Zhao Gao

Novel H5 clade 2.3.4.6 viruses with both α-2,3 and α-2,6 receptor binding properties may pose a pandemic threat

The emerging H5 clade 2.3.4.6 viruses of different NA subtypes have been detected in different domestic poultry in China. We evaluated the receptor binding property and transmissibility of four novel H5 clade 2.3.4.6 subtype highly pathogenic avian influenza viruses. The results show that these viruses bound to both avian-type (α-2,3) and human-type (α-2,6) receptors. Furthermore, we found that one of these viruses, GS/EC/1112/11, not only replicated but also transmitted efficiently in guinea pigs. Therefore, such novel H5 subtype viruses have the potential of a pandemic threat.

Adaptation of a natural reassortant H5N2 avian influenza virus in mice

It is reported that the H5N2 highly pathogenic avian influenza virus A/chicken/Hebei/1102/2010 (HB10) is a natural reassortant between circulating H5N1 and endemic H9N2 influenza viruses. To evaluate the potential of its interspecies transmission, the wild-type HB10 was adapted in mice through serial lung passages. Increased virulence was detectable in 5 sequential lung passages in mice and a highly virulent mouse-adapted strain (HB10-MA) with a 50% mouse lethal dose of 10(2.5) 50% egg infectious dose was obtained in 15 passages. The virulence and the replication efficiency of HB10-MA in mice were significantly higher than those of HB10 while HB10-MA grew faster and to significantly higher titers than HB10 in MDCK and A549 cells. Only five amino acid mutations in four viral proteins (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) of HB10-MA virus were found when compared with those of HB10, indicating that they may be responsible for the adaptation of the novel reassortant H5N2 avian influenza virus in mice with increased virulence and replication efficiency. The results in this study provide helpful insights into the pathogenic potential of novel reassortant H5N2 viruses to mammals that deserves further attentions.

Adaptive mutations in PB2 gene contribute to the high virulence of a natural reassortant H5N2 avian influenza virus in mice.

The highly pathogenic A/chicken/Hebei/1102/2010 (HB10) H5N2 virus is a natural reassortant derived from circulating H5N1 and endemic H9N2 avian influenza viruses (AIV). To evaluate the potential of its interspecies transmission, we previously serially passaged the non-virulent HB10 virus in the mouse lung and obtained a high virulence variant (HB10-MA). Genomic sequencing revealed five mutations (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) that distinguished HB10-MA virus from its parental HB10 virus. In this study, we further investigated the molecular basis for the enhanced virulence of HB10-MA in mice. By generating a series of reassortants between the two viruses and evaluating their virulence in mice, we found that both PB2 and PA genes contribute to the high virulence of HB10-MA in mice, whereas PB2 gene carrying the 591K and/or 701N had a dominant function. In addition, the two amino acids showed a cumulative effect on the virulence, virus replication, and polymerase activity of HB10 or HB10-MA. Therefore, our results collectively emphasized the crucial role of PB2 gene, particularly the paired mutations of Q591K and D701N in the host adaptation of the novel reassortant H5N2 AIV in mammals, which may provide helpful insights into the pathogenic potential of emerging AIV in human beings.

PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice.

PA-X is a novel discovered accessory protein encoded by the PA mRNA. Our previous study demonstrated that PA-X decreases the virulence of a highly pathogenic H5N1 strain A/Chicken/Jiangsu/k0402/2010 in mice. However, the underlying mechanism of virulence attenuation associated with PA-X is still unknown. In this study, we compared two PA-X-deficient mutant viruses and the parental virus in terms of induction of pathology and manipulation of host response in the mouse lung, stimulation of cell death and PA nuclear accumulation. We first found that down-regulated PA-X expression markedly aggravated the acute lung injury of the infected mice early on day 1 post-infection (p.i.). We then determined that loss of PA-X expression induced higher levels of cytokines, chemokines and complement-derived peptides (C3a and C5a) in the lung, especially at early time point’s p.i. In addition, in vitro assays showed that the PA-X-deficient viruses enhanced cell death and increased expression of reactive oxygen species (ROS) in mammalian cells. Moreover, we also found that PA nuclear accumulation of the PA-X-null viruses accelerated in MDCK cells. These results demonstrate that PA-X decreases the level of complement components, ROS, cell death and inflammatory response, which may together contribute to the alleviated lung injury and the attenuation of the virulence of H5N1 virus in mice.

Novel reassortant H5N5 viruses bind to a human-type receptor as a factor in pandemic risk

Highly pathogenic avian influenza A(HPAI) H5N1 viruses pose a serious pandemic threat due to their virulence and high mortality in humans, and their increasingly expanding host range and significant ongoing evolution could enhance their human-to-human transmissibility. Recently, various reassortant viruses were detected in different domestic poultry, with the HA gene derived from the A/goose/Guangdong/1/96-like (Gs/GD-like) lineage and the NA gene from influenza viruses of other subtypes. It is reported that some natural reassortant H5N5 highly pathogenic avian influenza viruses were isolated from poultry in China. And their HA genes were belonged to a new clade 2.3.4.4. We evaluated the receptor binding property and transmissibility in guinea pigs of these reassortant H5N5 HPAIVs. The results showed that these viruses bound to both avian-type (α-2,3) and human-type (α-2,6) receptors. In addition, we found that one of these viruses, 031, not only replicated but also transmitted efficiently in guinea pigs. Therefore, such reassortant influenza viruses may pose a pandemic threat.

iTRAQ-based quantitative proteomics reveals important host factors involved in the high pathogenicity of the H5N1 avian influenza virus in mice

Abstract We previously reported a pair of H5N1 avian influenza viruses which are genetically similar but differ greatly in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly lethal to mice, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is avirulent. In this study, to investigate the host factors that account for their virulence discrepancy, we compared the pathology and host proteome of the CK10- or GS10-infected mouse lung. Moderate lung injury was observed from CK10-infected animals as early as the first day of infection, and the pathology steadily progressed at later time point. However, only mild lesions were observed in GS10-infected mouse lung at the late infection stage. Using the quantitative iTRAQ coupled LC–MS/MS method, we first found that more significantly differentially expressed (DE) proteins were stimulated by GS10 compared with CK10. However, bio-function analysis of the DE proteins suggested that CK10 induced much stronger inflammatory response-related functions than GS10. Canonical pathway analysis also demonstrated that CK10 highly activated the “Acute Phase Response Signaling,” which results in a wide range of biological activities in response to viral infection, including many inflammatory processes. Further in-depth analysis showed that CK10 exacerbated acute lung injury-associated responses, including inflammatory response, cell death, reactive oxygen species production and complement response. In addition, some of these identified proteins that associated with the lung injury were further confirmed to be regulated in vitro. Therefore, our findings suggest that the early increased lung injury-associated host response induced by CK10 may contribute to the lung pathology and the high virulence of this virus in mice.

Characteristics of the emerging chicken-origin highly pathogenic H7N9 viruses: A new threat to public health and poultry industry

Two H7N9 chicken isolates showed amino acids insertion and mutation in the cleavage site of the hemagglutinin similar as those from the reported human cases. Phylogenetically, they formed a separate cluster. Animal infection test confirmed their high pathogenicity in chickens and varied virulence in mice, indicating the new threat to public health and poultry industry.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Generation and Comprehensive Analysis of Host Cell Interactome of the PA Protein of the Highly Pathogenic H5N1 Avian Influenza Virus in Mammalian Cells.

Accumulating data have identified the important roles of PA protein in replication and pathogenicity of influenza A virus (IAV). Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis. In this study, using immunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry, we identified 278 human cellular proteins that might interact with PA of H5N1 IAV. Gene Ontology annotation revealed that the identified proteins are highly associated with viral translation and replication. Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation, infectious disease, and signal transduction. In addition, Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival. Moreover, two cellular proteins (nucleolin and eukaryotic translation elongation factor 1-alpha 1) identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays. Therefore, this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells.

Host Interaction Analysis of PA-N155 and PA-N182 in Chicken Cells Reveals an Essential Role of UBA52 for Replication of H5N1 Avian Influenza Virus

PA-N155 and PA-N182 proteins were translated from the 11th and 13th start codon AUG of the RNA polymerase acidic protein (PA) mRNA of H5N1 influenza A virus (IAV), which plays an important role in viral replication. Little is known about the interactions between PA-N155 and PA-N182 and the host proteins. This study investigated the interaction landscape of PA-N155 and PA-N182 of H5N1 IAV in chicken cells while their interacting complexes were captured by immunoprecipitation and analyzed by mass spectrometry. A total of 491 (PA-N155) and 302 (PA-N182) interacting proteins were identified. Gene ontology and pathway enrichment analyses showed that proteins of the two interactomes were enriched in RNA processing, viral processing and protein transport, and proteins related to signaling pathways of proteasome, ribosome, and aminoacy1-tRNA biosynthesis were significantly enriched, suggesting their potential roles in H5N1 IAV infection. Comparative analysis of the interactome of PA, PA-N155, and PA-N182 identified UBA52 as a conserved host factor that interacted with all three viral proteins. UBA52 is a fusion protein consisting of ubiquitin at the N terminus and ribosomal protein L40 at the C terminus. Knockdown of UBA52 significantly decreased the titer of H5N1 IAV in chicken cells and was accompanied with attenuated production of proinflammatory cytokines. Our analyses of the influenza–host protein interactomes identified UBA52 as a PA interaction protein for virus replication.