The Role And Mechanism Of MDA5 Gene In CIV Infection

Canine influenza(CI)is a canine respiratory disease caused by the Canine influenza virus(CIV)of the Orthomyxoviridae family.The main clinical symptoms of sick dogs showed fever,cough,runny nose,loss of appetite,sneezing,depression and so on.It can cause respiratory failure and even death when severely infected.Although dogs are the natural host of CIV,they can spread across the host due to the high variability of influenza viruses,thus posing potential risks to human health and life.At present,there have been a lot of reports on the infection of CIV in dogs.However,the mechanism of continuous infection and replication of the virus in the host and the death of the dog is still unclear.In this study,the second generation of sequencing technology was used to collect tracheal and lung tissues in puppies infected with H3N2 and H5N1 on the third day and on the seventh day to establish transcriptome and mi RNA databases.What’s more,the bioinformatics method was used to analyze the differential genes.We found that after infected canine influenza viruses,there were 455 mi RNAs and 22516 m RNA were found.These genes are mainly involved in the regulation of immune and signaling-related pathways.Combined with H3N2 and H5N1 data,we categorized these different expressed genes,and found 151 genes are involved in immune response.Pathway analysis found these genes mainly involved in cytokine receptor interactions,influenza A virus and RIG-I-like receptor pathways.The biological processes involved mainly include response to stimuli,biomodulation,signaling and the immune system.Innate immunity,especially the innate immunity regulated by interferon,constitutes the host’s first line of defense against pathogen invasion.Toll-like receptors and RIG-I-like receptors in pattern recognition receptors are capable of activating interferon,and MDA5 is a receptor of RLRs.After viral infection,MDA5 activates interferon by recognizing ligands,allowing signals to be transmitted,thereby producing antiviral activity in the host.Therefore,we screened the MDA5 gene in the RIG-I pathway in differente expressed genes and further studied it.Canine MDA5 gene was mainly distributed in spleen,liver and lung.Sequence analysis revealed that MDA5 gene sequence of the dog was close to the felis.Through structural prediction,it was found that the canine MDA5 gene mainly consists of CARD domain,RD domain and DEx D/H anti-helical domain.To explore the functional domains of MDA5,We successfully cloned the MDA5 gene sequence and the functional domain sequences of MDA5 in dogs(p3x FLAG-MDA5,p3 x FLAG-MDA5-CARD,p3 x FLAG-MDA5△CARD+RD,p3 x FLAG-MDA5△RD and p3 x FLAG-MDA5△CARD),which were 3087 bp,603 bp,2103 bp,2706 bp and 2485 bp,respectively.These constructed plasmids were verified by IFA and Western Blot.The dual luciferase assay showed that the CARD region of the MDA5 gene activated the IFN-β promoter strongly(p<0.001)compared with other functional domains,suggesting that MDA5 mainly transmits signals downstream through the CARD region.By overexpressing the CARD region of MDA5,it was found that the MDA5-mediated signaling pathway can transmit signals downstream by activating IRF3 and NF-κB,and is more potent in activating IRF3.At the same time,the CARD region of MDA5 can significantly activate the expression of the other two pattern recognition receptors LGP2 and RIG-I,what’s more,RIG-I-like pathway and its downstream ISGs were also activated,induces the production of antiviral proteins and promotes the release of cytokines,thereby inhibiting the replication of influenza viruses.However,after silencing the MDA5 gene,the production of cellullar inflammatory factors is reduced,the antiviral activity of the organism is weakened,and the replication of influenza virus is enhanced.The interaction between the influenza virus protein and the host determines the adaptability and pathogenicity of the virus in different hosts.Through the Bi Fc and Co-IP experiments,it was found that MDA5 can interact with influenza virus RNPs.Further studies have found that PB2 and NP signals are mainly located in the nucleus.In addition,the Bi Fc assay found that MDA5 also interacts with the NS1 protein of influenza virus.To further validate the effect of NS1 protein on the interaction between v RNPs and MDA5,we co-transfected the subunits of NS1 and v RNPs,respectively,and found that NS1 had no significant effect on the expression of v RNPs.Numerous studies have shown that viral proteins can interact with host proteins to evade host immunity.Studies have shown that NS1 protein can inhibit the expression of interferon by binding to ds RNA,and NS1 protein can affect viral replication by enhancing the synthesis of viral proteins or preventing the translation of cellular m RNA.In this paper,eukaryotic expression of the NS1 gene was constructed and reverse genetics was used to analyze the regulation of NS1 on the MDA5 pathway.By double luciferase assay,it was found that CIV NS1 protein can antagonize the activation of interferon and IRF3 promoter in the CARD region of MDA5,and the RIG-I pathway and downstream ISGs of CIV NS1 protein regulated by CARD domain of MDA5 Genes also have antagonism.In summary,this paper establishes transcriptome databases after high-pathogenic H5N1 and low pathogenic H3N2 CIV-infected hosts,and explored the differential analysis of host m RNA and mi RNAs expression profiles in different disease processes,and related gene functional informatics.What’s more,the relationship between different mi RNAs and disease-related m RNAs were analyzed.In addition,the MDA5 gene of canine was successfully cloned for the first time,and the functional domains of MDA5 gene were verified.The results indicate that MDA5 functions primarily through the CARD region.MDA5 interacts with the v RNPs and NS1 of the virus.At the same time,it was found that the influenza virus evades the signaling pathway regulated by MDA5 through the NS1 protein to escape natural immunity.This article provides ideas for further study of the relationship between influenza virus and host natural immunity.