Molecular Characteristics Of H9N2Influenza Viruses Isolated From Zhejiang And The Immune Mechanisms Of Injury Mediated By Toll-like Receptors

Since the first human case of H9N2influenza virus infection reported in Hong Kong in1998, infected patients were also reported in mainland of China. H9N2viruses have a high infection rate (about15%) in people occupied in poultry fields, and they can cause mild to moderate disease. Previous studies have shown that H9N2virus has continuously circulated in domestic poultry in China, and undergone extensive reassortment, which indicated that the virus may be an important pathogen of human respire system diseases and a candidate human pandemic strain.For analyzing the molecular characteristics, several strains of H9N2influenza virus were isolated from poultry in Zhejiang Province in2011and whole genome sequencing and phylogenetic analyses were performed. By collecting the sequence information from the database of H9N2influenza virus in GenBank, we constructed a H9N2virus proteins date bank which was highly practical and efficient for analyzing H9N2virus biology characteristics. For exploring the mechanism of the H9N2virus infection in host cell, we studied the Toll-like receptor (TLR) induced immune responses by detecting levels of TLR4, TLR7, NF-κB and IL-6in mice and human cell lines.One hundred cloacal swabs were collected from apparently healthy poultry in live poultry markets in Zhejiang Province, January to June, in2011. Whole genome sequencing and phylogenetic analyses of the isolated three H9N2and nine H1subtypes influenza virus strains were performed. The result showed that these viruses clustered in the Eurasian lineage of influenza viruses. These H9N2viruses had the molecular characteristics associated with amantadine resistance. The results of phylogenetic analysis indicate that a reassortment event has occurred between H9N2and H1N2viruses, and the continued surveillance of influenza viruses in poultry is necessary. To our knowledge, this is the first report on the genomic sequence and phylogenetic analysis of H1N3influenza viruses isolated from China, and it will provide important information for prevention of H1N3epidemic.By collecting the sequence information from the database of H9N2influenza virus in GenBank and using Microsoft offices and BioEdit software, we constructed a H9N2viral proteins date bank including11viral proteins (PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1and NS2proteins) and8948piece sequences. The result showed that the poultry is the natural hosts (97.47%) and mainly come from Asia (96.37%) from1997to2012(96.32%). About78.10%of the H9N2viruses have hunman-like receptor and9.3%of them have S31N mutation. This date bank is a practical and efficient tool for analyzing H9N2viral biology characteristics, such as infection ability and drug resistance, with high effective.An appropriate animal model of acute respiratory distress syndrome (ARDS) infected by H9N2virus was made in mice, and the levels of IL-6, SOD, TLR4, TLR7and NF-κB in peripheral blood and lung were detected. The result showed that the H9N2virus (A/chicken/Zhejiang/329/2011, Zj329) isolated from Zhejiang had high virulence and pathogenicity in mice, characterized by a relatively acute clinical process in infected mice. The expression levels of TLR4, TLR7and NF-κB mRNA, and the content of IL-6were increased greatly in H9N2virus infected mice, and reached the highest level at day6after infection. The content of SOD were decreased greatly in H9N2virus infected mice from day2to day6, and reached the lowest level at day4after infection.The levels of IL-6, SOD, TLR4, TLR7and NF-κB were detected in human A549cell after infection by H9N2virus (Zj329). The results showed that the H9N2virus infected A549cell presented a typical cytopathic effect (CPE). The expression levels of TLR4and TLR7mRNA, and the contents of NF-κB and IL-6were increased greatly in H9N2virus infected human A549cell, and reached the highest level at24hours after infection.In summary, our data showed that,(i) the H9N2viruses prevailing in Zhejiang poultry were clustered in the Eurasian lineage of influenza viruses.(ii)Furthermore, a reassortment event was occurred between H9N2and H1N2viruses.(iii) H9N2viral infection induced typical ARDS in mice and typical CPE in human A549cells, and increased the levels of TLR4, TLR7, IL-6, and NF-κB. The H9N2viral proteins date bank can be used to analyze H9N2virus biology characteristics, such as infection and drug resistance, with high effectivity. Our results might give useful information to further studies of the pathogenesis of future potential avian H9N2disease in humans.