Profile Analysis Of CircRNA And Preliminary Study On Function Of CircRNA In Fowl Adenovirus Serotype 4 Infected LMH Cells

Fowl Adenovirus-4（FAdV-4）belongs to the family Adenovirdea and the genus Aviadenovirus,and is a double-stranded DNA virus without envelope.Aviadenovirus can be divided into 5 species（FAdV-A to FAdV-E）,and can be divided into 12 serotypes（FAdV-1 to 8A and 8B to 11）through serum cross neutralization tests.FAdV-4 infection mainly causes hydro pericardium syndrome（HPS）,which first broke out in Pakistan in 1987 and gradually spread to neighboring countries.In 2015,it broke out in most area in China,with the incidence rate and mortality rate as high as 30%～80%,which caused huge economic losses to China’s poultry industry.circRNA is a key member of noncoding RNA,which has attracted more and more attention in recent years and plays an important role in many biological processes.circRNA is a covalently closed cyclic molecule that is more resistant to exonyms and RNAse R than regular linear RNA,and thus it’s more stable.circRNA has functions such as acting as a sponge for microRNA（miRNA）,binding to proteins,regulating gene transcription,and encoding proteins.In addition,it can be used as diagnostic or predictive biomarkers of diseases,and provide new potential therapeutic targets.To investigate the circRNA expression profile and the function of circRNA after infecting Leghorn Male Hepatoma cell line（LMH）with FAdV-4,RNA-seq analysis was performed on FAdV-4 infected LMH.Identified 21258 candidate circRNAs,of which 19154 encoded by the host genome,the remaining 2104 were encoded by the viral genome,and the length was concentrated between 200 bp and 2000bp.After differential analysis of circRNA expression between the two groups,135 differentially expressed circRNAs were screened out,among which 86 were up-regulated and 45 were down-regulated.GO and KEGG enrichment analysis of the identified circRNAs showed that most circRNA played important roles in various biological processes such as cell division,metabolism and signal transduction.In order to further explore the function of some circRNAs,the accuracy of 10 cell-encoded circRNAs and 10 virus-encoded circRNAs was verified by semi-quantitative RT-PCR.The results showed that the RNA sequencing results were consistent with the verification results.In addition,seven circRNAs with the same 3’terminus were found in the pTP protein and DNA pol protein of FAdV-4 during the mapping of virus-encoded circRNAs,which was closely related to the selective splicing of circRNAs encoded by FAdV-4.What’s more,in order to further verify the relationship between the expression level of viral circRNA and viral replication,the expression level of viral circRNA was measured at different stages of virus infection.The results showed that virus-encoded circRNA played a role in different stages of virus entry into cells.Next,10 overexpression vectors of viral CricRNA were constructed,and the effects of circRNA on viral replicationwere measured by Western Blot and TCID50 after transfection and viral infection.The results showed that some virus-encoded circRNAs:circRNA₀23475,circRNA₀20380,circRNA₀29216,circRNA₀27083,and circRNA₀08682 promoted replication of FAdV-4,host encoded circRNA₀22182 can significantly inhibit virus replication.In summary,FAdV-4 infection induced differential expression of cell-encoded circRNAs,which were also encoded by FAdV-4,some of which affected the replication of FAdV-4.In summary,FAdV-4 infection induced the differential expression of circRNA from cell sources.Meanwhile,FAdV-4 itself encodes a large number of circRNAs,among which some virus-encoded circRNAs promote the replication of FAdV-4 while the host encoded circRNAs₀22182 can inhibit virus replication.These findings will provide new clues for further understanding of FAdV-4 and theoretical basis for studying virus-host interaction.