Complete Genome Sequences of a Novel Reassortant H4N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China

A/duck/Shanghai/28-1/2009(H4N2) (DK28) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the DK28 virus. The sequence analysis demonstrated that this H4N2 virus was a novel multiple-gene reassortant avian influenza virus (AIV) whose genes originated from H1N1, H1N3, H3N3, H4N2, and H4N6. Knowledge regarding the complete genome sequences of the DK28 virus will be useful for epidemiological surveillance.     

Origin of the 1918 Spanish influenza virus: a comparative genomic analysis

To test the avian-origin hypothesis of the 1918 Spanish influenza virus we surveyed influenza sequences from a broad taxonomic distribution and collected 65 full-length genomes representing avian, human and "classic" swine H1N1 lineages in addition to numerous other swine (H1N2, H3N1, and H3N2), human (H2N2, H3N2, and H5N1), and avian (H1N1, H4N6, H5N1, H6N1, H6N6, H6N8, H7N3, H8N4, H9N2, and H13N2) subtypes. Amino acids from all eight segments were concatenated, aligned, and used for phylogenetic analyses. In addition, the genes of the polymerase complex (PB1, PB2, and PA) were analyzed individually. All of our results showed the Brevig-Mission/1918 strain in a position basal to the rest of the clade containing human H1N1s and were consistent with a reassortment hypothesis for the origin of the 1918 virus. Our genome phylogeny further indicates a sister relationship with the "classic" swine H1N1 lineage. The individual PB1, PB2, and PA phylogenies were consistent with reassortment/recombination hypotheses for these genes. These results demonstrate the importance of using a complete-genome approach for addressing the avian-origin hypothesis and predicting the emergence of new pandemic influenza strains.     

Complete Genomic Sequence of an H5N1 Influenza Virus from a Parrot in Southern China

An H5N1 avian influenza virus (AIV) designated A/Parrot/Guangdong/C99/2005 (H5N1) was first isolated from a sick parrot in Guangdong in southern China in 2005. The complete genome of this strain was analyzed. Genome sequence analysis showed that all 8 gene segments of the virus nucleotide had 99.0% homology to A/chicken/Henan/12/2004 (H5N1). Phylogenetic analysis demonstrated that all 8 gene segments of the virus were derived from the Eurasian lineage. The availability of genome sequences is useful to investigate the host range and genetic evolution of the H5N1 avian influenza virus in Southern China.     

1株H6N6亚型禽流感病毒分子遗传特性分析

本研究采用无特定病原体(specific pathogen free,SPF)鸡胚,从某活禽市场环境中分离出1株H6N6亚型禽流感病毒(A/environment/Zhenjiang/zj18/2013,en/zj18)。通过二代测序技术进行全基因组测序,通过BLASTn 进行同源性检索,并采用MEGA5.0软件构建系统发生树。基因进化树分析表明,分离株en/zj18的所有8个基因节段(PB2、PB1、PA、HA、NP、NA、M和NS)均与近年来中国华东地区流行的H6N6亚型禽流感病毒的相应基因位于同一进化分支,与参考株的核苷酸同源性达96.7%～99.6%。分离株en/zj18的HA蛋白裂解位点为PQIETR↓GL,是低致病性禽流感病毒的分子特征。HA蛋白上关键受体结合位点190和228位(按H3亚型的HA蛋白序列排序)氨基酸分别是E和G,理论上更易与α2,3-半乳糖苷唾液酸受体结合。结果提示,需加强活禽市场禽流感病毒的持续监测,从而为有效应对禽流感病毒对公共卫生的持续威胁提供科学依据。     

Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.

We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.     

Genetic Characterization of a Novel Recombinant H5N2 Avian Influenza Virus Isolated from Chickens in Tibet

In this report, a novel H5N2 avian influenza virus (AIV) was isolated from chickens in Tibet in 2010, western China. Phylogenetic analysis demonstrated that it was a natural reassortant between H9N2 and H5N1 subtypes. It is of note that this virus has an HP genotype with HA, PB2, M, and NS genes homologous to those of A/peregrine falcon/Hong Kong/2142/2008(H5N1)-like HPAIV isolated from dead wild birds. Publishing this genome information will contribute to the investigation of avian influenza epidemiology and to further research of AIV''s biological properties.     

PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs

It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75:2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151:1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.     

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 new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry.     

Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009

Background

In April 2009, a novel triple-reassortant swine influenza A H1N1 virus (“A/H1N1pdm”; also known as SOIV) was detected and spread globally as the first influenza pandemic of the 21^st century. Sequencing has since been conducted at an unprecedented rate globally in order to monitor the diversification of this emergent virus and to track mutations that may affect virus behavior.

Methodology/Principal Findings

By Sanger sequencing, we determined consensus whole-genome sequences for A/H1N1pdm viruses sampled nationwide in Canada over 33 weeks during the 2009 first and second pandemic waves. A total of 235 virus genomes sampled from unique subjects were analyzed, providing insight into the temporal and spatial trajectory of A/H1N1pdm lineages within Canada. Three clades (2, 3, and 7) were identifiable within the first two weeks of A/H1N1pdm appearance, with clades 5 and 6 appearing thereafter; further diversification was not apparent. Only two viral sites displayed evidence of adaptive evolution, located in hemagglutinin (HA) corresponding to D222 in the HA receptor-binding site, and to E374 at HA2-subunit position 47. Among the Canadian sampled viruses, we observed notable genetic diversity (1.47×10⁻³ amino acid substitutions per site) in the gene encoding PB1, particularly within the viral genomic RNA (vRNA)-binding domain (residues 493–757). This genome data set supports the conclusion that A/H1N1pdm is evolving but not excessively relative to other H1N1 influenza A viruses. Entropy analysis was used to investigate whether any mutated A/H1N1pdm protein residues were associated with infection severity; however no virus genotypes were observed to trend with infection severity. One virus that harboured heterozygote coding mutations, including PB2 D567D/G, was attributed to a severe and potentially mixed infection; yet the functional significance of this PB2 mutation remains unknown.

Conclusions/Significance

These findings contribute to enhanced understanding of Influenza A/H1N1pdm viral dynamics.     

Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China

A total of 100 HIN1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang,Hubei and Guangdong between June and November 2009,were provided by local CDC laboratories.After MDCK cell culture,57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.A total of 39 HA sequences,52 NA sequences,36 PB2 sequences,31 PB1 sequences,40 PA sequences,48 NP sequences,51 MP sequences and 36 NS sequences were obtained,including 20 whole genome sequences.Sequence comparison revealed they shared a high degree of homology (96％～99％) with known epidemic strains (A/Califomia/04/2009(H1N1).Phylogenetic analysis showed that although the sequences were highly conserved,they clustered into a small number of groups with only a few distinct strains.Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences:A/Hubei/86/2009 PKVRDQEG→PKVRDQEA,A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER,A/Hubei/75/2009PKVRDQEG→PKVRDQGG,the A/Hubei/75/2009 was isolated from an acute case,while the other two were from patients with mild symptoms.Other key sites such as 119,274,292 and 294 amino acids of NA protein,627 of PB2 protein were conserved.Meanwhile,all the M2 protein sequences possessed the Ser32Asn mutation,suggesting that these viruses were resistant to adamantanes.Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.     

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96–99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.     

Complete genomic sequence of a novel natural recombinant H6N2 influenza virus from chickens in Guangdong, Southern China

Here, we reported the complete genome sequence of a novel H6N2 avian influenza virus (AIV) isolated from chicken in Guangdong, Southern China, in 2011 which was a natural recombinant virus between the H6N2 and H5N1 subtypes. It will help to understand the epidemiology and molecular characteristics of H6N2 influenza virus in Southern China.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

Origin and molecular characterization of the human-infecting H6N1 influenza virus in Taiwan

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain’s pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.     

一株人感染高致病性禽流感(H5N1)病毒基因组分子特征分析

【背景】H5N1禽流感病毒可以感染人类导致重症呼吸道感染,致死率高。【目的】研究我中心确认的一例人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015的可能起源及基因组分子特征。【方法】对病人痰液样本中的H5N1病毒进行全基因组测序,使用CLC Genomics Workbench 9.0对序列进行拼接,使用BLAST和MEGA 5.22软件进行同源性比对和各片段分子特征分析。【结果】该株禽流感病毒属于H5亚型的2.3.2.1c家系,其8个片段均与江浙地区禽类中分离的病毒高度同源,未发现有明显的重配。分子特征显示,该病毒血凝素(Hemagglutinin,HA)蛋白裂解位点为PQRERRRR/G,受体结合位点呈现禽类受体特点,但出现D94N、S133A和T188I氨基酸置换增强了病毒对人类受体的亲和性。神经氨酸酶(Neuraminidase,NA)蛋白颈部在49-68位缺失20个氨基酸,非结构蛋白1 (Non-structure protein,NS1)存在P42S置换和80-84位氨基酸的缺失。其他蛋白中也存在多个增强病毒致病力和对人类细胞亲和力的氨基酸突变。对耐药位点分析发现存在对奥司他韦的耐药突变H_274Y,病毒对金刚烷胺仍旧敏感。【结论】人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015属于2.3.2.1c家系,禽类来源,关键位点较保守,但仍出现了多个氨基酸的进化与变异使其更利于感染人类。H5N1禽流感病毒进化活跃,持续动态监测不能放松。     

Genomic Signatures for Avian H7N9 Viruses Adapting to Humans

An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans.     

PB2 E627K对A/H6N1亚型禽流感病毒致病性的影响分析

目的利用A/H6N1亚型禽流感病毒的反向遗传平台,评估PB2 E627K对A/H6N1亚型禽流感病毒的致病性,探究A/H6N1流感病毒的致病性分子基础。方法通过A/H6N1亚型禽流感病毒A/Mallard/San-Jiang/275/2007株反向遗传操作系统和点突变技术拯救病毒rA/H6N1和PB2 E627K位点发生突变的rA/H6N1-627,两株拯救病毒分别以101EID50～106EID50的攻毒剂量人工感染BALB/c小鼠,通过体重变化、死亡率、病毒滴定等方面进行致病性分析。结果成功构建A/H6N1亚型禽流感病毒的反向遗传平台,rA/H6N1的8个基因片段完全源于A/H6N1的基因组,核苷酸序列及生物学特性与A/H6N1完全一致。rA/H6N1能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠呈现低致病性(MLD50>106.5EID50),病毒在小鼠体内的分布情况及各个脏器中的病毒滴度与A/H6N1保持一致;rA/H6N1-627能感染小鼠,引起小鼠体重下降,但不能引起所有106EID50组小鼠死亡,病毒能在小鼠的肺脏和脑部进行增殖。结论实验结果表明,在H5N1禽流感中发挥重要作用的PB2-E627K位点并非A/H6N1流感病毒的毒力决定因子。A/H6N1流感病毒致病性的分子基础还有待继续研究,该反向遗传操作系统和点突变技术的建立为研究该亚型流感病毒致病机制、传播机制及病毒基因功能奠定了基础,同时也为A/H6N1亚型禽流感病毒新型疫苗的研制开辟了新途径。     

The highly pathogenic H5N1 influenza A virus down‐regulated several cellular MicroRNAs which target viral genome

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3′UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR‐584‐5p and miR‐1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down‐regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR‐1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host‐mediated inhibition of viral replication through down‐regulation of cellular miRNAs, which target its viral genome.     

Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.