禽流感病毒H5N1亚型RNA聚合酶蛋白表达纯化及晶体生长

通过使用原核表达载体大量表达H5N1病毒RNA聚合酶亚基PA-C257,PB1_N25,再经过GST亲和层析和Sephadex G-200层析柱纯化,获得了高纯度的蛋白复合体.采用悬滴气相扩散法筛选蛋白晶体,在1～1.5mol/L乙酸钠和pH7.9条件下获得了理想的晶体,为解析禽流感病毒RNA聚合酶三维结构并进一步认识其生物功能奠定了基础.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA

Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 A crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.     

Molecular characterization of avian influenza virus (H7N8) isolated from poultry in Central China in the mid-1980s

The molecular and pathogenic properties of avian influenza virus (A/duck/Hubei/216/1985/H7N8) isolated from Hubei Province of China in 1985 were characterized.The hemagglutinin gene (HA) of Dk/Hb/216/85/H7N8 had the multiple amino acid se-quences (-PEIPKGRG-) at the connecting peptide between HA1 and HA2, which is considered to be a distinguishing molecular characteristic of low pathogenicity.The key sites of host markers among the genes (M, NP, NS, PA and PB2) of Dk/Hb/ 216/85/H7N8 were similar to those of...     

甲型H1N1流感超敏感诊断方法的建立及优化

利用几种助沉剂和核酸纯化方法富集病毒RNA,建立甲型H1N1流感超敏感诊断方法。收集31份甲型H1N1流感确诊病人提取的病毒RNA,稀释至1000分之一倍后分别应用酵母tRNA、糖原、AcrylCarrier助沉剂和磁珠、硅胶颗粒和离心柱的9种组合进行病毒RNA的富集,富集后分别进行RealtimePCR检测。并对检测结果进行统计学分析。利用几种助沉剂和核酸纯化方法的组合时,发现磁珠+AcrylCarrier助沉剂方法是最佳的核酸富集方法,能够大大提高病毒核酸的检出效率。建立了甲型H1N1流感超敏感诊断方法,该方法能够大大提高甲型H1N1流感的检出率。     

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.     

Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.     

Genome evolution of novel influenza A （H1N1 ） viruses in humans

The epidemic situation of A H1N1 flu arose in North America in April 2009, which rapidly expanded to three continents of Europe, Asia and Africa, with the risk ranking up to 5. Until May 13th, the flu virus of A H1N1 had spread into 33 countries and regions, with a laboratory confirmed case number of 5728, including 61 deaths. Based on IRV and EpiFluDB database, 425 parts of A H1N1 flu virus sequence were achieved, followed by sequenced comparison and evolution analysis. The results showed that the current predominant A H1N1 flu virus was a kind of triple reassortment A flu virus： （i） HA, NA, MP, NP and NS originated from swine influenza virus; PB2 and PA originated from bird influenza virus; PB1 originated from human influenza virus. （ii） The origin of swine influenza virus could be subdivided as follows： HA, NP and NS originated from classic swine influenza virus of H1N1 subtype; NA and MP originated from bird origin swine influenza virus of H1N1 subtype. （iii） A H1N1 flu virus experienced no significant mutation during the epidemic spread, accompanied with no reassortment of the virus genome. In the paper, the region of the representative strains for sequence analysis （A/California/04/2009 （H1N1） and A/Mexico/4486/2009 （H1N1）） included USA and Mexico and was relatively wide, which suggested that the analysis results were convincing.     

H5N1亚型禽流感病毒血凝素基因的克隆与表达

 根据已知H5N1亚型禽流感病毒血凝素(HA)基因序列设计、合成克隆引物.自灭活的云南地方H5N1亚型病毒阳性临床组织样品中提取总RNA,反转录后采用高可信度DNA聚合酶(Pyobest^TMDNA Polymerase)扩增HA基因,采用Invitrogen定向表达系统(Champion^TMpET directional TOPO expression system)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组HA,分子质量约78ku.采用阳性血清经免疫印迹及ELISA分析重组HA的免疫反应性,结果表明重组HA能与H5N1亚型病毒抗血清发生特异性结合,具有良好的免疫反应性.     

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin （HA） cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.     

流感分子病毒学研究进展

流感病毒繁殖周期很短,基因结构又非常简单,病毒RNA分成8个独立的片段。每个基因片段编码一个蛋白,有利于宿主发生双重感染后不同毒株之间基因交换重组。流感病毒为了自身生存,适应外界环境,频繁发生变异。每次新亚型出现,都引起世界性大流行。人类与流感病毒将长期共存。     

X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains-a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker-is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-A-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.     

Architecture of ribonucleoprotein complexes in influenza A virus particles

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.     

Avian flu: isolation of drug-resistant H5N1 virus

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.     

甲型流感病毒PB1-PA蛋白作用位点的发现与分析

 基于共进化理论,探究了甲型流感病毒PB1蛋白与PA蛋白上具有共同进化可能性的保守九聚片段 (C9MP)。结构信息显示PB1蛋白的第1-15位氨基酸与PA蛋白的第239-716位氨基酸具有相互作用域;对该区域变异分布的分析发现,PA蛋白第670位氨基酸Q所在的C9MP与PB1蛋白的第9位氨基酸F、第12位氨基酸V和第13位氨基酸P所在的C9MP在PB1-MP1相互作用面上具有最低的共进化值。结合DSSP程序的分析表明,由PA蛋白第670位氨基酸Q与PB1蛋白的第9位氨基酸F、第12位氨基酸V与第13位氨基酸P构成的区域可能成为潜在的相互作用位点。     

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.