Identification And Functional Analysis Of S Rna Derived From JEV

Japanese Encephalitis virus(JEV)belongs to the genus Flavivirus of the Flaviviridae.It is a serious zoonosis pathogen and causes Japanese encephalitis(JE)in human and animal.JEV can target central nervous system and causing neuroinflammation.sRNA is a umbrella term for a large group of regulatory molecules with a length of 18~30nt,mainly including miRNA,siRNA,piRNA,snoRNA and so on.sRNA almost exists in all beings and plays an important role in cell proliferation,differentiation and apoptosis,cancer and virus infection.But few report is available about sRNAs derived from JEV and the interaction between JEV and host cells at the sRNA level.Thus,related research of sRNAs derived from JEV is really important for deep understanding of the pathogenic mechanism of JEV.The specific content of papers is as follows:A new strain of JEV named JD-15 was isolated from mosquitoes samples from Jiading District's pigpens in Shanghai through RT-PCR and IFA detection.All BHK21 cells and C6/36 cells showed cytopathic effects at 72 h after inoculation of the isolated strain.The titer was 1.96×10~6 PFU/0.1mL.The median lethal dose of the isolate measured by intracerebral injection in mice was 4.2×10~1PFU/0.1mL,which indicated the isolate possessed strong neurovirulence.The cloning and sequence analysis based on the amplified E gene indicated that the isolate belonged to Genotype?of JEV.Meanwhile,homology of the nucleotide and amino acid sequences between the isolated JD-15 and vaccine stain SA-14-14-2 was 98.1%and 97.5%,respectively.Deep sequencing was conducted on total RNA extracted from mice brain infected with and without JEV.The total number of mappable reads was about 31,543,891.Among them,more than 55 thousands small RNAs sequences were aligned to JEV viral genome.To explore the possibility of any sRNAs derived from JEV in sequencing result,paper information and RNAfold sofeware was used.Four such precursors containing small RNA sequences found in the deep sequencing data were selected.To further confirm the existence of JEV-encoded sRNAs,ploy A quantitative RT-PCR(qRT-PCR)was performed for all four small RNAs using RNA derived from BHK-21,PIEC and A549 cells infected with JEV virus(MOI=5).The expression levels of viral small RNAs were normalized to levels of U6 snRNA,and values were presented relative to RNA derived from mock-infected cells.One sRNA were readily detected in all cells,while the other viral sRNAs were expressed at very low levels,so we named the small RNA with relative higher read number as JEV-32.Subsequently,we performed a stem-loop reverse transcription followed by quantitative PCR along with northern blot analysis to further confirmed the expression of JEV-32 in total RNAs isolated from BHK-21,PIEC and A549 cells infected with JEV.Meanwhile we examined the evolutionary conservation of JEV-32 and found that the predicted sRNA was highly conserved.Thus,JEV-32 was not limited to a particular isolate but was highly conserved among JEV viruses.To find out the molecular synthesis pathway of JEV-32,siRNA of Drosha,DCR1 and Ago2 were transfected into A549 cells followed by JEV infection.The results revealed Drosha,DCR1 and Ago2participate in the molecular synthesis pathway of JEV-32.To find out the impact of the JEV-32 on viral RNA replication,synthetic inhibitors or mimics were transfected into BHK-21 cells followed by JEV infection.The results revealed of up-regulation in viral gRNA levels with JEV-32.In the study,sRNAs derived from JEV were selected and determined.Identification and functional analysis of sRNAs showed that microRNA-like small RNA named JEV-32 plays significant role in viral replication,which provided the basic data for further exploring the mechanisms of interaction between virus and host at small RNA level.