Pathogenicity And Aerosol Infection Of H9N2 Subtype Avian Influenza Virus

Avian influenza?AI?is an infectious disease caused by influenza A virus that poses a serious threat to the health of poultry.Avian influenza virus?AIV?is a common cause of respiratory diseases in poultry.Low pathogenic avian influenza?LPAI?virus of H9N2subtype is the most frequently detected subtype in poultry.After infection,the disease usually does not occur,and the fatality rate is relatively low.But when it is mixed with H5,H7subtype influenza virus or other pathogens,it will cause a significant increase in poultry morbidity and mortality.In recent years,the gene structure of H9N2 subtype AIV has also changed.After adaptive evolution,some strains can adapt well to mammals,mice or guinea pigs.According to research,H9N2 subtype AIV can not only directly break the interspecies barrier to infect humans,but also provide part or even the entire internal gene cassette to produce new human influenza viruses,such as H5N1,H7N9,H10N8 and H5N6 viruses.Therefore,we must pay attention to the adaptive evolution of H9N2 subtype AIV in mammals,strengthen the research on the pathogenic mechanism and horizontal transmission ability of H9N2 subtype AIV,and formulate effective prevention measures for assessing influenza pandemic trends and the risk of harm to public health.Control measures have important guiding significance.Based on this,this study tested the newly isolated avian SD18 strain in 2018.In order to understand the pathogenicity of the SD18 strain,the nasal drip group and the lung delivery group were infected with BALB/C mice to clarify its effect on mammals.?Mice?In vivo replication,lung pathological damage,expression of lung-related immune factors and changes in serum antibody levels will be comprehensively evaluated for its pathogenicity.In order to understand the airborne transmission ability of the SD18 strain,this study conducted guinea pig pathogenicity experiments and aerosol generation,transmission and infection experiments to clarify its effects on mammalian?guinea pig?in vivo replication,serum antibody levels and isolator airborne AIV,To evaluate its horizontal communication ability.In addition,this study also conducted in vitro experiments to infect human bronchial epithelial cells with SD18 strain to evaluate the replication of SD18 strain on Beas-2B cells and the expression of related immune factors and the immune response of host cells.It is mainly divided into the following three parts:1.Study on H9N2 AIV infection of BALB/C miceIn order to evaluate the effects of the SD18 strain on the pathological damage of the lungs,the expression of lung-related immune factors and the level of serum antibodies after the infection of BALB/C mice through different routes of infection,the mouse challenge experiment was conducted in this study.The SD18 strain was inoculated into BALB/c mice by means of nose drops and lung delivery?quantitative aerosolization of lung liquid to form aerosol?,15 mice in each group,the inoculation dose was 10⁷EID₅₀/50?L,each 50?L.The results of throat swabs and anal swabs showed that the throat swabs and anal swabs of mice in the nose drop group and lung delivery mice had basically the same detoxification rules,and the detoxification was detected on the third day and reached a peak.The amount of throat swab detoxification of mice in the lung delivery group was higher than that of the nasal drip group.The amount of throat swabs in the two groups was higher than that of anal swabs at the same time.Compared with anal swabs,the difference is not significant.The results of lung histopathology showed that both nasal drops and lung delivery methods would cause different degrees of pathological damage to the lungs of mice,and the lung delivery group mice had more severe lung pathological damage than the nasal drops group mice.The detection results of Toll-like receptors?TLR3,TLR7?showed that the expression of the nose drop group and the lung delivery group was significantly up-regulated compared with the control group?P<0.01?;for the cytokines IL1-?,IL-6,IL-8,The test results of IFN-?,IFN-?,IFN-?,MX,and OAS showed that the expression of the nasal drip group and the lung delivery group compared with the control group was also significantly up-regulated?P<0.01?.The expression of related immune factors induced by the lung delivery group was higher than that of the nasal drip group,indicating that the infection of mice through lung delivery can cause a high level of immune response in the lung tissue of the mice,and exert a more obvious antiviral effect on the lungs of mice The damage is the most serious.Serum antibody levels in mice showed that both the nasal drip group and the lung delivery group could detect the presence of AIV antibodies on the 6th day,and the antibody level showed an upward trend.Among them,the lung delivery group produced higher antibody levels than the nasal drip group.Under the same conditions as other factors,the delivery of challenge through the lungs can induce the body to produce higher antibody levels.2.Study on SPF guinea pigs infected with H9N2 AIVIn order to evaluate the influence of SD18 strain on the pathogenicity and aerosol transmission ability of guinea pigs after being infected with guinea pigs,this study conducted pathogenicity experiments in guinea pigs.Inoculate 5 SPF guinea pigs with the SD18 strain at a dose of 10⁷EID₅₀/0.3 m L,0.3 m L each.The virus titer in the tissue showed that the virus can be detected in the lung tissue and trachea of guinea pigs,and the virus titer in the lung tissue of the guinea pig is higher than that in the trachea,indicating that the SD18 strain can infect guinea pigs and can multiply in guinea pigs Replication,and the replication and proliferation efficiency in guinea pig lung tissue is higher than that in trachea.In the SPF mouse isolator,aerosol generation,transmission and infection experiments were carried out.The guinea pigs were divided into 4 groups,the vaccination group,the direct contact group,the droplet transmission group and the aerosol passive infection group,with 7 in each group.The results showed that after the SD18 strain was infected with guinea pigs,AIV could be detected in the nasal washes of the inoculation group,the direct contact group and the droplet transmission group.AIV antibodies were also present in the serum,but the aerosol passive infection group was not detected in the nasal wash of the guinea pigs.When AIV was detected,no AIV antibody was detected in the serum,and the airborne AIV result in the isolator was negative.It shows that the SD18 strain can spread the virus and infect guinea pigs through direct contact and droplet transmission,but currently it does not have the ability to transmit the virus through aerosol to infect guinea pigs.3.Study on H9N2 AIV infection of Beas-2B cellsIn order to evaluate the intracellular replication of SD18 strain after infection of human bronchial epithelial cells and the expression of related immune factors.It is detected by indirect immunofluorescence?IFA?and fluorescent quantitative PCR.The results of indirect immunofluorescence showed that the virus could be detected in Beas-2B cells at 12 h after inoculation with the virus,and a small number of cells showed fluorescence in the field of view.At 24 h,the fluorescence in the field of view increased,and the fluorescence intensity reached the strongest.At 36 h The decrease indicates that the SD18 strain has the ability to infect Beas-2B cells and the virus infection ability reaches the strongest at 24 h.The detection results for Toll-like receptors?TLR3,TLR7?showed that compared with the control group,the expression of SD18 strain was significantly up-regulated after infection?P<0.01?.The detection results of inflammatory cytokines IL-6,IL-8,IFN-?and antiviral proteins MX,OAS showed that compared with the control group,the expression of SD18strain was also significantly up-regulated after infection?P<0.01?.The above results indicate that the SD18 strain can induce high-level expression of related immune factors in cells after infecting cells,and play an important role in resisting viral infections in cells.