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。     

Inefficient splicing of segment 7 and 8 mRNAs is an inherent property of influenza virus A/Brevig Mission/1918/1 (H1N1) that causes elevated expression of NS1 protein

Influenza A virus encodes two segments (7 and 8) that produce mRNAs that can be spliced. We have investigated if naturally occurring sequence polymorphisms in the influenza A virus family affects splicing of these viral mRNAs, as that could potentially alter the NS1/NS2- and/or M1/M2-protein ratios. We compared splicing efficiency of segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) and A/Netherlands/178/95 (H3N2), as well as various H5N1 avian strains. Results revealed that both segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) were inefficiently spliced compared to other influenza virus segment 7 and 8 mRNAs. This resulted in production of higher levels of functional NS1 protein, which could potentially contribute to the pathogenic properties of the A/Brevig Mission/1918/1 (H1N1). We also show that A/Brevig Mission/1918/1 (H1N1) segment 8 mRNAs responded differently to overexpression of SR proteins than A/Netherlands/178/95 (H3N2).     

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.     

Genetic stability and linkage analysis of the 2009 influenza A(H1N1) virus based on sequence homology

The 2009 swine-origin Influenza A virus subtype H1N1 (S-OIV) is generally believed to be a mixture of human, bird and swine viruses, resulting from multiple reassortments. The evolutionary origin of the S-OIV is of high interest but still remains obscure. In order to understand the evolution of the new virus, we performed sequence homology, segment stability and segment linkage analysis, as well as analysis of the host and geographic distribution of the evolutionarily related viruses. Stability analysis demonstrated that segment 6 (NA) was the most unstable one, followed by segment 4 (HA), while the other 6 segments were relatively stable. Host and geographic distribution analysis indicated that all 8 segments of the new virus were closely related to those of swine influenza viruses circulating either in North America or in Eurasia. Segment linkage analysis showed that segments 1 (PB2), 2 (PB1), 3 (PA), 4 (HA), 5 (NP), and 8 (NS) are in linkage disequilibrium exclusively with North American swine influenza viruses, and segments 6 (NA) and 7 (M) are evolutionarily linked solely with Eurasian swine influenza viruses. Two North American swine strains and 2 Eurasian swine strains were identified as possible ancestors of S-OIV 2009. Based on the most recent linkage analysis with the updated influenza sequences, South Dakota avian strains were found to be the closest known relatives of S-OIV 2009.     

流感病毒A/广州/333/99(H9N2)毒株基因组特性的研究

目的 了解一株再次从流感患儿中分离出禽H9N2流感毒株的基因组特性，并弄清它的来源。方法 病毒在鸡胚中传代，从收获的尿囊液中提取RNA，通过逆转录合成cDNA，cDNA用PCR扩增。PCR产物用纯化试剂盒纯化，接着做核苷酸序列测定，然后用Meg Align(Version 1.03)和Editseg(Version 3.69)软件进行基因进化树分析。结果 A／广州／333／99（H9N2）毒株的基因组属于禽流感病毒，但它明显不同于A／Duck/Hong Kong/Y439/97毒株。同时不含有任何人流感病毒基因节段，其基因组中有4个基因节段（分别编码HA、NA、NP和NS蛋白）来自G9毒株基因系，而其余4个基因节段（分别编码PB2、PB1、PA和M蛋白）来自G1毒株基因系。结论 A／广州／333／99（H9N2）病毒是G9和G1毒株通过基因重配而来的重配株，它最大可能性直接来自禽。进一步证实了禽H9N2毒株能感染人，同时首次证实了H9N2不同基因系毒株间，在自然界中也能发生基因重配。     

两株京科猪H1N1亚型流感病毒内部基因来源的研究

目的通过内部基因研究了解两株猪(H1N1)亚型流感病毒内部基因是否含有禽流感病毒基因节段及是否与猪群中H9N2亚型毒株发生了基因重配。方法病毒在鸡胚中传代,从收获的尿囊液中提取RNA,通过逆转录合成cDNA,cDNA用PCR扩增。PCR产物用纯化试剂盒纯化,接着进行核苷酸序列测定,然后用MegAlign(Version1.03)和Editseq(Version3.69)软件进行基因进化树分析。结果两株京科猪H1N1病毒内部基因除PB2基因节段有所不同外,其余5个基因节段均相同,但与猪H1N1流感病毒内部基因相近,然而,与古典型猪H1N1毒株有差异。结论两株京科猪H1N1病毒不是基因重配株,它们的内部基因均属猪H1N1流感病毒基因系。     

Correlation of amino acid residues in the M1 and M2 proteins of influenza virus with high yielding properties

The ability of influenza A viruses to replicate to high titer in the allantoic sac of the chicken embryo has been mapped to the matrix protein gene (RNA 7). Because influenza A/WSN/33 (H1N1) virus grows poorly in this host but contains a matrix protein gene with a sequence similar to sequences from viruses that grow well in eggs, we derived a single gene reassortant containing only the M gene from A/WSN/33 (H1N1) in a background of the other 7 RNA segments from A/Philippines/2/82 (H3N2) (a low yielding virus, hy-). This reassortant replicated 10 times better than the A/WSN parent itself, indicating that the high yielding (hy+) phenotype of the A/WSN/33 M gene may be suppressed by one of the other genes of A/WSN/33. Comparison of M gene sequences between hy+ (including A/WSN/33) and hy- strains allowed us to correlate specific amino acid positions in M1 and M2 proteins with the growth properties of influenza viruses.     

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.     

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.     

动物源性和人源性甲型流感病毒(H1N1)血凝素(HA)的特征分析

目的 研究动物源性和人源性甲型H1N1流感病毒(influenza A virus)血凝素(hemagglutinin,HA)的特征,以探讨动物源性和人源性甲型H1N1流感病毒血凝素之间的关系.方法 从美国生物信息中心(NCBI)下载禽(鸟)源、猪源、人源的甲型H1N1流感病毒血凝素氨基酸序列,使用Clustal W2.0生物学软件比较上述血凝素氨基酸序列,并建立甲型H1N1流感病毒血凝素氨基酸序列的进化树.结果 2009年分离的人源性甲型流感病毒血凝素氨基酸序列同源性非常高,达到了99%～100%,而2009年分离的人源性甲型流感病毒血凝素氨基酸序列和禽(鸟)源,猪源的甲型流感病毒血凝素氨基酸序列之间的同源性非常低,只有77%～90%(只有猪源ABW36355和2009年分离的人源甲型流感病毒血凝素氨基酸序列同源性为90%,余同源性为77%～83%);蛋白生物进化树表明禽源(鸟)、猪源、人源的甲型流感病毒血凝素氨基酸序列明显分为3个大的分支.2009年分离的人源性甲型流感病毒血凝素氨基酸序列(ADA71154除外)与疫情前分离得到的人源的血凝素氨基酸序列同源性很低(79%～80%),并且进化树分为3个分支.结论 2009年流行的甲型H1N1流感病毒是一种新的流感病毒,病毒的血凝素氨基酸序列之间的同源性非常高,而和猪、禽(鸟)源的甲型H1N1流感病毒的血凝素氨基酸序列之间的同源性非常低,从这一层面上来讲,目前流行的甲型流感病毒的血凝素的基因并不是猪源和禽源,和疫情前人源的血凝素比对的结果也表明,2009年流行的甲型H1N1流感病毒也并不是直接源于2009年以前的人源流感H1N1病毒,而应该是另有来源.     

Evolution of influenza polymerase: nucleotide sequence of the PB2 gene of A/Chile/1/83 (H1N1)

Summary The complete nucleotide sequence of the PB2 gene of influenza virus A/Chile/1/83 (H1N1) is presented. Sequence comparison between A/Chile PB2 protein and the known PB2 sequences of the influenza strains A/WSN/33 (H1N1), A/PR/8/34 (H1N1), A/NT/60/68 (H3N2), A/Kiev/59/79 (H1N1), A/FPV/Rostock/34 (H7N1), and B/Ann Arbor/1/66 indicates extensive amino acid homology for the influenza A virus PB2 proteins. Small clusters of basic amino acids are conserved in all PB2 proteins including the influenza B PB2 protein which has only 39% sequence homology overall to the PB2 polypeptides of type A influenza viruses. The evolutionary rate of 5.7 × 10^–3 nucleotide substitutions per site per year and 0.25% amino acid changes per year between the A/Chile/1/83 and A/NT/60/68 PB2 appears to be higher than that calculated earlier for A/NT, A/PR/8 and A/WSN. An unusually high degree of sequence change between A/Chile/1/83 and A/Kiev/59/79 PB2 polymerase was revealed and this is discussed in terms of its probable origin.     

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.     

Highly pathogenic avian influenza (H7N1) in ostriches (Struthio camelus)

The clinical, virological and pathological findings observed in a natural outbreak of highly pathogenic avian influenza in intensively farmed ostriches (Struthio camelus) are reported. Clinical signs characterized by anorexia, depression, nervous and enteric signs were observed in young birds, which resulted in death of 30% of the affected birds. Virus isolation performed in accordance with the guidelines listed in European Union Directive 92/40/EEC yielded an influenza A virus of the H7N1 subtype with a deduced cleavage site motif containing multiple basic amino acids, typical of highly pathogenic viruses. Gross lesions, mainly haemorrhagic enteritis and liver degeneration and necrosis, were confirmed by histopathology and immunohistochemistry, resulting in the detection of necrotic lesions and influenza A nucleoprotein in selected organs. The findings reported indicate that ostriches are susceptible to highly pathogenic avian influenza.     

Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "DIVA" vaccination strategies, enabling the Differentiation of Infected from Vaccinated Animals have been recommended by international organisations. A system, based on the detection of antibodies to the Non-Structural 1 (NS1) protein of AI has been proposed but the success of such a system lies in the conservation of the NS1 protein among different AI isolates. With this in mind, the ns1 gene of 40 influenza A viruses isolated from a spectrum of avian species was sequenced and compared phylogenetically. The isolates included both low pathogenicity (LPAI) (n=22) and highly pathogenic (HPAI) (n=18) viruses of the H7 subtype and were representative of the avian influenza viruses that circulated in Northern Italy from 1999 to 2003. Size variation in the predicted amino acid sequence of each NS1 was revealed with two different levels of carboxy-terminal truncation being observed. Of the 40 isolates analysed, 16 had a full-length NS1 protein of 230 aa, 6 had a truncated protein of 220 aa and 18 had an intermediate truncation resulting in a protein of 224 aa. All of the H7N1 HPAI isolates possessed the intermediate carboxy-terminal truncation. In addition, all of the H7N1 LPAI viruses circulating at the beginning of the epidemic had a full length NS1 while those circulating towards the end of the period had a truncated protein. To determine whether modifications to NS1 could be a result of laboratory manipulation, two strains (A/ty/Italy/977/99 and A/ck/Italy/1082/99) with a full length NS1 were inoculated into 10-day-old embryonated chicken and 12-day-old embryonated turkey eggs via the allantoic route for 20 blind passages and sequenced at passages 3, 10, and 20. No truncation was observed following these serial passages. To determine whether the truncation involved an immunogenic region of the NS1 protein a peptide spanning residues 219 aa to 230 aa was synthesized and tested in an indirect ELISA against sera obtained from turkeys experimentally infected with a virus strain known to have a full length NS1 protein. The peptide proved to be immunogenic highlighting the fact that the variations of the NS1 protein presented in this work must to be taken into consideration when developing a diagnostic test based on the identification of antibodies to the NS1 protein.     

Genetic characterisation of an influenza A virus of unusual subtype (H1N7) isolated from pigs in England

Summary.  An H1N7 influenza A virus, isolated from pigs in England in 1992, was examined genetically to determine the characteristics and probable origin of the eight gene segments. Six of the RNA segments encoding PB2, PB1, PA, HA, NP and NS were related most closely to those of human viruses, whilst two of the RNA segments (NA and M) were related most closely to those of equine viruses. The HA gene was most similar to that of A/USSR/90/77 (H1N1) but amino acid differences suggested independent genetic drift. In contrast, there were relatively few changes in the NA and M genes compared to those of A/equine/Prague/1/56 (H7N7). Accepted November 12, 1996 Received November 12, 1996     

Genetic relatedness between A/Swine/Iowa/15/30(H1N1) and human influenza viruses

The nucleotide sequences of the M and NS1 genes of influenza virus A/Swine/Iowa/15/30 (A/SW/IW/30)(H1N1) were determined with cloned DNAs and compared with reported sequences of human and avian influenza viruses. A/SW/IW/30 virus was found to be closely similar to A/PR/8/34(H1N1) virus in the nucleotide sequences of the M and NS1 genes, the base differences between the two strains being 64 out of 1027 nucleotides in the M gene and 52 out of 740 in the NS1 gene. Based on the assumptions that these two viruses were derived from a common ancestor and that the rate of base changes per year was the same in man and in swine, it was estimated that the progenitor virus was in circulation during the period from 1915 to 1920. This estimation was compatible with the epidemiological findings suggesting that the progenitor of the swine influenza virus was the agent of the 1918 influenza pandemic. Furthermore, the M and NS1 gene sequences of A/FPV/Rostock/34(H7N6) virus were much closer to those of A/SW/IW/30 and A/PR/8/34 viruses than to A/duck/Alberta/60/76(H12N5) virus, but not as close as the A/SW/IW/30 virus was to A/PR/8/34 virus.     

Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health

Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.     

The nucleoprotein is responsible for intracerebral pathogenicity of A/duck/Mongolia/47/2001 (H7N1) in chicks

Avian influenza viruses A/duck/Mongolia/47/2001 (H7N1) (47/01) and A/duck/Mongolia/867/2002 (H7N1) (867/02) were defined as low-pathogenic avian influenza viruses (LPAIVs) using an intravenous pathogenicity test in chickens. On the other hand, the intracerebral pathogenicity indices of 47/01 and 867/02 were 1.30 and 0.00, respectively. A series of reassortant viruses were generated between 47/01 and 867/02, and their intracerebral pathogenicity was compared in one-day-old chicks to identify the protein(s) responsible for the intracerebral pathogenicity of 47/01. The results indicate that the amino acids at positions 50 and 98 of the nucleoprotein are related to the pathogenicity of 47/01 in chicks by intracerebral inoculation. A significant association was found between mortality of the chicks inoculated intracerebrally with 47/01 and virus replication in the lungs and/or brain. These results indicate that the NP of avian influenza viruses may be responsible for intracerebral pathogenicity in the host.