Isolation And Identification Of Avian Reovirus And Preliminary Study On Effect Of Artificial MiRNA Against Avian Reovirus

Avian reovirus(ARV)is one of the important pathogens among poultry diseases in the world.With a wide host spectrum,ARV can not only cause diseases in the host,but also cause immune suppression in the host and aggravate the infection of other pathogens.It is a class of avian infectious pathogens capable of horizontal and vertical transmission.There have been cases of ARV infection all over the world.Since the mid-1980s,many provinces and cities have been found in our country,which seriously affected the development of poultry industry.The significant economic loss of post-infection ARV to the poultry industry underscores the importance of studying the prevalence,genetic characteristics,and pathogenicity evolution of emerging ARV isolates.At present,there are no direct anti-ARV drugs,and the mainstream vaccine strains S1133,attenuated S1133 and 1733 on the market are all chicken strains,which have poor immune effect against waterfowl strains.Therefore,it is of great scientific significance and application value to isolate and identify ARV strains,search for novel treatment strategies after ARV infection,and explore new prevention and control technologies of the disease.In this study,we isolated and identified ARV and preliminarily studied the inhibition of replication of ARV by RNA interference.1.Isolation and Identification of Avian ReovirusIn order to understand the infection situation of ARV,this study used RT-PCR to detect the infection situation of ARV in the abrasive fluid of liver and spleen of the disease material,and inoculated the PCR-positive disease material with chicken embryos,CEF and Vero cells to isolate the virus and measure the hemagglutination characteristics and infectivity of the virus.All the 8 isolated ARV strains could infect CEF and produce cytopathic changes,while only ARV-SD-2021-1 and ARV-SD-2021-3 could infect Vero cells and produce cytopathic changes with the highest titer at 48h.Whole gene sequencing was performed for ARV-SD2021-1 and ARV-SD-2021-3.Homology,genetic evolution and bioinformatics analysis were performed with the published ARV sequences in GenBank.The results showed that the mainstream vaccine strains S1133,attenuated S1133 and 1733 on the market were all chicken strains,and the immune effect against waterfowl strains was not good.ARV-SD-2021-1 isolated from chickens belongs to ARV from waterfowl,indicating that ARV from waterfowl can infect chickens as well,and there is a phenomenon of cross infection,which poses a huge challenge for the prevention and control of ARV.In summary,8 strains of ARV were successfully isolated in this study,and genetic evolution and bioinformatics analysis of the whole genes of some isolates were carried out,which is helpful to understand the genetic variation of prevalent strains of ARV,and provide basis for subsequent prevention and control of ARV and research and development of vaccines.2.AmiRNA inhibits ARV replication in Vero cellsIn this study,according to the design principle of amiRNA,12 amiRNA with conserved sequences of L2 gene targeting ARV were designed and chemically synthesized,which were correctly identified by sequencing.Vero cells were transfected with ARV-SD-2021-1 at 48h after transfection,and cells and cell supernatants were collected at 48h after infection.The inhibitory effect of amiRNA on ARV was verified by TCID50 and Real-time PCR.The results showed that the constructed amiRNA could effectively inhibit the replication of ARV in Vero cells to varying degrees.The four amiRNA with the best inhibitory effect(amiRNA-420,amiRNA-1107,amiRNA-1979,amiRNA-2863)were further verified.Cells and cell supernatant were collected at 24,48 and 72h after infection.The inhibitory effect of amiRNA on ARV was detected by CPE,TCID50,Real-time PCR and Western blot.The results showed that amiRNA-420 had the best inhibitory effect on ARV at 24h and 48h,followed by amiRNA1107.At 72h,amiRNA-1979 had the best inhibitory effect on ARV,followed by amiRNA-420.In conclusion,this study successfully constructed amiRNA targeting the conserved region of L2 gene of ARV,which provided a foundation for the application of RNA interference technology in the field of anti-ARV therapy and the study of anti-ARV infection in animals.3.AmiRNA multi-target tandem expression vector inhibited ARV replication in Vero cellsIn order to achieve the inhibition of different genotypes of ARV strains,avoid virus mutation escape and play a lasting antiviral effect,this study combined the above single amiRNA sequences with good ARV inhibition effect to construct amiRNA multi-target tandem expression vectors amiRNA-420+1107 and amiRNA-420+1979.Vero cells were transfected with single-target and multi-target amiRNA.ARV were infected at 48h after transfection.Cells and cell supernatant were collected at 24,48 and 72h after infection.The inhibitory effect of amiRNA on ARV was detected by CPE,TCID50,Real-time PCR and Western blot.The results showed that amiRNA-420+1107 and amiRNA-420+1979 could stably inhibit ARV replication in Vero cells at 24,48 and 72h,and the inhibitory effect was similar to that of amiRNA,the single target with the strongest inhibitory effect,at all three time periods.In conclusion,this study successfully constructed a multi-target amiRNA expression vector in series,and proved its inhibitory effect on ARV replication in Vero cells,which will lay a theoretical and material foundation for the use of RNAi technology to inhibit ARV isolates of different genotypes,provide a potential approach for the prevention and treatment of ARV,and provide materials for subsequent animal experiments.