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.     

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.     

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.     

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

新世纪流感大流行的思考

2009年从墨西哥开始暴发了一场席卷全世界的流感疫情．此次大流行的毒株,甲型H1N1病毒,包含了猪源、禽源和人源流感病毒的基因片段．研究该毒株的基因重配、进化历程及其生物学特性,将对防控此次流行具有重要意义．目前,该毒株的遗传进化关系已明确,通过遗传性状分析可获知该毒株可能的生物学性状,但流感大流行动向、毒株遗传变化、毒力及致病性变化仍在密切监控中．流感病毒生态系统具有复杂性,其基因组易突变、易重配、易在自然宿主保存,使得流感大流行存在一定的必然性．正视流感大流行的威胁,积极提高流感病毒在生态系统中的监控,加强流行病学调查,发展疫苗与药物,建立有效公共卫生保障体系,才能降低流感大流行的破坏性．     

Development and diagnostic application/evaluation of pandemic (H1N1) 2009 influenza virus-specific monoclonal antibodies

We prepared mAb specific to the H1N1 2009 virus (H1N1 2009) to facilitate development of an RDT with enhanced sensitivity and specificity. Among these antibodies, we identified two clones--hybridomas 1H7E1 and 3A3H7-that specifically bound to H1N1 2009 (non-seasonal) and were very suitable for application to a diagnostic kit. The affinity constants (K(a)) of 1H7E1 and 3A3H7 were 1.10 × 10(10) and 2.35 × 10(10), respectively. To identify the antibodies, we performed ELISA and immunoblot analyses and found that 1H7E1 recognized a conformational epitope of HA while 3A3H7 recognized a linear epitope. In clinical evaluations using specimens from 215 patients, a lateral flow rapid testing kit comprising these mAb showed a sensitivity of 81.5% (75/92) and a specificity of 96.7% (119/123). Results using the RDT kit were well correlated with conventional RT-PCR methods as commonly and commercially used. Based on our findings, we believe that use of these mAb with a rapid evaluation kit could serve as a good diagnostic tool for H1N1 2009.     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

Hepatitis C Virus NS5A Activates the Mammalian Target of Rapamycin (mTOR) Pathway,Contributing to Cell Survival by Disrupting the Interaction between FK506-binding Protein 38 (FKBP38) and mTOR

Hepatitis C virus (HCV) often establishes a persistent infection that most likely involves a complex host-virus interplay. We previously reported that the HCV nonstructural protein 5A (NS5A) bound to cellular protein FKBP38 and resulted in apoptosis suppression in human hepatoma cell line Huh7. In the present research we further found that NS5A increased phosphorylation levels of two mTOR-targeted substrates, S6K1 and 4EBP1, in Huh7 in the absence of serum. mTOR inhibitor rapamycin or NS5A knockdown blocked S6K1 and 4EBP1 phosphorylation increase in NS5A-Huh7 and HCV replicon cells, suggesting that NS5A specifically regulated mTOR activation. Overexpression of NS5A and FKBP38 mutants or FKBP38 knockdown revealed this mTOR activation was dependent on NS5A-FKBP38 interaction. Phosphatidylinositol 3-kinase (PI3K) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment in NS5A-Huh7 showed that the mTOR activation was independent of PI3K. Moreover, NS5A suppressed caspase 3 and poly(ADP-ribose) polymerase activation, which was abolished by NS5A knockdown or rapamycin, indicating NS5A inhibited apoptosis specifically through the mTOR pathway. Further analyses suggested that apoptotic inhibition exerted by NS5A via mTOR also required NS5A-FKBP38 interaction. Glutathione S-transferase pulldown and co-immunoprecipitation showed that NS5A disrupted the mTOR-FKBP38 association. Additionally, NS5A or FKBP38 mutants recovered the mTOR-FKBP38 interaction; this indicated that the impairment of mTOR-FKBP38 association was dependent on NS5A-FKBP38 binding. Collectively, our data demonstrate that HCV NS5A activates the mTOR pathway to inhibit apoptosis through impairing the interaction between mTOR and FKBP38, which may represent a pivotal mechanism for HCV persistence and pathogenesis.     

B cell epitopes in the intrinsically disordered regions of neuraminidase and hemagglutinin proteins of H5N1 and H9N2 avian influenza viruses for peptide-based vaccine development

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were −1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were −1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were −0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were −0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use.     

Differential miRNA expression profiling of highly pathogenic avian influenza virus H5N1 infected chicken lungs reveals critical microRNAs,biological pathways and genes involved in the molecular pathogenesis

Highlights
· Highthrouput sequencing of small RNA of H5N1 infected and mock infected chicken lungs.
· 297 miRNAs identified in mock-infected and 201 miRNAs identified in AIV infected chicken lungs.
· 36 miRNAs were upregulated and 90 were downregulated during H5N1 infection.
· Functional analysis and gene ontology of predicted target genes of expressed miRNAs.
· MAPK pathway, NF-κB, IGF and gga-let-7b might play important role during H5N1 pathogenesis.