The NS1 gene contributes to the virulence of H5N1 avian influenza viruses

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.     

Paramyxovirus accessory proteins as interferon antagonists

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co-evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.     

Highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.     

Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs

It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-alpha/beta) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-alpha/beta synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-alpha/beta induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

H5N1亚型禽流感病毒NS第263—277位核苷酸缺失提高病毒对鸡的致病力

为研究2000年以来绝大多数H5N1亚型禽流感病毒分离株在非结构基因的第263—277位发生15个碱基缺失现象的生物学意义,构建H5N1A／D／SD／04株HA、NA、NS的全基因表达／转录载体,以及NS的删除突变载体（m248）,A/D/YZ/04株的NS基因表达／转录载体（848）和其补加15个核苷酸的NS突变载体（m848）。构建的载体分别与编码WSN（H1N1）内部基因载体进行组合转染,拯救获得4个具不同NS的重组的H5N1亚型流感病毒：RWSN-848和RWSN—m248在263-277位缺失15个碱基。RWSN-m848和RWSN-248则在相同位置不发生缺失。4个重组病毒的平均鸡胚繁殖效价（HA）、鸡胚的平均死亡时间（MDT）和鸡胚半数感染量（EID50）均无显著差异;但RWSN-848和RWSN-m248对6周龄SPF鸡的致病力明显高于RWSN—m848和RWSN-248。结果说明H5N1的NS基因在263～277位核苷酸发生缺失后,不影响重组H5N1在鸡胚中的繁殖性能,但提高了病毒对鸡的致病力。     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) Virus from an Adult Case in Fujian Province,China

正Dear Editor,As we known,pigs play a vital role as genetic mixing vessels for human and avian influenza viruses as their tracheal epitheliums possess both sialic acid a-2,6-Gal and a-2,3-Gal receptors(Ma et al.2008),and swine influenza viruses occasionally infect humans(Shinde et al.2009).The Eurasian avian-like swine influenza A(H1N1)virus     

A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice

In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.     

Amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene

Avian influenza virus (AIV) A/turkey/Oregon/71-SEPRL (TK/OR/71-SEPRL) (H7N3) encodes a full-length NS1 protein and is a weak inducer of interferon (IFN). A variant, TK/OR/71-delNS1 (H7N3), produces a truncated NS1 protein and is a strong inducer of IFN. These otherwise genetically related variants differ 20-fold in their capacities to induce IFN in primary chicken embryo cells but are similar in their sensitivities to the action of IFN. Furthermore, the weak IFN-inducing strain actively suppresses IFN induction in cells that are otherwise programmed to produce it. These phenotypic differences are attributed to the enhanced IFN-inducing capacity that characterizes type A influenza virus strains that produce defective NS1 protein. The pathogenesis of these two variants was evaluated in 1-day-old and 4-week-old chickens. The cell tropisms of both viruses were similar. However, the lesions in chickens produced by the weak IFN inducer were more severe and differed somewhat in character from those observed for the strong IFN inducer. Differences in lesions included the nature of inflammation, the rate of resolution of the infection, and the extent of viral replication and/or virus dissemination. The amelioration of pathogenesis is attributed to the higher levels of IFN produced by the variant encoding the truncated NS1 protein and the antiviral state subsequently induced by that IFN. The high titer of virus observed in kidney tissue ( approximately 10(9) 50% embryo lethal doses/g) from 1-day-old chickens infected intravenously by the weak IFN-inducing strain is attributed to the capacity of chicken kidney cells to activate the hemagglutinin fusion peptide along with their unresponsiveness to inducers of IFN as measured in vitro. Thus, the IFN-inducing capacity of AIV appears to be a significant factor in regulating the pathogenesis, virulence, and viral transmission of AIV in chickens. This suggests that the IFN-inducing and IFN induction suppression phenotypes of AIV should be considered when characterizing strains of influenza virus.     

Attenuation of equine influenza viruses through truncations of the NS1 protein

Equine influenza is a common disease of the horse, causing significant morbidity worldwide. Here we describe the establishment of a plasmid-based reverse genetics system for equine influenza virus. Utilizing this system, we generated three mutant viruses encoding carboxy-terminally truncated NS1 proteins. We have previously shown that a recombinant human influenza virus lacking the NS1 gene (delNS1) could only replicate in interferon (IFN)-incompetent systems, suggesting that the NS1 protein is responsible for IFN antagonist activity. Contrary to previous findings with human influenza virus, we found that in the case of equine influenza virus, the length of the NS1 protein did not correlate with the level of attenuation of that virus. With equine influenza virus, the mutant virus with the shortest NS1 protein turned out to be the least attenuated. We speculate that the basis for attenuation of the equine NS1 mutant viruses generated is related to their level of NS1 protein expression. Our findings show that the recombinant mutant viruses are impaired in their ability to inhibit IFN production in vitro and they do not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in MDCK cells, or in vivo in a mouse model. Therefore, these attenuated mutant NS1 viruses may have potential as candidates for a live equine influenza vaccine.     

Generation of High-yield Vaccine Strain Wholly Derived from Avian Influenza Viruses by Reverse Genetics

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the haemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 were generated by cell transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with haemagglutination unit of 1:2 048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce haemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and the maximum geometric mean HI-titers were observed 4–5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1-infected and H5N3-vaccinated animals.     

利用反向遗传学技术构建H5亚型禽流感高产疫苗株

采用RT-PCR技术分别扩增了鹅源高产禽流感病毒的6条内部基因片段,近期分离的H5N1亚型禽流感病毒的血凝素基因以及N3亚型参考毒株的神经氨酸酶基因,分别构建了8个基因的转录与表达载体,利用反向遗传学技术拯救出了全部基因都源于禽源的重组流感病毒疫苗株rH5N3。通过对血凝素蛋白HA1和HA2连接肽处的5个碱性氨基酸(R-R-R-K-K)基因缺失与修饰,从而消除了病毒基因的毒力相关序列,拯救的rH5N3疫苗株对鸡和鸡胚均无致病性,病毒在鸡胚尿囊液和细胞培养上清的HA效价得到极大提高,分别为12048和1512。制备的禽流感疫苗免疫动物后4~5周即可诱导产生高效价的HI抗体,鸡免疫后18周依然保持高水平的HI抗体。重组疫苗不论是对于国内早期分离的禽流感病毒A/Goose/Guangdong/1/96还是近期分离的A/Goose/HLJ/QFY/04都能够产生完全的免疫保护作用,免疫鸡攻毒后不发病、不排毒、不死亡。带有N3鉴别诊断标记禽流感疫苗株的研制为H5N1高致病性禽流感的防治提供了新的技术保障。     

Characterization of a human H5N1 influenza A virus isolated in 2003

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.     

Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.     

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

Lethal H5N1 influenza viruses escape 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 antiviral 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.     

人H5N1亚型禽流感病毒安徽株NS1基因的克隆及在原核系统的表达

利用RT-PCR方法,从人H5N1亚型禽流感病毒安徽株扩增到了NS1基因,对其进行了克隆、序列测定和分析,并在原核系统高效表达和纯化了NS1蛋白。进化分析表明,A/Anhui/01/2005毒株与近些年国内分离的水禽H5N1病毒进化关系更为接近。NS1与福建、湖南分离的禽流感病毒同源性最高,分别达到99.1%和98.2%。序列分析表明,与病毒的致病性相关的92位氨基酸为Asp,与病毒的细胞因子抗性相关的80~84位氨基酸发生缺失,与断裂/多聚腺苷酸化特异性因子结合的基序改变为GFEWN,和病毒致死性相关的PL基序为ESEV。随后在大肠杆菌高效表达并纯化了NS1蛋白。NS1基因及其编码产物的特性分析以及在原核系统的表达,为进一步研究NS1的致病机制和抗病毒药物研制奠定了基础。