Genetic Evolution Analysis Of H5N8 And H4N6 Subtype AIV Of Wild Bird Origin And Innate Immune Response Mediated By RIG-I In Ducks After Infection

In recent years,various emerging avian influenza viruses have been reported to gradually show trends such as wide spread,expanded host range,and cross-species spread,which has attracted widespread attention.As a natural host,wild ducks play an important role in the epidemiological cycle of avian influenza viruses.In particular,wild ducks can carry highly pathogenic avian influenza asymptomatic for long-distance transmission across regions.Although the research on the carrying and transmission of highly pathogenic and low pathogenic avian influenza by wild ducks has been gradually carried out,the research on the innate immune response of ducks infected with influenza virus is very limited.In recent years,the newly circulating H5N8 subtype highly pathogenic avian influenza virus has caused the death of wild swans,and has even threatened human health.However,as a common low pathogenic avian influenza virus,H4N6 can often be detected in wild bird disease surveillance.In order to study the differences of innate immune responses in ducks infected with avian influenza viruses with different virulence,we analyzed the genetic evolution trend of two wild bird-derived H5N8 and H4N6 avian influenza viruses with different virulence,and use them for infection experiments in ducks.Then Quantitative Real-time PCR was used to detect the RNA loads of the two viruses,as well as retinoic acid-inducible gene I(RIG-I),nuclear factor kappa B(NF-κB)and interferon beta(IFN-β)in the host innate immune response and analyze its dynamic change rule.The results show that(1)the H5N8 subtype avian influenza virus has dominated the2.3.4.4 branch,and the mutation and active evolution may form a new evolutionary branch.The evolution of H4N6 is relatively conservative.(2)The mortality of ducks after Ln/d59 virus strain infection was 0.The mortality rate of ducks infected with Hn/h4 virus strain was 6.67%,and there were no obvious clinical symptoms.(3)Quantitative Real-time PCR detection showed that the expressions of RIG-I,NF-κB and IFN-β all fluctuated.The innate immune response started rapidly within 6 hours after infection,and was subsequently suppressed by the virus to varying degrees,so that the expressions of RIG-I,NF-κB and IFN-β decreased rapidly within 1-2 days after infection.This suggests that avian influenza virus elicits a transient and effective innate immune response early in duck infection.(4)By comparison,it was found that the RNA load of Hn/h4 virus was basically negatively correlated with the expression changes of duck RIG-I,NF-κB and IFN-β.Except for the spleen,Hn/h4 virus strain induced duck RIGI,NF-κB and IFN-β expression up-regulated peaks higher than Ln/d59 virus strain,and the expression fluctuated greatly.The highly pathogenic avian influenza virus H5N8 elicited stronger innate immune responses than the low pathogenic avian influenza virus H4N6,and the highly pathogenic avian influenza virus H5N8 had a stronger inhibitory ability on the innate immune response mediated by duck RIG-I.Ducks were infected with the highly pathogenic avian influenza Hn/h4 virus strain on 2dpi,the viral RNA load was the highest in the lungs,and the comparative analysis of the data at the same time showed that the expression of RIG-I was the lowest in the lungs.This suggests that H5N8 virus may have a stronger inhibitory effect on RIG-I-mediated innate immune response in duck lung than in other tissues.On the second day after infection with low pathogenic avian influenza H4N6 virus,the number of virus copies in the rectum was the largest followed by the lung,and the least in the spleen,while the expression of RIG-I was the highest in the rectum and much higher than that in the lung and spleen.This indicated that the low pathogenicity avian influenza virus H4N6 had no obvious inhibitory effect on the innate immune response mediated by RIG-I.In conclusion,this study revealed the dynamic changes of viral RNA load and RIG-I and other innate immune genes in ducks infected with H5N8 and H4N6 avian influenza viruses.It provides reference data for exploring the innate immune mechanism of wild ducks against avian influenza virus,and lays a foundation for studying the mechanism of wild birds in the spread and epidemic of avian influenza.