Identification And Functional Evaluation Of MiRNA Expression Profile In A.albopictus And PST Cells Infected With Bluetongue Virus

Bluetongue is a non-contagious infectious disease caused by Bluetongue virus(BTV) which is mainly transmitted by biting midges(Culicoides spp). The susceptible animals of Bluetongue include sheep, goat, cattle, deer and other ruminant animals. Due to the huge economic losses in the global livestock industry, bluetongue is listed as one of the notifiable diseases by the World Organization for Animal Health(OIE). BTV belongs to the genus Orbivirus of the family Reoviridae. microRNA(miRNA) is a kind of small single-stranded non-coding RNA with lengths of 19 to 25 nucleotides and often functions as regulatory molecules in many processes. In recent years, studies suggested that miRNA plays an important role in pathogen-vector-host interactions, innate antiviral immune response in host cells. It can affect virus infection and replication through regulating the expression of target gene.Nowadays, knowledge about interaction between BTV and its target cell is still limited and the role of cellular miRNA in BTV infection is not well understood. In this study, the Solexa high-throughput sequencing was used to detect the miRNA in BTV-infected and mock-infected Aedes albopictus(A. albopictus) and primary sheep testis(PST) cells. The differentially expressed miRNAs and their target genes were analyzed by bioinformatics technology and subsequently validated by qRT-PCR. Three differentially expressed cellular miRNAs were further selected and their regulatory functions on BTV replication were studied. The results are as follows:1.High-throughput sequencing data of A. albopictus cells resulted in 11,206,854 and 12,125,274 clean reads respectively in BTV-infected and mock-infected A. albopictus cells, with 89 and 92 known miRNAs, as well as successfully predicted 266 and 181 novel miRNAs in individual groups. Compared with mock-infected A. albopictus cells 15 known and 125 novel miRNAs differentially expressed in BTV-infected A. albopictus cells. GO function and KEGG pathway analysis showed that the target genes were mainly involved in metabolic pathways, endocytosis as well as the FoxO, Hippo, Jak-STAT and MAPK signaling pathways. The results indicated that miRNAs in vector may play widely regulatory roles in BTV infection.2.The regulatory functions of 2 differentially expressed miRNAs including aae-miR-1889-5p and aae-miR-2940-5p were further studied by transfecting their mimics, inhibitor and controls into A. albopictus cells and analyzing the efficiency of BTV multiplication via qRT-PCR target BTV NS3 gene and plaque formation assay. The results showed that the expression levels of BTV NS3 gene were 1.55-2.88 and 2.00-3.22 fold increased in transfected group of aae-miR-1889-5p mimic and aae-miR-2940-5p mimic. The virus titers were consistently higher in transfected group of each miRNA mimic. However, the expression levels of BTV NS3 gene were 0.54-0.64 and 0.55-0.86 fold down regulated in inhibitor-transfected groups of aae-miR-1889-5p and aae-miR-2940-5p. Consistently, virus titers in both inhibitor-transfected groups were lower than control groups. Taken together, the data indicated that aae-miR-1889-5p and aae-miR-2940-5p can promote BTV replication in A. albopictus cells.3.The high-throughput sequencing of BTV-infected and mock-infected PST cells revealed that the clean tags in two groups were respectively 10,908,164 and 11,920,794, including 87 and 90 known miRNAs as well as 182 and 155 novel miRNAs in BTV-infected PST cells and mock-infected PST cells. Differential analysis indicated that two known and 70 novel miRNAs expressed differentially between the two groups. GO function and KEGG pathway analysis showed that target genes were mainly involved in catalytic activity, viral infection, endocytosis, metabolic pathway and other signaling pathways, indicating that mi RNAs play potential regulatory roles in multiple steps of the cells infection with BTV.4.The novel-mir-105 was selected and its mimic, inhibitor and control were transfected into PST cells, followed by BTV infection and multiplication analysis through qRT-PCR targeted BTV NS3 and plaque formation assay. The results showed that the expression level of BTV NS3 gene was 0.32-0.80 fold down regulated and the virus titers was lower in PST cells transfected with novel-mir-105 mimic, while the expression level of BTV NS3 gene was 2.09-2.41 fold up regulated and the virus titers was higher in PST cells transfected with novel-mir-105 inhibitor, indicating that novel-mir-105 suppresses the replication of BTV in PST cell.