Sujuan Chen

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.

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.

Hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the H5N1 avian influenza virus

The location and number of glycosylation in HA proteins exhibit large variations among H5 subtype avian influenza viruses (AIVs). To investigate the effect of glycosylation in the globular head of HA on the pathogenicity and antigenicity of H5N1 AIVs, seven rescued AIVs differing in their glycosylation patterns (144N, 158N and 169N) within the HA globular head of A/Mallard/Huadong/S/2005 were generated using site directed mutagenesis. Results showed that loss of glycosylation 158N was the prerequisite for H5 AIV binding to the α2,6-linked receptor. Only in conjunction with the removal of the 158N glycosylation, the H5 AIVs harboring both 144N and 169N glycosylations obtained an optimal binding preference to the α2,6-linked receptor. Compared with the wild-type virus, growth of viruses lacking glycosylation at either 158N or 169N was significantly reduced both in MDCK and A549 cells, while replication of viruses with additional glycosylation 144N was significantly promoted. Mutant viruses with loss of 158N or 169N glycosylation sites showed increased pathogenicity, systemic spread and pulmonary inflammation in mice compared to the wild-type H5N1 virus. In addition, chicken studies demonstrated that inactivated de-glycosylation 169N mutant induced cross-reaction HI and neutralization antibody against various clades of H5N1 AIVs. Moreover, this type of glycan pattern vaccine virus provided better cross-protection in chickens compared to wild-type vaccine virus. Thus, the glycosylation alteration of HA should be considered in the global surveillance and vaccine design of H5 subtype AIVs.

A 20-Amino-Acid Deletion in the Neuraminidase Stalk and a Five-Amino-Acid Deletion in the NS1 Protein Both Contribute to the Pathogenicity of H5N1 Avian Influenza Viruses in Mallard Ducks

Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A−) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S−) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A−S− and three variants (A−S+ with a five-amino-acid insertion in the NS1 protein, A+S− with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A− and/or S− replicated in DEF cells were higher than that of A+S+, and the A−S− virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A−S− induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S−, the A−S+ and A−S− viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.

The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1 highly pathogenic avian influenza virus and chicken interleukin 6 gene in ducks

Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.

Comparison of biological characteristics of H9N2 avian influenza viruses isolated from different hosts

The pathogenicity and transmissibility of H9N2 influenza viruses has been widely investigated; however, few studies comparing the biological characteristics of H9N2 viruses isolated from different hosts have been performed. In this study, eight H9N2 viruses, isolated from chickens (Ck/F98, Ck/AH and Ck/TX), pigeons (Pg/XZ), quail/(Ql/A39), ducks (Dk/Y33) and swine (Sw/YZ and Sw/TZ) were selected, and their biological characteristics were determined. The results showed that all H9N2 viruses maintained a preference for both the avian- and human-type receptors, except for Sw/TZ, which had exclusive preference for the human-type receptor. The viruses replicated well in DF-1 and MDCK cells, whereas only three isolates, Ck/F98, Ck/TX and Sw/TZ, could replicate in A549 cells and also replicated in mouse lungs, resulting in body weight loss in mice. All H9N2 viruses were nonpathogenic to chickens and were detected in the trachea and lung tissues. The viruses were shed primarily by the oropharynx and were transmitted efficiently to naïve contact chickens. Our findings suggest that all H9N2 viruses from different hosts exhibit efficient replication and contact-transmission among chickens, and chickens serve as a good reservoir for the persistence and interspecies transmission of H9N2 influenza viruses.

Characterisation and haemagglutinin gene epitope mapping of a variant strain of H5N1 subtype avian influenza virus.

H5N1 avian influenza virus (AIV) undergoes rapid evolution, and its antigenicity needs to be constantly evaluated in order to update the vaccine strain. In this report, a clade 7.2 AIV isolate named A/Chicken/Huadong/4/2008 (DT) is identified. Antigenic analysis revealed that DT had a significant low cross-reactive HI titre with antiserum against a clade 7 representative AIV, A/Chicken/Shanxi/2/2006 (Re-4). Animal experiments demonstrated that the Re-4+Re-5 vaccine provided 80% protection against DT challenge in chickens. Antisera cross-reactivity showed that a mutant with a change of residues 129, 139, 140 in site A in the HA protein had reduced reactivity with DT antiserum and increased reactivity with Re-4 antiserum. Furthermore, residue Leu129 in site A of the HA protein was confirmed to be critical for maintenance of the reactivity with the DT antiserum, and Asn140, possessing a new glycosylation site, was confirmed to be critical for reducing reactivity with the Re-4 antiserum. These results imply that there is an antigenic drift within clade 7 viruses, and insertion and glycosylation of amino acid residues in site A of the HA protein may contribute to the antigenic variation.

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

There are three conserved N-linked glycosites, namely, Asn10, Asn23 and Asn286, in the stem region of haemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF cells in comparison with that of the WT virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role in HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

Cross-clade protective immune responses of NS1-truncated live attenuated H5N1 avian influenza vaccines

BACKGROUND H5N1 highly pathogenic avian influenza (HPAI) has raised global concern for causing huge economic losses in poultry industry, and an effective vaccine against HPAI is highly desirable. Live attenuated influenza vaccine with trunctated NS1 protein as a potential strategy will be extremely useful for improving immune efficacy. METHODS A series of H5N1 avian influenza virus reassortants harboring amino-terminal 48, 70, 73, and 99 aa in NS1 proteins, along with a modified low pathogenic HA protein was generated, and named as S-HALo/NS48, S-HALo/NS70, S-HALo/NS73, and S-HALo/NS99, respectively. In addition, their biological and immunological characteristics were further analyzed. RESULTS The viruses S-HALo/NS70, S-HALo/NS73, and S-HALo/NS99, but not S-HALo/NS48, had a comparable growth property with the full-length NS1 virus, S-HALo/NSFu. Mice and chickens studies demonstrated that the viruses with truncated NS1 protein were further attenuated when compared to the virus S-HALo/NSFu. Vaccination with the virus S-HALo/NS73 in chickens induced significant cross-protection against homologous clade 2.3.4 H5 virus and heterologous clade 7.2, 2.3.2.1, and 2.3.4.4 H5 viruses. CONCLUSION A 70-aa amino-terminal fragment of NS1 protein may be long enough for viral replication. The recombinant virus S-HALo/NS73 is a broad-spectrum live attenuated H5N1 avian influenza vaccine candidate in chickens.

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.