Avian Influenza Virus H9N2Adapted To Mice Induce Lung CD4~+T Cells Th17Response

Avian influenza viruses (AIV) are segmented, negative-strand RNA virus belonging to the Orthomyxoviridae family, which have the capacity to infect not only birds, but also various mammals such as mink, pigs and horses under natural circumstances. Avian-to-human transmission of AIV posed the concern about their pandemic potential to human.Host immune response against AIV and the molecular mechanisms by which AIV transmit across species barrier to humans remained not fully understood. Based on this, we focus on AIV H9N2, establishment of mouse model of AIV H9N2, and investigated the molecular mechanism and host immune response of enhanced virulence of AIV H9N2to mice.1. Multiple amino acid substitutions involved in enhanced pathogenicity of H9N2Human infection of avian influenza H9N2virus highlighted the need to better understand the mechanism of interspecies transmission. In this study, we generated mouse-adapted influenza virus (ma01) through serial lung-to-lung passages of a wild-type H9N2(A/chicken/Hubei/01/1999). Ma01caused highly lethal infection in mice with severe lung pathology and extended tissue tropism. Nine amino acid substitutions of ma01were observed in five viral genes (those for PB2, PA, NA, M1, and NS1). Of these mutations, substitutes of PB2627, PA349, PA605, NA88, and NA356were absent in influenza H9N2. Furthermore, the targets of wild-type virus responding to mouse micro RNA mmu-mir-1940and mmu-mir-1904were eliminated in ma01. The mutation PB2627of ma01confirmed as a key virulence determinant of influenza H5N1was responsible for the altered recognition of mmu-mir-1904. Real-time PCR analysis of cytokines revealed increased expression of IL-lb, IL-6, TNF-a, and MIP-la in ma01infected mouse lung than parental strain. In addition, induction of IL-1b, IL-6, TNF-a, and IFN-b was found in significantly higher levels in ma01infected mouse peripheral blood than parental strain. These results demonstrate that multiple amino acid substitutions and avoidance of microRNA recognitions may be essential for lethal infection and high speed of virus growth can outcompete the antiviral response of infected host.2. Influenza neuraminidase contribute to CD4+T cells Th17response in mouse lungAvian influenza virus infection induce cytokine dysregulation contribute to disease severity. In this study, based on mouse model of AIV H9N2, we revealed H9N2infection induce high levels of TGF-β1and IL-17A in lung, subsequent flow cytometry analysis of CD4+T Thl7cells in mouse lung, revealed AIV infection expanded CD4+/IL-17A cells up to16.3%compared with mock7.87%. Furthermore, influenza neuraminidase contribute to CD4+T cell Thl7response through induce dendritic cells maturation upregulate surface marker CD40,CD80,CD83and MHCII, release cytokines TGF-β1, TNF-a and MIP-1α. Neuraminidase enhance LPS, Poly(I:C) and HMGB1induced dendritic cell maturation. Neuraminidase cannot directly induce CD4+T cell Th17response, this effect need dendritic cells.