Pathogenicity Of A Duck-origin H9N2Avian Influenza Virus In Cherry Valley Ducks

Avian influenza viruses (AIV) belong to the Orthomyxoviridae, are of the Type A genus,which show high pathogeniticity to poultry. Since the first isolation from terrestrial birds(chiken) in1994, H9N2became prevalent in China and occasionally, they infected a widerange of species. Although H9N2are indentified as low pathogenic avian influenza viruses(LPAIVs) based on their virulence in domestic galliformes and the amino acid sequence at thehemagglutinin cleavage site, they have caused huge economic loss to poultry industry inChina. Of special interest to medicine and agriculture, ducks are considered the principalnatural reservoir for AIV and play a critical role in the transmission and evolution of AIV.Currently, H9N2subtype AIVs have been isolated from numberous ducks flocks from manyfarms, moreover, the current knowledge of H9N2infection of ducks is still very limited.Therefore, study on the pathogenicity of H9N2gains high priority, which will be of greatimmortance to virology, veterinary research and public health.To investigate the outcome of H9N2subtype AIV infection in Cherry Valley ducks,ducks at the age of two weeks of age were intravenously and intranasally, respectively,inoculated with a duck-origin avian H9N2influenza virus. After infection, clinical signs,dynamics of viral shedding and histological lesions were recorded. In addition, flowcytometric analysis of CD4+and CD8+T-lymphocytes in peripheral blood mononuclear cells,quantitative real-time RT-PCR analysis of immune-related genes and serology test were alsoperformed.After infection, the primary clinical signs presented by ducks were mild depression,respiratory symptoms,decreased feed and water intake and significant decrease in bodyweight gain in IV group, however, no apparent clincal signs were observed in IN group. Inaddition, based on the results of virus shedding presented in OP and CL swabs, ducks in IVgroup shed significant more virus (104.2EID50/mL)presented by OP swabs at2dpi in relativeto ducks in IN group (102.4EID50/mL). OP viral shedding in IN group showed its peak at4dpi up to103.0EID50/mL. Moreover, OP swabs collected from IV group showed a peak at2dpi (104.2EID50/mL) and on4dpi, CL swabs from IN group displayed significant highertiters than those from IV group. In addition, there appeared greater positive recovery rates andlonger persistence of viral shedding from CL swabs.The microscopic lesions were mainly characterized by mild to moderate lymphocyticinfiltration after infection with H9N2. Duck in IV group displayed lesions present in thetracheas consisting of mild to moderate lymphocytic infiltration and mild edema in the submucosa and loss of cilia. Lesions in the lung were congestion and mild to moderatelymphocytic infiltration mixed with red blood cell. In pancreas and liver, lesions were mainlycell degeneration and lymphocytic infiltration. As for ducks in IN group, the microscopiclesions were mainly confined to the respiratory tracts consisting of lymphocytic infiltrationand congestion. In addition, immunohistochemical staining showed postive staining wereobserved in all examined tissues from ducks in IV group and in IN group postive stainingwere confined to the respiratory system.Flow cytometric analysis revealled the proportion of CD4+T cells and CD8+T cells inthe peripheral blood in the uninfected group were20.68±2.8%and30.35±4.1%, respectively.The infection seemed to result in increase the proprotions of CD4+T cells although statisticaldifferences were recorded. On the other hand, CD8+T cells in the peripheral blood of bothgroups were significantly elevated on day4and7post-infection in comparison with the ducksin the uninfected control group.On1dpi, upregulation of IFNα, IFNβ, IFNγ and IL2mRNA in both groups wereobserved in the spleen of infected ducks, moreover, IFNβ, IFNγ, IL1B and IL6showed higherexpression levels in intravenously infected ducks determined by quantitative real-timeRT-PCR. On4dpi, increased expression levels of IFNγ, IL1B and IL2were recorded in bothgroups while upregulation of IL1B and IL6mRNA were only recorded in intravenouslyinfected ducks. On7dpi, IFNγ and IL2mRNA were both upregulated in the spleen of bothgroups, while expression levels of IFNγ mRNA were elevated in both groups on10dpi. Inaddition, expression levels of IFNγ mRNA in the trachea were higher than those in ilea fromducks infected by intranasal route, which may provide an explanation for higher positiverecovery rate in CL swabs and longer viral shedding observed in ducks.Serology test showed a weak HI antibody response and shorter persistence of antibodieswas observed in intravenously and intranasally infected ducks, which indicated ducks couldbe reinfected with H9N2within a shorter interval. Therefore, the authors of our study suggestthat the vaccination procedures should be carefully planned for ducks in order to efficientlycontrol transmisson and outbreaks of AIV.