南京市2011年乙型流感血凝素基因分子特征分析

[目的]分析2011年南京市乙型流感病毒的血凝素(HA)分子学特征.[方法]选择7株2011年南京市不同时间段有代表性的乙型流感毒株进行HA基因序列测定,通过生物信息学方法对HA分子学特征进行分析.[结果]7株乙型流感毒株分为两个系,4株为Victoria,3株为Yamagata;与2011年度疫苗株相比,Victoria和Yamagata系毒株分别在抗原位点146、197和116、198发生了氨基酸替换;其中197和198位点分别是Victoria和Yamagata毒株的受体结合位点,由于上述位点的替换使得Victoria系/Yamagata系毒株分别在197/196位增加了一个潜在的糖基化位点.[结论]2011年南京市乙型流感Victoria 系和Yamagata系病毒同时存在,Victoria/Yamagata毒株197/198位点的氨基酸替换,值得做进一步的探讨.     

Emergence and genetic variation of neuraminidase stalk deletions in avian influenza viruses

When avian influenza viruses (AIVs) are transmitted from their reservoir hosts (wild waterfowl and shorebirds) to domestic bird species, they undergo genetic changes that have been linked to higher virulence and broader host range. Common genetic AIV modifications in viral proteins of poultry isolates are deletions in the stalk region of the neuraminidase (NA) and additions of glycosylation sites on the hemagglutinin (HA). Even though these NA deletion mutations occur in several AIV subtypes, they have not been analyzed comprehensively. In this study, 4,920 NA nucleotide sequences, 5,596 HA nucleotide and 4,702 HA amino acid sequences were analyzed to elucidate the widespread emergence of NA stalk deletions in gallinaceous hosts, the genetic polymorphism of the deletion patterns and association between the stalk deletions in NA and amino acid variants in HA. Forty-seven different NA stalk deletion patterns were identified in six NA subtypes, N1-N3 and N5-N7. An analysis that controlled for phylogenetic dependence due to shared ancestry showed that NA stalk deletions are statistically correlated with gallinaceous hosts and certain amino acid features on the HA protein. Those HA features included five glycosylation sites, one insertion and one deletion. The correlations between NA stalk deletions and HA features are HA-NA-subtype-specific. Our results demonstrate that stalk deletions in the NA proteins of AIV are relatively common. Understanding the NA stalk deletion and related HA features may be important for vaccine and drug development and could be useful in establishing effective early detection and warning systems for the poultry industry.     

Growth control of influenza A virus by M1 protein: analysis of transfectant viruses carrying the chimeric M gene.

Analysis of fast-growing reassortants (AWM viruses) of influenza A virus produced by mixed infection with a fast-growing WSN strain and a slowly growing Aichi strain indicated that the M gene plays a role in the regulation of virus growth rate at an early step of infection (J. Yasuda, T. Toyoda, M. Nakayama, and A. Ishihama, Arch. Virol. 133:283-294, 1993). To determine which of the two M gene products, M1 or M2, is responsible for the growth rate control, one recombinant WSN virus (CWA) clone possessing a chimeric M gene (WSN M1-Aichi M2) was generated by using an improved reverse genetics and transfection system. The recombinant CWA virus retained the phenotype of both large plaque formation and early onset of virus growth. This indicates that the WSN M1 protein is responsible for rapid virus growth.     

Susceptibility of influenza A viruses to amantadine is influenced by the gene coding for M protein.

Influenza A virus recombinants derived from "resistant" and "sensitive" parental viruses were examined for susceptibility to inhibition by amantadine. Correlation of gene constellation and amantadine susceptibility revealed that the gene coding for M protein influences sensitivity or resistance to amantadine. All recombinants which derived an M protein from an amantadine-resistant parent were found to be resistant to amantadine. All amantadine-sensitive recombinants derived an M gene from the amantadine-sensitive parent. However, a few amantadine-resistant recombinants which derived an M gene from the sensitive parent were also isolated, suggesting that the expression of amantadine sensitivity in these recombinants may be influenced by other genes.     

Nucleotide sequence of the influenza virus A/USSR/90/77 neuraminidase gene

The complete nucleotide sequence of the N1 neuraminidase gene of influenza virus A/USSR/90/77 was determined. Comparison of its predicted amino acid sequence with other N1 and N2 neuraminidases indicates that the N1 neuraminidases share most of the antigenic determinants mapped on the N2 neuraminidase but display at least one additional potentially antigenic region probably as a result of intersubtypic differences in glycosylation.     

A universal influenza A vaccine based on the extracellular domain of the M2 protein.

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.     

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterization and phylogenetic analysis of H3N2 human influenza A viruses in Cheongju, South Korea

To investigate the genetic characteristics of human influenza viruses circulating in Chungbuk province, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Genetic characterization of the HA genes of H3N2 isolates indicated the relative higher similarity to A/Virginia/04/07 (99.6%) rather than that of A/Wisconsin/67/2005 (98.4%), a Northern Hemisphere 2007∼2008 vaccine strain, based on amino acid sequences. We found several altered amino acids at the H3 HA1 antigenic sites compared with the vaccine strain; K140I at site A, K158R at site B, and K173N (H471) or K173Q, and S262N at site E, but there was no antigenic shift among the H3N2 viruses. Interestingly, A/Cheongju/H383/08 and A/Cheongju/H407/08 isolates had single amino acid substitution at D151G on the catalytic site of the N2 NA while A/Cheongju/H412/08 and A/Cheongju/ H398/07 isolates had one amino acid deletion at residue 146. Furthermore, we found that 25% (3 out of 12 isolates) of the H3N2 subtype viruses had the amino acid substitution at position 31 on the M2 protein (Aspartic acid to Asparagine) and confirmed their drug-resistance by biological assays. Taken together, the results of this study demonstrated continuous evolutions of human H3N2 viruses by antigenic drift and also highlighted the need to closely monitor antigenic drug resistance in influenza A viruses to aid in the early detection of potentially pandemic strains, as well as underscore the need for new therapeutics.     

Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene

Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5′ and 3′ remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.     

Molecular cloning and sequence analysis of the mouse kallikrein-binding protein gene.

A genomic clone (MKBP-10) encoding the mouse kallikrein-binding protein (MKBP) was isolated from a mouse genomic DNA library by screening with a rat kallikrein-binding protein (RKBP) cDNA probe. The total sequenced region of the MKBP gene spans 8615 base pairs. The exon and intron locations of the RKBP gene were identified by similarity with the RKBP gene. The MKBP gene encodes a prepeptide of 417 amino acid residues which exhibits 71% homology with RKBP. A TATA box sequence was located in the 5' flanking region of the MKBP gene by similarity with the consensus sequence TATAAAA.     

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3′, suggesting a similar protease specificity. Also the putative heparin binding sites and `hinge' regions are highly homologous in mouse and hPCI.     

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.     

Viral replication, persistence in water and genetic characterization of two influenza A viruses isolated from surface lake water

Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats.     

Shift in oligosaccharide specificities of hemagglutinin and neuraminidase of influenza B viruses resistant to neuraminidase inhibitors

Influenza virus neuraminidase inhibitors (NAIs), currently used as anti-influenza drugs, can lead to the appearance of drug-resistant variants. Resistance to NAIs appears due to mutations in the active site of the neuraminidase (NA) molecule that decrease the NA enzymatic activity and sometimes in the hemagglutinin (HA) that decrease its affinity for cell receptors and, therefore, reduce the requirement for NA activity in releasing mature virions from infected cells. Using a set of sialo-oligosaccharides, we evaluated changes in the receptor-binding specificity of the HA and substrate specificity of the NA of influenza B viruses that had acquired resistance to NAIs. The oligosaccharide specificity of two pairs of field influenza B viruses, namely: i) B/Memphis/20/96 and its NAI-resistant variant, B/Memphis/20-152K/96, containing mutation R152K in the NA and 5 amino acid substitutions in the HA1, and ii) B/Hong Kong/45/2005 and its NAI-resistant variant B/Hong Kong/36/2005, containing a single R371K mutation in the NA, was evaluated. Wild type viruses bound strictly to a “human type” receptor, α2-6-sialo-oligosaccharide 6`SLN, but desialylated it is approximately 8 times less efficiently than the α2-3 sialosaccharides. Both drug-resistant viruses demonstrated the ability to bind to “avian type” receptors, α2-3 sialo-oligosaccharides (such as 3`SLN), whereas their affinity for 6`SLN was noticeably reduced in comparison with corresponding wild type viruses. Thus, the development of the NAI resistance in the studied influenza B viruses was accompanied by a readjustment of HA-NA oligosaccharide specificities.     

Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses

Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes.     

草地贪夜蛾PP2A的克隆、序列分析及在大肠杆菌中的表达

为建立斑蝥素等PP2A抑制剂的体外活性测定方法,用于以杀虫剂为目的的药物快速活性评价,本研究首次同源克隆了鳞翅目昆虫草地贪夜蛾Spodoptera frugiperda(J.E.Smith)的PP2A催化亚基cDNA全长序列,探索了该基因在大肠杆菌中的表达。结果显示,S.frugiperda的PP2A编码一条309个氨基酸的肽链,预测其蛋白质分子量为35.46 ku,等电点5.37。分析蛋白质氨基酸序列,没有发现信号肽和跨膜结构,推测该PP2A主要存在于胞质中。多重比对分析S.frugiperda及其它昆虫的PP2A,表明PP2A的保守性较高,可以用S.frugiperda PP2A作为研究斑蝥素等抑制剂的药物筛选靶标酶,用于杀虫剂的筛选。利用该基因构建pET30a-PP2A原核表达载体转化至E.coli BL21(DE3)中,在16³0℃,0.230℃,0.2⁰.8 mmol/L的IPTG下均能成功诱导PP2A-His表达,经Ni-琼脂糖柱纯化后可以得到SDS-PAGE呈现单一条带的纯化蛋白,电泳纯度大于90%,为进一步活性测定奠定了基础。