Qunhui Li

Enzootic genotype S of H9N2 avian influenza viruses donates internal genes to emerging zoonotic influenza viruses in China

Avian influenza viruses of subtype H9N2 are widely prevalent in poultry in many Asian countries, and the segmented nature of the viral genome results in multiple distinct genotypes via reassortment. In this study, genetic evolution of H9N2 viruses circulating in eastern China during 2007-2013 was analyzed. The results showed that the diversity of the gene constellations generated six distinct genotypes, in which a novel genotype (S) bearing the backbone of A/chicken/Shanghai/F/98-like viruses by acquiring A/quail/Hong Kong/G1/97-like polymerase basic subunit 2 and matrix genes has gradually established its ecological niche and been consistently prevalent in chicken flocks in eastern China since its first detection in 2007. Furthermore, genotype S possessed the peculiarity to donate most of its gene segments to other emerging influenza A viruses in China, including the novel reassortant highly pathogenic avian influenza H5N2, the 2013 novel H7N7, H7N9 and the latest reassortant H10N8 viruses, with potential threat to poultry industry and human health.

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.

Molecular mechanism of the airborne transmissibility of H9N2 avian influenza A viruses in chickens

ABSTRACT H9N2 avian influenza virus has been prevalent in poultry in many parts of the world since the 1990s and occasionally crosses the host barrier, transmitting to mammals, including humans. In recent years, these viruses have contributed genes to H5N1 and H7N9 influenza viruses, threatening public health. To explore the molecular mechanism for the airborne transmission of H9N2 virus, we compared two genetically close strains isolated from chickens in 2001, A/chicken/Shanghai/7/2001(SH7) and A/chicken/Shanghai/14/2001 (SH14). SH7 is airborne transmissible between chickens, whereas SH14 is not. We used reverse genetics and gene swapping to derive recombinant SH7 (rSH7), rSH14, and a panel of reassortant viruses. Among the reassortant viruses, we identified segments HA and PA as governing the airborne transmission among chickens. In addition, the NP and NS genes also contributed to a lesser extent. Furthermore, the mutational analyses showed the transmissibility phenotype predominantly mapped to the HA and PA genes, with HA-K363 and PA-L672 being important for airborne transmissibility among chickens. In addition, the viral infectivity and acid stability are related to the airborne transmissibility. Importantly, airborne transmission studies of 18 arbitrarily chosen H9N2 viruses from our collections confirmed the importance of both 363K in HA and 672L in PA in determining their levels of transmissibility. Our finding elucidates the genetic contributions to H9N2 transmissibility in chickens and highlights the importance of their prevalence in poultry. IMPORTANCE Our study investigates the airborne transmissibility of H9N2 viruses in chickens and the subsequent epidemic. H9N2 virus is the donor for several prevalent reassortant influenza viruses, such as H7N9/2013 and the H5N1 viruses. Poultry as the reservoir hosts of influenza virus is closely associated with human society. Airborne transmission is an efficient pathway for influenza virus transmission among flocks and individuals. Exploring the mechanism of the airborne transmission of the H9N2 virus in chickens could provide essential data regarding prevention and control of influenza endemics and pandemics.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process.

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.

Virulence Determinants in the PB2 Gene of a Mouse-Adapted H9N2 Virus

ABSTRACT The molecular bases of adaptation and pathogenicity of H9N2 influenza virus in mammals are largely unknown. Here, we show that a mouse-adapted PB2 gene with a phenylalanine-to-leucine mutation (F404L) mainly contributes to enhanced polymerase activity, replication, and pathogenicity of H9N2 in mice and also increases the virulence of the H5N1 and 2009 pandemic H1N1 influenza viruses. Therefore, we defined a novel pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.

Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein

Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein.

The antigenic drift molecular basis of the H5N1 influenza viruses in a novel branch of clade 2.3.4.

H5N1 subtype influenza A virus has evolved into many HA clades since late 1990 s. Six circulating H5N1 influenza viruses clustered to a novel branch in clade 2.3.4 and could escape vaccine protection, indicating their antigenic drift. Eleven amino acids substitutions in three antigenic sites of the hemagglutinin of these isolates were found when compared with the hemagglutinin of the primary viruses in clade 2.3.4. On the backbone of the novel isolates A/chicken/Northern China/k0602/2010, we generated a panel of recombinant viruses with HA mutations of restoring the primary vaccine strain Re-5s amino acid and homologous antisera to determine the role of these substitutions. The results of cross-HI assay, micro-neutralization assay and the antigen map of the mutated recombinant viruses showed that three substitutions in antigenic site B, especially D205K, are the major contributors to the antigenic drift of the novel branch of clade 2.3.4. Our study highlights the importance of surveillance of antigenic drift of H5N1 viruses for the control and preparedness of pandemic threats.

Reassortant H5N1 avian influenza viruses containing PA or NP gene from an H9N2 virus significantly increase the pathogenicity in mice.

Reassortment between different influenza viruses is a crucial way to generate novel influenza viruses with unpredictable virulence and transmissibility, which may threaten the public health. As currently in China, avian influenza viruses (AIVs) of H9N2 and H5N1 subtypes are endemic in poultry in many areas, while they are prone to reassort with each other naturally. In order to evaluate the risk of the reassortment to public health, A/Goose/Jiangsu/k0403/2010 [GS/10(H5N1)] virus was used as a backbone to generate a series of reassortants, each contained a single internal gene derived from the predominant S genotype of the A/Chicken/Jiangsu/WJ57/2012 [WJ/57(H9N2)]. We next assessed the biological characteristics of these assortments, including pathogenicity, replication efficiency and polymerase activity. We found that the parental WJ/57(H9N2) and GS/10(H5N1) viruses displayed high genetic compatibility. Notably, the H5N1 reassortants containing the PA or NP gene from WJ/57(H9N2) virus significantly increased virulence and replication ability in mice, as well as markedly enhanced polymerase activity. Our results indicate that the endemicity of H9N2 and H5N1 in domestic poultry greatly increases the possibility of generating new viruses by reassortment that may pose a great threat to poultry industry and public health.