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.     

The NS1 protein of H5N6 feline influenza virus inhibits feline beta interferon response by preventing NF-κB and IRF3 activation

Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-β promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-β suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.     

Non-structural protein 1 from avian influenza virus H9N2 is an efficient RNA silencing suppressor with characteristics that differ from those of <Emphasis Type="Italic">Tomato bushy stunt virus</Emphasis> p19

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional dimeric protein that contains a conserved N-terminal RNA binding domain. Studies have shown that NS1 suppresses RNA silencing and the NS1 proteins encoded by different influenza A virus strains exhibit differential RNA silencing suppression activities. In this study, we showed that the NS1 protein from avian influenza virus (AIV) H9N2 suppressed systemic RNA silencing induced by sense RNA or dsRNA. It resulted in more severe Potato virus X symptom, but could not reverse established systemic green fluorescent protein silencing in Nicotiana benthamiana. In addition, its systemic silencing suppression activity was much weaker than that of p19. The local silencing suppression activity of AIV H9N2 NS1 was most powerful at 7 dpi and was even stronger than that of p19. And the inhibition ability to RNA silencing of NS1 is stronger than that of p19 in human cells. Collectively, these results indicate that AIV H9N2 NS1 is an effective RNA silencing suppressor that likely targets downstream step(s) of dsRNA formation at an early stage in RNA silencing. Although NS1 and p19 both bind siRNA, their suppression mechanisms seem to differ because of differences in their suppression activities at various times post-infiltration and because p19 can reverse established systemic RNA silencing, but NS1 cannot.     

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。     

A／鹅／广东／2／96（H5N1）流感毒株7和8RNA节段核苷酸？…

目的 弄清Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株ＲＮＡ７和８核苷酸全序列及它们与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ７和８之间的内胡关系,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。     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

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.     

Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides

An influenza A virus, A/turkey/Oregon/71, was shown by protein gel analysis to code for an NS1 protein approximately half the size of those of other influenza A viruses. Sequence analysis of the NS gene of this virus revealed a 10 nucleotide deletion resulting in an NS1 protein of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by indirect immunofluorescence analysis of virus infected cells. Also, A/turkey/Oregon/71 virus grew to high titer in embryonated chicken eggs comparable to other influenza A viruses. We also identified a laboratory variant of an influenza B virus, clone 201, which codes for a truncated NS1 protein. Sequence analysis revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids as compared to other influenza B virus NS1 proteins possessing a length of 281 amino acids. Again as shown for the NS1 proteins of other influenza B viruses the NS1 polypeptide of B virus clone 201 was found to localize in the nucleus of infected cells. It appears that large deletions in the carboxyl terminus of the NS1 proteins of influenza A and B viruses can be tolerated without affecting the functional integrity of the NS1 polypeptide.     

1994～1997年深圳地区流感病毒活动及H3N2亚型毒 …

目的 了解几年流感病毒在深圳地区活动的特点及甲３（Ｈ３Ｎ２）亚型毒株ＨＡ１基因演变概况。方法 病毒分离采用常规的鸡胚双腔接种,毒株检和常量半加敏ＨＩ测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取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 近几年来深圳地区流感活动概况与全国情况相一致;在人群中仍同时流行Ｈ３Ｎ２,Ｈ１Ｎ１ 型和乙型毒     

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.     

Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

甲1(H1N1)亚型流感病毒相变异分子生物学基础的研究

目的 阐明甲１（Ｈ１Ｎ１）亚型株相变异的分子生物学基础。方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ,利用聚合酶链反应（ＰＣＲ）进行扩增,产物纯化,采用双脱链末端终止法进行核苷酸序列测定,最后用ＤＮＡ ＳＴＡＲ公司出口的分析软件ＭｅｇＡｌｉｎｇ（１．０３版）和Ｅｄｉｔｓｅｑ（３．６９版０对核苷酸序列进行分析。结果 见不到“Ｏ”、“Ｄ”相毒株ＨＡ１蛋白分子间有特殊氨基酸的差异。但１９９５年前后毒株在－２,－     

1994～1997年深圳地区流感病毒活动及H3N2亚型毒株HA1基因演变特点

目的　了解近几年流感病毒在深圳地区活动的特点及甲３（Ｈ３Ｎ２） 亚型毒株ＨＡ１ 基因演变概况。方法　病毒分离采用常规的鸡胚双腔接种,毒株检定用常量半加敏ＨＩ测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取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　近几年来深圳地区流感活动概况与全国情况相一致：在人群中仍同时流行Ｈ３Ｎ２,Ｈ１Ｎ１ 亚型和乙型毒株,当甲型毒株活动减弱时,乙型毒株活动就增强,反之,甲型毒株增强时,乙型毒株就减弱。随着时间的推移,Ｈ３Ｎ２ 亚型毒株ＨＡ１ 基因不断地发生点突变,这种突变严重受人群免疫压力所影响,１９９６ 年的毒株与１９９５ 的毒株相比,不仅氨基酸替换点中多数是位于抗原决定簇区或受体结合部位上,并增加两个糖基化位点,故导致Ｈ３Ｎ２ 毒株於１９９６ 年活动明显增强。结论　近来在深圳地区人群中仍同时流行着Ｈ３Ｎ２,Ｈ１Ｎ１ 亚型和乙型流感病毒。然而,不同年其优势毒株是不一样的。１９９６ 年Ｈ３Ｎ２ 毒株活动增强是由于其ＨＡ１ 区氨基酸序列发生替换所造成。     

深圳地区甲1(H1N1)亚型流感病毒基因特性的研究

目的　了解近几年H1N1亚型毒株在深圳地区人群中活动加强及“O”相特性出现的分子生物学基础及其基因演变的特性。方法　病毒粒RNA经逆转录合成cDNA,用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并推导出其所编码的氨基酸序列。进化树分析用DNASTAR公司出品的序列分析软件,MegAlign(103版)的Editseq(369版)。结果　根据病毒粒HA1基因特性,至少1995年以来深圳地区人群中同时流行着基因特性不同的三系毒株;1995～1997年H1N1毒株与A新加坡686(H1N1)病毒相比较,其HA1蛋白分子上第54和155位上分别插入和缺失一个糖基化点,同时氨基酸序列发生了替换。结论　近年来深圳地区人群中同时流行着HA1基因不同的三系H1N1亚型毒株。由于HA1蛋白分子上氨基酸序列发生了替换,尤其糖基化位点插入和缺失,造成1995年以来H1N1毒株活动加强,这些可能与毒株“O”相特性再现密切相关。     

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。     

Electrophoretic migration rate differences of polypeptides of human influenza A viruses: Partial analysis of the genome of influenza vaccine recombinant viruses

Summary Electrophoretic migration rate differences were detected in high resolution SDS polyacrylamide gels for nucleoprotein (NP), matrix protein (M), non structural protein (NS1), haemagglutinin (HA) and, less regularly, for the polymerase polypeptides P1, P2 and P3 induced by different influenza A viruses. The technique allowed parental assignation of the corresponding genes in certain recombinant viruses including A/PR/8/34 (H0N1)—A/HK/117/77 (H1N1), A/Okuda/57 (H2N2)—A/HK/119/77 (H1N1) and A/Leningrad/76 (H3N2)—A/Leningrad/46 (H0N1) recombinants, thus considerably extending the technique which had been applied previously to A/PR/8/34—A/HK/68 (H3N2) only. Agreement in gene assignment between three recombinants of the former group and 11 of 17 recombinants in the A/Okuda/57—A/HK/119/77 group was noted when the data obtained using the polypeptide method was correlated with a direct genetic analysis by others using RNA:RNA hybridisation techniques. The polypeptide method appears to have wide application for the initial rapid analysis of influenza A virus recombinants obtained using parents of different influenza subtypes although complete analysis of the total genome requires the use of RNA hybridisation techniques. Two additional virus induced proteins are described, a phosphorylated form of NS 1 and a non structual polypeptide with a molecular weight of 16 K daltons.With 6 Figures     

The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian, and swine influenza viruses, are resistant to the anti-viral effects of interferons and tumor necrosis factor alpha The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever, and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans and provide insight into the virulence of 1918 Spanish influenza.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.