Shunlin Hu

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.

A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses.

To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.

Surveillance for avirulent Newcastle disease viruses in domestic ducks (Anas platyrhynchos and Cairina moschata) at live bird markets in Eastern China and characterization of the viruses isolated.

We isolated and identified 201 Newcastle disease viruses (NDVs) from domestic ducks in a 5-year surveillance study at live bird markets in Eastern China. Seventy-three of these isolates were characterized biologically and genetically. Fusion protein (F) genes of these isolates were amplified by reverse transcription-polymerase chain reaction and sequenced. Intracerebral pathogenicity index tests in 1-day-old specific-pathogen-free chickens and the mean death time of embryonated fowl eggs in addition to the cleavage site analysis of the F-protein precursor for these viruses showed that they were all avirulent NDVs. Phylogenetic analysis based on partial sequences of the F gene showed that 30 isolates clustered into the class I clade and the other 43 isolates clustered into genotype I of class II, but diverged from the vaccine virus Queensland V4, which is extensively used in China. Most class I viruses (18/30) formed a separate branch closest to the Hong Kong live bird market strains that have been recently designated as genotype 3, while the rest (12/30) were closely related to some European viruses within genotype 2. All of the 43 class II genotype I viruses diverged from viruses originally assigned to genotype Ia and formed a separate sublineage designated as Ib with water bird isolates from the Far East, suggesting the possible transmission between the wild and domestic waterfowl. The results in the present study clearly showed that the domestic duck population carries avirulent NDVs with genetic divergence regularly and may act as one of the important reservoirs.

Generation of a velogenic Newcastle disease virus from cDNA and expression of the green fluorescent protein

SummaryNewcastle disease virus (NDV) is a pathogen that is important in the poultry industry worldwide. Specifically, the virulent (velogenic) NDV is aparticular threat because it has now occurred frequently worldwide. The outbreaks caused by highly virulent NDV in waterfowl and especially in goose flocks, have led to greater concern in recent years as aquatic birds were previously resistant to most virulent NDV strains from chickens. The molecular determinants of host tropism, virulence and emergence of NDV isolated from diseased goose flocks are poorly understood. In the present study, we rescued a highly virulent NDV isolated from a goose using the reverse genetics approach. Infectious virus was successfully generated by cotransfection of a full-length cDNA clone of the NDV strain ZJ1 with helper plasmids. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth kinetics in cell culture as well as its biological properties. A recombinant NDV expressing green fluorescent protein (GFP) was generated, and GFP was subsequently detected in cells and various organs from the infected chickens.

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.

Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005–2008

Seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons (Columba livia), geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. The results showed genetic diversity of these viruses: three class I viruses and one genotype I and 12 genotype II viruses of class II circulating in chickens were avirulent; four genotype VIb viruses isolated from pigeons were moderately virulent; and two genotype III viruses and 57 genotype VIId viruses were highly virulent. The three class I viruses were further classified as genotypes 2 and 3. The very high F protein sequence identity of one genotype I virus with strain Queensland V4 and 12 genotype II viruses with strain La Sota indicated that these viruses originated from the two vaccine strains. Two genotype III viruses shared greater than 99% sequence identity with the moderately virulent vaccine strain Mukteswar but exhibited significantly higher virulence, suggesting that they evolved from the vaccine virus and that the Mukteswar vaccine should be banned in China. Fifty-seven of the 63 virulent NDVs in this study belonged to genotype VIId, indicating its predominance in Eastern China. Genotype VIId viruses could be further classified into two subgroups. Four of the five NDVs isolated from pigeons belonged to genotype VIb, indicating its host-specific preference. Both the genotype VIb and VIId NDVs showed low amino acid similarity to the vaccine strains currently used in China, implying the urgent need to develop better vaccines against the most prevalent NDVs in China.

Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs

SUMMARY. To generate a genotype VII Newcastle disease virus (NDV) vaccine with high yield in embryonated chicken eggs, we selected genotype VII NDV strain JS5/05, which possesses a high virus titer in embryos as the parental virus. Using reverse genetics, we generated a genetically tagged derivative (NDV/AI4) of JS5/05 by changing the amino acid sequence of the cleavage site of the F0 protein. Pathogenicity tests showed that NDV/AI4 was completely avirulent. NDV/AI4 was genetically stable and replicated efficiently during 10 consecutive passages in embryos. More importantly, serologic assays showed that oil-emulsion NDV/AI4 induced higher hemagglutination inhibition (HI) titers against the prevalent virus than oil-emulsion LaSota vaccine in chickens and geese. Moreover, NDV/AI4-induced HI titers rose faster than those elicited by LaSota in chickens. Both NDV/AI4 and LaSota provided protection against clinical disease and mortality after the challenge with the genotype VII NDV strain JS3/05. However, NDV/AI4 significantly reduced virus shedding from the vaccinated birds compared to LaSota. Taken together, these results suggest that NDV/AI4 can provide better protection than LaSota and is a promising vaccine candidate against genotype VII NDV.

Identification of a variable epitope on the Newcastle disease virus hemagglutinin-neuraminidase protein.

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin-neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345-353) on the HN protein.

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.

Apoptin Enhances the Oncolytic Properties of Newcastle Disease Virus

Objective: Naturally occurring strains of Newcastle disease virus (NDV) have demonstrated the potential to kill cancer cells in both preclinical and clinical studies. Previous studies showed that apoptin, the VP3 protein of chicken infectious anemia virus, is a p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in transformed cells. In this study, we tested the hypothesis that apoptin enhances NDV-mediated tumor cell death. Methods: Reverse genetics was used to engineer an oncolytic NDV strain, FMW, to express apoptin. The antitumor effects of the recombinant virus (rFMW/AP) were also evaluated in the tumor cell lines and tumor-bearing mice. Results: Compared to the parental strain FMW, rFMW/AP was more potent in killing A459 and SMMC7721 tumor cells. Recombinant NDV also exhibited higher efficacy in suppressing tumor growth in mice bearing A549-induced tumors. Furthermore, rFMW/AP did not display apparent toxic effects in either normal cells or control mice. Conclusion: Our results suggest that the recombinant NDV expressing apoptin is a promising novel antitumor agent.