Xiaorong Zhang

A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has been circulating in poultry in eastern China since 1998.

ABSTRACT Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.

Characterization of duck H5N1 influenza viruses with differing pathogenicity in mallard (Anas platyrhynchos) ducks

A number of H5N1 influenza outbreaks have occurred in aquatic birds in Asia. As aquatic birds are the natural reservoir of influenza A viruses and do not usually show clinical disease upon infection, the repeated H5N1 outbreaks have highlighted the importance of continuous surveillance on H5N1 viruses in aquatic birds. In the present study we characterized the biological properties of four H5N1 avian influenza viruses, which had been isolated from ducks, in different animal models. In specific pathogen free (SPF) chickens, all four isolates were highly pathogenic. In SPF mice, the S and Y isolates were moderately pathogenic. However, in mallard ducks, two isolates had low pathogenicity, while the other two were highly pathogenic and caused lethal infection. A representative isolate with high pathogenicity in ducks caused systemic infection and replicated effectively in all 10 organs tested in challenged ducks, whereas a representative isolate with low pathogenicity in ducks was only detected in some organs in a few challenged ducks. Comparison of complete genomic sequences from the four isolates showed that the same amino acid residues that have been reported to be associated with virulence and host adaption/restriction of influenza viruses were present in the PB2, HA, NA, M and NS genes, while the amino acid residues at the HA cleavage site were diverse. From these results it appeared that the virulence of H5N1 avian influenza viruses was increased for ducks and that amino acid substitutions at the HA cleavage site might have contributed to the differing pathogenicity of these isolates in mallards. A procedure for the intravenous pathogenicity index test in a mallard model for assessing the virulence of H5/H7 subtype avian influenza viruses in waterfowl is described.

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.

Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

OL-004 Sequence analysis of hepatitis C virus 5′ non-coding region

needed to increase the cure rate. In vitro experiments show strong antiviral effects of fluvastatin against HCV. Aim of the study: To asses the clinical outcome of fluvastatin addition to the standard regimen for treatment of chronic HCV in Egypt. Subjects and Methods: The study included 80 patients with chronic hepatitis C virus infection fulfilled clinical, laboratory and histo-pathological criteria to be ready for interferon therapy, divided into two groups: Group I (n = 40) received standard treatment for HCV (pegylated interferon and ribavirin) and group II (n = 40) received standard treatment plus fluvastatin (80 mg/daily). Before and after 6 months of treatment liver function tests and HCV-RNA were evaluated. Results: Addition of fluvastatin to the standard HCV treatment (pegylated interferon and ribavirin) significantly increased SVR from (55% to 62.5%; P< 0.01) and significantly decreased viral load in relapser patients (P < 0.001). No significant differences and correlations were found between serum levels of LDL-cholesterol and viral load before and after treatment in both groups. Conclusion: Fluvastatin can be used to increase SVR when added to standard treatment (pegylated interferon and ribavirin) of chronic HCV.

OL-044 Molecular evolution of H9N2 influenza A viruses in Eastern China (1996 to 2008): implications for the origin of highly pathogenic H5N1 viruses

cine to prepare anti-H5N1 IgGs. The F(ab’)2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for ’enhanced pepsin digestion’. The protective effect of the F(ab’)2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE) assay and in a BALB/c mouse model by survival rate assay. Results: By the protocol for “enhanced pepsin digestion”, total 16 g F(ab’)2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab’)2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab’)2 reached 1:2048. The in vivo assay showed that 100 μg of the F(ab’)2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion: The availability of highly purified H5N1-specific F(ab’)2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.