Guo Zhao

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.

Novel Reassortant Highly Pathogenic Avian Influenza (H5N5) Viruses in Domestic Ducks, China

In China, domestic ducks and wild birds often share the same water, in which influenza viruses replicate preferentially. Isolation of 2 novel reassortant highly pathogenic avian influenza (H5N5) viruses from apparently healthy domestic ducks highlights the role of these ducks as reassortment vessels. Such new subtypes of influenza viruses may pose a pandemic threat.

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.

Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk.

BACKGROUND The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus. OBJECTIVE Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased. METHODS We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks. RESULTS In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA. CONCLUSION These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.

Isolation and phylogenetic analysis of pandemic H1N1/09 influenza virus from swine in Jiangsu province of China

To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.

Characterisation of one H6N6 influenza virus isolated from swine in China.

There is multiple evidence that swine may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of one strain of H6N6 influenza virus isolated from swine with clinical signs of infection in China. Although phylogenetic analysis revealed that this virus might originate from domestic ducks, pathogenicity experiments indicated that the virus replicated in mice without prior adaptation. These findings suggest that the virus has transmitted from ducks to swine in China, and replicated in mammalian hosts without prior adaptation, which may pose a threat to both veterinary and public health.

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.

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

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.