Zhiqiang Duan

Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China.

One H5N8 and three H5N5 highly pathogenic avian influenza (HPAI) viruses which derived their HA genes from the Asian H5N1 lineage were isolated from poultry during 2009-2010 in mainland China. Pathogenicity studies showed that these viruses were all highly virulent to chickens, while they varied from moderate to high virulence in mice and from mild to intermediate virulence in mallards. Phylogenetic analyses showed that these viruses were reassortants bearing the H5N1 backbone while acquiring PB1, NP and NA genes from unidentified non-H5N1 viruses, and had developed into three distinct genotypes (B-D). Molecular characterization indicated that all these viruses might resist to antiviral agents. Our findings highlight the emergence and development of HPAI H5 viruses of other NA subtypes in H5N1 endemic areas and their potential threat to poultry industry and public health.

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

Molecular evolution of the H6 subtype influenza a viruses from poultry in eastern China from 2002 to 2010

BackgroundAlthough extensive data demonstrates that the majority of H6 duck isolates belonged to a single H6N2 virus lineage with a single gene constellation in southern China from 2000 to 2005, the prevalence of H6N2 virus in poultry in Eastern China is largely unknown.ResultsEpidemiology revealed that H6N2 viruses were the most frequently detected influenza subtypes in live bird markets from 2002 to 2008 in Eastern China, but from 2009 onwards, they were replaced with novel H6N6 viruses. We phylogenetically and antigenically analyzed 42 H6 viruses isolated mainly in domestic ducks from 2002 to 2010 in Eastern China. Surprisingly, none of these isolates grouped with the previously described H6N2 viruses which belonged to a single H6N2 virus lineage with a single gene constellation in domestic ducks in southern China from 2000 to 2005. Two distinct hemagglutinin lineages were identified and they all underwent frequent reassortment with multiple virus subtypes from the natural gene pool, but few reassortants were persistent or prevalent.ConclusionsFive subtypes of H6 influenza viruses (H6N1, H6N2, H6N5, H6N6 and H6N8) cocirculated in Eastern China, which form a significant part of the natural influenza virus reservoir in domestic ducks, and significant viral reassortment is still ongoing in this species.

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.

Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

BackgroundThe goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial.ResultsIn this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33.ConclusionsThere is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.

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.

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.

Characterization of signal sequences determining the nuclear export of Newcastle disease virus matrix protein

The Newcastle disease virus (NDV) matrix (M) protein has been demonstrated to be a nuclear-cytoplasmic trafficking protein. Previous studies have shown that the M protein localizes in the nucleus through a bipartite nuclear localization signal. Here, we report that the ability of the M protein to shuttle to the cytoplasm is mediated by three nuclear export signal sequences (NESs). Using leptomycin B (LMB), a specific inhibitor of CRM1, we found that the nuclear export of the three NESs was LMB insensitive and thus was CRM1 independent. In addition, inactivation of these NESs led to nuclear accumulation of the M protein. Our results highlight the significance of these NESs to the nuclear export of the NDV M protein.

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.