N1N2）亚型毒株血凝素和神经氨酸酶基 …

目的 研究新分离到的Ｈ１Ｎ２亚型毒株血凝素（ＨＡ）和神经氨酸酶（ＮＡ）基因的来源。方法 病毒通过鸡胚增殖后提取其ＲＮＡ,通过逆转录合成ｃＤＮＡ,经ＰＣＲ扩增和产物纯化,用双脱氧链终止法进行核苷酸序列测定,并用ＭｅｇＡｌｉｇｎ（１．０３版）和Ｅｄｉｔｓｅｑ（３．６９版）软件进行种系发生学分析。结果 新分离到Ｈ１Ｎ２毒株ＨＡ１区氨基酸序列与Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）和Ａ／Ｇｕａｍｇｄｏｎｇ／６／９     

H5N1亚型流感病毒M2与NA基因真核表达载体的构建与鉴定

目的 构建表达H5N1亚型流感病毒M2和NA基因的多种真核表达载体.方法 以我国分离的首株人H5N1亚型禽流感病毒（A/Anhui/1/2005）作为研究对象,PCR扩增全长M2与NA基因.将M2基因克隆入真核表达载体pStar的IRES上游或下游的MCS,构建重组pStar-M2/和pStar-/M2.将NA基因克隆人pStar载体IRES上游或下游的MCS,构建重组pStar-NA/和pStar-/NA.将M2和NA基因先后克隆到pStar载体IRES序列的上游和下游MCS,分别构建双基因共表达重组pStar-M2/NA和pStar-NA/M2.将重组质粒转染293细胞,使用IFA方法 检测各重组质粒相应外源基因的表达.结果 通过酶切鉴定,各重组质粒构建正确.将其转染293细胞后,使用IFA方法 检测到了各重组质粒中相应外源基因的表达.结论 构建了多种表达H5NI亚型流感病毒M2和（或）NA基因的真核表达载体,为研究开发流感DNA疫苗奠定了基础.     

丙型肝炎病毒（HCV）E2／NS1相对保守区多肽抗原在检？…

目的 研究（ＨＣＶ）Ｅ２／ＮＳ１相对保守区多肽抗原在检测抗－ＨＣＶ中的意义。方法 利用ＨＣＶＥ２／ＮＳ１基因编码的膜区糖蛋白合成Ｅ２／ＮＳ１相对保守区多肽抗原,建立酶免疫试验（ＥＩＡ）,对９６例ＨＣＶ感染者及４０例正常献血员进行ＨＣＶＥ２／ＮＳ１抗体的检测,同时平行检测ＨＣＶＲＮＡ肝炎为１３．５５％,慢性丙型肝炎为２５．０４％,无症状感染者为２．０８％;正常献血员中发现３例抗ＨＣＶＥ２／ＮＳ１抗体     

1997—1998年在香港从人群中分离出禽流感病毒A（H5N1）的最新情况

在香港，到1998年1月6日为止，已明确16例确诊和3例疑似禽流感病毒A（H5N1）感染的病例。确诊病例为分离到流感A（H5N1）病毒或是通过对流感病毒A（H5N1）进行中和试验发生血清学反应的病例。疑似病例为有类流感样疾病和初步的实验室证据的病例。本文报道了香港卫生部门和美国疾病控制中心对流感A（H5N1）进行流行病学调查和实验室研究的结果。首发病例是一个3岁小孩，已于1997年5月死于呼吸衰竭。其余的15个确诊病例当中有5例在11月发病，其余10例在12月发病，3例疑似病例则均在12月份发病。确诊病例的年龄分布介于1～60岁之间（平均1…     

A549细胞和Hep-2细胞表面糖链类型鉴定及其与H5N1型禽流感病毒结合的特性

目的 研究H5N1病毒对A549细胞和Hep-2细胞的黏附和进入特性。方法 用凝集素染色技术和流式细胞术检测A549细胞和Hep-2细胞表面SAa2,3Gal 和SAa2,6Gal的表达;用间接免疫荧光法检测H5N1病毒进入细胞情况;用Western blot法分析病毒进入A549细胞和Hep-2细胞的效率。结果 A549细胞和Hep-2细胞表面有大量SAa2,3Gal,但SAa2,6Gal含量却很少。Hep-2细胞表面SAa2,3Gal受体的表达水平高于A549细胞。H5N1病毒能够进入A549细胞和Hep-2细胞,而且H5N1病毒对A549细胞的亲嗜性更强。与A549细胞相比,Hep-2细胞对H5N1病毒诱导的细胞死亡更敏感。结论 细胞表面唾液酸-a2,3-半乳糖的表达与H5N1病毒的黏附一致。然而,唾液酸受体可能不是介导病毒进入的唯一因素。     

Nucleotide sequence of RNA segment 7 and the predicted amino sequence of M1 and M2 proteins of FPV/Weybridge (H7N7) and WSN (H1N1) influenza viruses

Since the gene products (M1 and M2) of influenza virus RNA segment 7 have been implicated in host range restriction, sensitivity to the drug amantadine, virus yield in chicken embryos as well as in virus assembly and morphology, we have determined the nucleotide sequence of this RNA segment for an avian [A/FPV/Weybridge (H7N7)] and a human [A/WSN/33 (H1N1)] virus and compared it to that of the other influenza A virus strains. The results show that all ten strains of influenza A virus contain an identical number of nucleotides (1027 bases) in RNA segment 7 and an identical number of amino acids in M1 (252 aa) and M2 (97 aa) proteins. The observed amino acid changes are conservative in nature suggesting the requirement of a critical structure of both proteins in virus assembly. Furthermore, the presence of some consistent amino acid substitutions among different human and avian strains also supports the possible existence of host range and drug resistance determinants in M1 and M2 proteins.     

Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.

Analysis of the sequences of all eight RNA segments of the influenza A/G oose/Guangdong/1/96 (H5N1) virus, isolated from a sick goose during an outbreak in Guangdong province, China, in 1996, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997. However, the remaining genes showed greater similarity to other avian influenza viruses. Notably, the neuraminidase gene did no have the 19-amino-acid deletion in the stalk region seen in the H5N1 Hong Kong viruses and the NS gene belonged to allele B, while that of the H5N1 Hong Kong viruses belonged to allele A. These data suggest that the H5N1 viruses isolated from the Hong Kong outbreaks derived their HA genes from a virus similar to the A/Goose/Guangdong/1/96 virus or shared a progenitor with this goose pathogen.     

Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: Conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment

The complete nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS gene was determined. A direct sequence comparison of this NS gene with that of influenza A/PR/8/34 virus showed a difference of 27.3%. In contrast, the differences among the previously sequenced NS genes of the A/PR/8/34, A/FPV/Rostock/34, and A/Udorn/72 viruses were much lower and ranged from 8–11%. The NS genes of the latter three strains contain open reading frames in their virion (?) RNAs which potentially code for polypeptides of 167 and 216 amino acids. Although the NS segment of the A/duck/Alberta/60/76 virus lacks such an agnogene in its negative strand sequence, it conserves the overlapping NS1 and NS2 gene arrangement identified in the other NS RNA segments. A comparison of the deduced amino acid sequences of the A/duck/Alberta/60/76 and A/ PR/8/34 virus NS genes showed that the NS2 polypeptides are more highly conserved (18.2% differences) than the NS1 polypeptides (33.0% differences). Surprisingly, the NS1 polypeptides of the two strains show this high degree of variation over their entire lengths, including the amino acids encoded by the conserved RNA domain of the NS1 gene and NS2 gene overlap.     

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000–2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005–2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.     

Conservation of the influenza virus membrane protein (M1) amino acid sequence and an open reading frame of RNA segment 7 encoding a second protein (M2) in H1N1 and H3N2 strains

The complete sequence of a full-length cloned DNA copy of the influenza virus A/Udorn/72 (H3N2) RNA segment 7 has been determined. A second open reading frame has been found which overlaps the membrane protein (M₁) sequence by 68 nucleotides. This second reading frame could code, in the +1 reading frame, for a protein (M₂) with a maximum of 97 amino acids depending on whether there is splicing of the mRNA and the methionine residue used for initiation of protein synthesis. Comparison of the present H3N2 sequence with the previously published sequence (18., 19.) of RNA segment 7 of A/PR/8/34 (H1N1), a strain isolated 38 years earlier, has shown that the amino acid sequence for the M₁ protein has been greatly conserved. In a total of 252 amino acids, only seven amino acid changes have occurred, of which only one results in a change in charge. In the amino acid sequences coded by the second open reading frame, changes were more common between strains, 11 out of a total of 96 amino acids.     

Preparative isolation of A/Hong Kong/1/68 (H3N2) virus hemagglutinin]

Hemagglutinin of A/Hong Kong/1/68 virus was isolated by electrophoresis in acetate-cellulose of a recombinant 12/13 (H3N1) strain destroyed by sodium dodecyl sulphate. Electron microscope examinations were carried out and molecular weights of hemagglutinin polypeptides were determined. A monospecific serum containing no antibody to neuraminidase was prepared.     

Heterogeneity of A/Singapore/1/57 (H2N2) influenza virus.

The population of A/Singapore/1/57 (H2N2) influenza virus was found to contain two types of infectious particle. "Normal" virions with a diameter from 100-130 nm, a buoyant density in sucrose of 1.21 g/cm3 and a sedimentation coefficient 770 S represented about 90 percent of the population. In addition, a considerable amount of larger particles (up to 1000 nm) with a buoyant density in sucrose of 1.18 g/cm3 and a sedimentation coefficient of over 1000 S were present.