Investigation Of Synergetic Pathogenicity Of Co-infection With Infectious Bronchitis Virus And H9N2 Avian Influenza Virus

Infectious Bronchitis（IB）caused by Infectious Bronchitis Virus（IBV）,is an acute,highly contagious respiratory disease in chickens.Avian H9N2 influenza virus（H9N2 AIV）is a widely prevalent and low pathogenic pathogen in poultry.Both pathogens are widely prevalent in chicken flocks and can be transmitted through respiratory tract,which provide possibility for the co-infection of the two pathogens in chicken.Although co-infection of IBV and H9N2 AIV in chicken has been reported,the interaction between IBV and H9N2AIV during infection and virus synergistic pathogenesis have not been clearly explained in the existing research.Thus,this study aims to explore the relationship between co-infection of IBV and H9N2 AIV orderly and synergistic pathogenicity,analyzing the mechanism of synergistic pathogenesis by co-infection of SPF chickens and SPF chicken embryos with two viruses simultaneously and orderly.The main findings of this research are as follows:Firstly,two pairs of primers were designed based on IBV and H9N2 AIV sequences published in Gen Bank.The N gene of IBV and the M gene of H9N2 AIV were amplified by RT-PCR with reference to the primers in literature.The amplified products were cloned into PMD-19T vector to obtain positive standard plasmids,and the IBV-N and H9N2-M standard curves were successfully established by q PCR.Secondly,105 SPF chickens were randomly divided into 6 groups,IBV-H9N2 AIV ordinal co-infection group（I-H）,H9N2 AIV-IBV ordinal co-infection group（H-I）,IBV+H9N2 AIV simultaneous infection group（I+H）,IBV infection group,H9N2 AIV-infection group and the control group;The clinical symptoms and pathological changes of the animals were observed.Virus shedding and the viral load of IBV and H9N2 AIV in the trachea,lung,kidney,thymus,spleen and bursa of Fabricius were detected by q PCR;The titer of the HI antibody and the index of the animal’s immune organs were analyzed after 14days.These findings suggested that the pathogenicity of I-H ordinal co-infection group is more severe than that of other testing groups.Morbidity and mortality of this group is 70%and 20%respectively.In the I-H ordinal co-infection group and I+H simultaneous infection group,The pathological changes were more serious and the level of virus shedding and the virus load in the organs were higher than the other three groups.The immune organ indexes（thymus and bursa）of the I-H group and the I+H group were significantly lower than those of the IBV or H9N2 AIV groups after 14 days of challenge.The titer of HI antibody against H9N2 in serum of I-H group and I+H group was significantly lower than that of H-I group14 days after challenge.The results showed that the I-H ordinal co-infection group displayed the most severe disease.Thirdly,the inflammatory cytokines was detected by the q PCR,the inflammatory protein IL1βand NLRP3 were tested by ELISA and WB,respectively.The results showed that the m RNA expression levels of MDA5 and TLR7 in I-H ordinal co-infection group and the inflammatory factors m RNA expression levels of IFNβ,IFNα,IRF7,IL-1β,TNFα,IL-6,IL-8,IL-18,NLRP3 and Caspase1 in I-H group and I+H group were significantly higher than those in the single infected group and H-I group.The expression of IL1βand NLRP3 protein in tissues of I-H group and I+H group were significantly higher than those in the infected group,and H-I group.These results showed that the expression of inflammatory factors in the I-H group is higher.Lastly,SPF chicken embryos were co-infected with IBV and H9N2 AIV with 10¹EID₅₀,10³EID₅₀ and 10⁵EID₅₀ at different doses and in different infection sequences（orderly and simultaneously）.The results showed that the IBV titer was significantly higher than that of IBV alone.when IBV was inoculated into chicken embryos before infected with H9N2 AIV after 12 h or 24 h.In summary,this study adopted two viruses to infect SPF chicken and SPF chicken embryos simultaneously and orderly.The results showed that mortality,morbidity,the level of virus shedding and the virus load in the organs were significantly increased in IBV-H9N2AIV ordinal co-infection group.IBV first infection creates conditions for H9N2 infection,and subsequent infection with H9N2 AIV promotes IBV replication,which caused more intense inflammatory factor expression and more severe tissue damage,and lead to more serious disease.This research lays a foundation for further studies of the host immune response to virus and the interaction between virus and virus under the condition of co-infection.Furthermore it provides a scientific basis for disease prevention and control for IBV and H9N2 AIV co-infections.