Study On Innate Immune Response Of MAVS^-/- Cell And RIG-I^-/- Mice To H9N2 Subtype Avian Influenza Virus

H9N2 subtype avian influenza virus(AIV)spreads in poultry around the world and causes sporadic infection in humans,which pose a persistent and serious threat to human health.Mitochondrial antiviral signaling proteins MAVS play a crucial role in the anti-RNA virus infection process,and can receive the signal of upstream protein RIG-I and activate the signaling pathway.Retinoid-induced gene I(RIG-I),a key cytoplasmic pathogen recognition receptor,serves as the primary sensor for RNA viruses and facilitates the recognition of certain DNA viruses.In this study,MAVS^-/-MDCK cell lines were constructed using CRISPR/Cas9 technology,and MAVS^-/-MDCK cell lines and RIG-I^-/-mice were infected with H9N2subtype avian influenza virus to preliminarily explore the role of RIG-I-MAVS signaling pathwayin the replication of influenza virus and in the innate immune response of the body.Mitochondrial antiviral signaling protein(MAVS)plays a central role in congenital immunity,and its mediated signaling pathway activation is an important antiviral response.In this study,the sg RNA expression vector targeting MAVs gene was designed and constructed,which was co-transfected with pcag-Cas9-EGFP expression vector into MDCK cells.MAVS knockout cell lines were screened by flow cytometry sorting,PCR and gene sequencing,and cell proliferation rate was detected by CCK-8 method.The H9N2 subtype avian influenza virus was used to infect MAVS knockout cell lines,and the changes of viral copy number and transcription levels of downstream signal molecules,interferons and ISGs of MAVS were detected by Real-time PCR.The results showed that a MDCK cell with MAVS gene knockout was successfully obtained,and its proliferation rate was not significantly different from that of normal cells.36h after virus infection,the virus copy number in MAVS^-/-MDCK cells was significantly higher than that in WT cells(p<0.01),the copy number of influenza virus was 1.82 times that of WT,indicating that the replication of influenza virus increased after MAVS gene knockout.The m RNA expression levels of IRF3,IFN-?and MX1 were significantly decreased,indicating that MAVS blocked the IFN-I signaling pathway and promoted the replication of H9N2 subtype avian influenza virus in MDCK cells.Twelve 6-week-old RIG-I^-/-mice and Twelve 6-week-old SPF grade ICR WT mice were randomly divided into immune challenge group and non-immune challenge group,respectively.The mice in RIG-I^-/-and WT immunized groups were inoculated with the inactivated H9N2 virus vaccine twice by percutaneous multi-point injection,and then were inoculated with CK/HLJ/3/00 strain of H9N2 subtype avian influenza virus by nasal drops in the unimmunized group.Blood samples were collected from the tail vein and lung tissue was taken aseptically on the 3rd and 6th day after infection.The pathological changes of lung tissue of mice were observed by H&E staining.The titer of virus in lung tissue was detected by inoculating chicken embryos.The titer of specific antibody against influenza virus in peripheral blood of RIG-I^-/-mice and WT mice in the immunized group was detected by HI.Real-time PCR PCR was used to detect the expression of signal molecules downstream of RIG-I.Results:the titers of specific antibodies against influenza virus in RIG-I^-/-mice were significantly lower than those in WT group before challenge,3 days and 6 days after challenge.on the 3rd day after the virus infection,the titer of lung tissue virus in RIG-I^-/-unimmunized group was significantly higher than that of WT control group,which was close to 8 times of that of control group,which indicated that RIG-I could inhibit the replication of H9N2 influenza virus in mice;on the 3rd day after infection,the m RNA expression levels of MAVS,IRF3 and NF-?B were significantly or significantly decreased,and the expression level of IL-1?m RNA of proinflammatory factor was significantly decreased.The expression levels of IL-10 and IFN-?,anti-viral protein Mx1 and ISG15m RNA were significantly lower than those of WT group,which indicated that RIG-I played an important role in anti-influenza virus infection.After its deletion,it inhibited the expression of downstream signal molecules,the expression of inflammatory factors was disturbed,and the expression levels of interferon and anti-viral signal proteins were significantly reduced,which was beneficial to the release and replication of virus.In this study,RIG-I-/-mouse model infected with H9N2 subtype avian influenza virus was used to demonstrate that RIG-I knockout inhibited the activation of innate immune signals of RIG-I-MAVS-IRF3/NF-?B,decreased the expression of interferon and antiviral proteins,enhanced viral replication in mice,and significantly decreased the titer of H9N2 specific antibody.This study lays a foundation for exploring the role of RIG-I in the innate immune response against influenza virus and the interaction between innate immunity and adaptive immunity.