Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

An overview of the highly pathogenic H5N1 influenza virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.     

Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies

We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) ΔHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants’ HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.     

The role of nuclear NS1 protein in highly pathogenic H5N1 influenza viruses

The non-structural protein (NS1) of influenza A viruses (IAV) performs multiple functions during viral infection. NS1 contains two nuclear localization signals (NLS): NLS1 and NLS2. The NS1 protein is located predominantly in the nucleus during the early stages of infection and subsequently exported to the cytoplasm. A nonsense mutation that results in a large deletion in the carboxy-terminal region of the NS1 protein that contains the NLS2 domain was found in some IAV subtypes, including highly pathogenic avian influenza (HPAI) H7N9 and H5N1 viruses. We introduced different mutations into the NLS domains of NS1 proteins in various strains of IAV, and demonstrated that mutation of the NLS2 region in the NS1 protein of HPAI H5N1 viruses severely affects its nuclear localization pattern. H5N1 viruses expressing NS1 protein that is unable to localize to the nucleus are less potent in antagonizing cellular antiviral responses than viruses expressing wild-type NS1. However, no significant difference was observed with respect to viral replication and pathogenesis. In contrast, the replication and antiviral defenses of H1N1 viruses are greatly attenuated when nuclear localization of the NS1 protein is blocked. Our data reveals a novel functional plasticity for NS1 proteins among different IAV subtypes.     

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。     

An H5N1 highly pathogenic avian influenza virus isolated from a local tree sparrow in Indonesia

The isolation of an H5N1 influenza A virus from a tree sparrow (Passer montanus) captured in East Java, Indonesia in 2010 is reported here. Its hemagglutinin and neuraminidase were genetically similar to those of human isolates from 2006-2007 in Indonesia. The finding of a tree sparrow H5N1 virus that possesses genetically similar surface molecules to those of human viruses highlights the importance of monitoring resident wild birds, as well as migratory birds, for pandemic preparedness.     

Inhibition of highly pathogenic avian H5N1 influenza virus replication by RNA oligonucleotides targeting NS1 gene

H5N1 avian influenza virus (AIV) has caused widespread infections in poultry and wild birds, and has the potential to emerge as a pandemic threat to human. In order to explore novel approaches to inhibiting highly pathogenic H5N1 influenza virus infection, we have developed short RNA oligonucleotides, specific for conserved regions of the non-structural protein gene (NS1) of AIV. In vitro the hemagglutination (HA) titers in RNA oligonucleotide-treated cells were at least 5-fold lower than that of the control. In vivo, the treatment with three doses of RNA oligonucleotides protected the infected chickens from H5N1 virus-induced death at a rate of up to 87.5%. Plaque assay and real-time PCR analysis showed a significant reduction of the PFU and viral RNA level in the lung tissues of the infected animals treated with the mixed RNA oligonucleotides targeting the NS1 gene. Together, our findings revealed that the RNA oligonucleotides targeting at the AIV NS1 gene could potently inhibit avian H5N1 influenza virus reproduction and present a rationale for the further development of the RNA oligonucleotides as prophylaxis and therapy for highly pathogenic H5N1 influenza virus infection in humans.     

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure—a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.     

中国H5N1禽流感病毒HA基因的分子进化分析

为了阐明中国H5N1禽流感病毒血凝素(HA)基因的分子进化规律,从GenBank下载中国近年来分离的27株禽流感H5N1病毒的HA基因,利用LaserGene软件包中的EditSeq程序剪切共同序列并翻译成氨基酸序列,与DNAMAN软件进行比对,并以LaserGene软件包中的MegAlign程序构建分子进化树,结合背景资料分析其传播流行规律。结果显示:①在HA裂解位点上游毗邻的位置,gdch04、gddu04、gxch0405、gxdu05、gxqu0502、hkcph05、stch05、stqu05、ynch0502株都缺失了三个碱基;②27株毒株在HA裂解位点上游毗邻的位置存在多个连续的碱性氨基酸;③来自广西的gxch0405、gxdu05、gxqu0502和来自香港的hkcph05、hkgh04的序列同源性很高,很可能与苍鹭的迁移有关;④来自广西的gxqu0502和来自香港的hkcph05亲源关系很近,很可能来自同一祖先。     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using α‐agglutinin for production of oral vaccines

Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol‐anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C‐terminal fusion with the Saccharomyces cerevisiae GPI‐anchored cell wall protein, α‐agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast‐based vaccine for protection against highly pathogenic strains of avian influenza. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.     

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1–3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

H5N8亚型高致病性禽流感病毒的全球流行概况

H5N8亚型高致病性禽流感病毒(highly pathogenic avian influenza virus,HPAIV)随候鸟的迁徙活动及商业贸易活动现已蔓延至亚洲、欧洲、非洲、美洲等国家和地区.2014-2015和2016-2019年H5N8亚型HPAIV已引发两波全球疫情,现正经历第三波疫情,导致家禽及野生鸟类...     

H5N1亚型禽流感病毒HA基因核酸序列的变异分析

从GenBank上获得194株不同来源的H5N1亚型禽流感病毒HA基因核酸序列,利用MEGA3分析了HA基N核酸序列碱基突变的特点。并通过比较序列同源性,构建NJ系统进化树,探讨了不同来源的H5N1病毒的系统进化关系。序列分析结果表明,被研究的194条H5N1亚型禽流感病毒HA基因核酸序列,碱基长度大约在1700bp左右,共发现了757个可变位点,其中Parsimony—informative sites有537个,Singleton sites有220个;病毒的变异速率很快,平均变异率为3．23％;病毒的序列变异具有显著的地区特点和时间特点;同时,全球化的贸易以及候鸟的迁徙在传播病毒过程中起一定作用。