Huimou Liu

Genomic sequence of an isolate of Newcastle disease virus isolated from an outbreak in geese: a novel six nucleotide insertion in the non-coding region of the nucleoprotein gene

Summary.We have completely sequenced the genome of an isolate of Newcastle disease virus (NDV) obtained following an outbreak in geese. The genomic sequence consists of 15192 nt, which is six nt longer than the published full length genome of the NDV strains La Sota, B1, Clone-30, Beaudette C and HB V4. The six nt insertion was located in the non-coding region of the nucleoprotein (NP) gene between nt 1646 and nt 1647 of the NDV genome (numbered according to the genomic sequence of the La Sota strain). An additional 22 NDV strains were searched for the existence of this six nt insertion. NDVs in genotypes VI, VII, VIII and IX had this insertion while NDV’s in genotypes I, II, III, IV, and V did not. The significance of this insertion in NDV evolution is discussed.

Novel Variants of Clade 2.3.4 Highly Pathogenic Avian Influenza A(H5N1) Viruses, China

We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009–2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.

Characterization of H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern China during a 5 year period (1998-2002).

In the current study, we characterized H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation during a 5 year period (1998-2002). Phylogenetic analysis of the 8 genes of 11 representative viruses showed that they all shared high similarity to that of the first isolate, A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages. Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of the neuraminidase gene. These genetic characteristics strongly suggest that these viruses are descendants of the first isolate. In addition, our study also showed that the H9N2 viruses circulating in the operation during this 5 year period were evolving, as shown by antigenic variations between viruses manifested by reactivity with polyclonal antisera and monoclonal antibodies, by haemagglutination with erythrocytes from different animals, by amino acid differences in haemagglutinin and neuraminidase proteins, and by variation in their ability to replicate in the respiratory and intestinal tract and to be transmitted by aerosol. Phylogenetic analysis revealed that the internal genes from some H5N1 viruses of duck origin clustered together with those from H9N2 virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more recent reassortment events between these two subtypes of viruses. Continuous surveillance of influenza virus in poultry and waterfowl is critical for monitoring the genesis and emergence of potentially pandemic strains in this region.

The PA-Gene-Mediated Lethal Dissemination and Excessive Innate Immune Response Contribute to the High Virulence of H5N1 Avian Influenza Virus in Mice

ABSTRACT Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

PA-X Decreases the Pathogenicity of Highly Pathogenic H5N1 Influenza A Virus in Avian Species by Inhibiting Virus Replication and Host Response

ABSTRACT PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus. IMPORTANCE Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode as many as 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly identified protein in segment 3 that acts to decrease the virulence of the 1918 H1N1 virus in mice by modulating host gene expression. Our study extends these functions of PA-X to H5N1 HPAIV. We demonstrated that loss of PA-X expression increases the virulence and replication of an H5N1 virus in mice and avian species and alters the host innate immune and cell death responses. Our report is the first to delineate the role of the novel PA-X protein in the pathogenesis of H5N1 viruses in avian species and promotes our understanding of H5N1 HPAIV.

The PA and HA gene-mediated high viral load and intense innate immune response in the brain contribute to the high pathogenicity of H5N1 avian influenza virus in mallard ducks.

ABSTRACT Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks.

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.

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.

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.

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.