The Distribution Of RIG-I Gene In Anas Platyrhynchos Tissues And The Effect Of Avian Influenza Virus Infection With Different Pathogenicity On The Expression Of Key Molecules Of RIG-I Pathway In The Spleens Of SPF Ducks

Avian influenza virus(Avian influenza virus,AlV)is widespread in poultry(chicken,quail,domestic goose,domestic duck,etc.),wild fowl(wild duck,wild goose,swan,etc.)and other birds around the world.According to the different pathogenicity of AIV to chickens through artificial infection,it can be divided into Highly pathogenic avian influenza Virus(HPAIV)and Low pathogenic avian influenza Virus(LPAIV).Moreover,interspecies transmission has become the main trend of AIV in recent years.Retinoic acid-inducible genel(RIG-I),as an important component of the innate immune system,many studies have shown that RIG-I plays an important role in the influenza resistance in humans and mammals.However,there are limited research data on RIG-I in poultry.Some studies have found that ducks have the RIG-I gene while chickens lack it,which is speculated to one of the main reasons for the obvious difference between chickens and ducks after AIV infection.Why do ducks have relatively strong resistance to AIV?To solve this problem,this study firstly amplified the full length of RIG-I gene in wild mallards(Anas platyrhynchos)and analyzed its expression and distribution in specific organs and tissues.Then,SPF ducks(Shaoxing Ma Ducks),as the model animal,were artificially inoculated different virulence influenza viruses.We explored the expression of innate immune factors in the RIG-I pathway changing with time,the role in anti-AIV and the molecular mechanism of the RIG-I pathway so as to provide the theoretical and practical guidance for the prevention and control of AIV.1?After preliminary screening of avian influenza virus,Newcastle disease virus,Escherichia coli,salmonella et al,samples of wild mallards with better health were selected and their hearts,liver,spleen,lungs,kidneys,and duodenum were collected.The wild mallards RIG-I gene(MdRIG-I)was amplified by RT-PCR and bioinformatics analysis was performed.Then,we used fluorescent quantitative PCR to detect its relative expression level in specific organs.The results showed that the CDS sequence of the MdRIG-I gene with a total length of 2802 bp,which encodes 933 amino acids,has a strong interspecies specificity.The protein structure was typical and the mRNA expression was different among different parts,with the highest expression in liver,followed by the duodenum,with low expression in the lungs and kidneys.2?90 4-week-old SPF ducks(Shaoxing Ma Ducks)were divided into 3 groups and inoculated with PBS,LPAIV H4N6 and HPAIV H5N8 by nasal drip and oropharyngeal methods.Every 6h,Id,2d,3d,5d,7d,10d,14d after inoculation,three ducks were randomly selected from each group and their spleens were collected after euthanasia.We extracted the total RNA and used Fluorescence quantitative RT-PCR to detect the relative expression of key innate immune factors RIG-I,IPS-1,IKK-?,NF-?B,TBK-1,IFN-?,IL-8.The results show that H4N6 can induce multiple immune peaks in the spleen of SPF ducks(Shaoxing Ma Ducks),the first time is in 6h-2d,the second time is in 5d-10d,there is also an upward trend in 10d-14d,indicating that there will be a next peak;H5N8 can induce two distinct peaks of expression in the spleen of SPF ducks(Shaoxing Ma Ducks).The first expression peak usually occurs during 6h-2d/3d,and in 5d/7d-14d can induce a high expression with constantly increased.Both of them in 2d/3d-5d/7d appear a low expression.The innate immune factors RIG-I,IPS-1,IKK-?,NF-?B,TBK-1,IFN-?,IL-8 are all involved in the RIG-I pathway mediated anti-influenza response and are highly correlated with each other.10-14d after SPF ducks(Shaoxing Ma Ducks)were infected with AIV,HPAIV H5N8 induced a higher level of innate immune response than LPAIV H4N6.In conclusion,this study successfully cloned the mallard duck RIG-I gene,and the SPF ducks(Shaoxing Ma Duck),whether infected with LPAIV H4N6 or HPAIV H5N8,can induce the body's innate immune response through the RIG-I signaling pathway to play an important role in anti-AIV..These findings provide theoretical and practical guidance for the research on innate immunity of birds and human prevention and control of AIV.