ABSTRACT
H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-ß, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-ß, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.
Subject(s)
Immunity, Innate , Influenza A Virus, H5N1 Subtype/physiology , Influenza A Virus, H9N2 Subtype/physiology , Influenza in Birds/genetics , Poultry Diseases/genetics , Animals , Cell Line, Transformed , Chickens , China , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza A Virus, H9N2 Subtype/genetics , Influenza A Virus, H9N2 Subtype/pathogenicity , Influenza in Birds/immunology , Influenza in Birds/virology , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Interferons/genetics , Interferons/immunology , Phylogeny , Poultry Diseases/immunology , Poultry Diseases/virologyABSTRACT
An avian influenza virus (AIV) strain belonging to the H4 subtype and provisionally designated as A/duck/China/J1/2012(H4N6) was isolated from diseased ducks with respiratory disease at a commercial poultry farm in Shandong, China, in 2012. The genomic coding sequences of all eight segments of this J1 isolate were determined and used for subsequent analysis. Phylogenetic analysis of all eight segments showed that this duck H4N6 virus was of Eurasian lineage and not American lineage. The results show that the virus probably emerged because of a reassortment event involving other avian H4N6 and H6N1 viruses. Interestingly, this H4N6 virus had all the conserved features common to low-pathogenic AIVs, including the HA cleavage sequence, receptor-binding sequences for the 2,3-linked sialic acid receptor in avian species, and the PB2 627E motif. These results suggest that the duck H4N6 isolate could not cross the species barrier to infect and replicate in mammals, including humans. In addition, screening of the duck serum samples showed that only 0.57 % (2/352) of the individuals had weak but measurable hemagglutination inhibition (HI) antibody titers. The low antibody prevalence data were also supported by the failure to detect H4N6 virus (0/56) in clinical nasal swabs of the ducks. These data indicate an alternate reservoir for the H4N6 virus.
Subject(s)
Influenza A virus/isolation & purification , Influenza in Birds/virology , Animals , China/epidemiology , Ducks , Genome, Viral , Influenza A virus/classification , Influenza A virus/genetics , Influenza in Birds/epidemiology , PhylogenyABSTRACT
Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early, late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid, a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10(5) PFU and 2 x 10(5) PFU respectively. After 28 days,antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.
