Part ? Full-length Genome And Molecular Characterization Of Dengue Virus Serotype ? And Serotype ? Isolated From Imported Patients From Myanmar Part ? The Discovery Of Polyomavirus SV40-derived Circular RNA

Background:Dengue is the most common mosquito-borne infection worldwide and a serious threat to global public health.Sporadic dengue virus imported cases from Myanmar have been documented almost every year in Yunnan Province of China since 2005.However,the complete genome sequences of DENV-1 and DENV-2 isolates imported from Myanmar are not available.Methods:The full-length genome of the new DENV-1?YNPE1?and DENV-2?YNPE2?strain,isolated from two imported cases from Myanmar,were identified by the next-generation sequencing.The extreme ends of the viral genome of the YNPE2 were validated by 5'/3' RACE and Sanger sequencing.Furthermore,phylogenetic analysis was used to reveal the genetic relationship of YNPE1 and YNPE2 isolates,respectively.Recombination and selection pressure analyses were conducted for the molecular characterization of YNPE2 strain.Results:The complete genome sequence of strain YNPE1 is 10,821 nucleotides?nt?in length with an ORF encoding a polyprotein of 3,392 amino acids.Whole-genome sequencing revealed that the full-length sequence of YNPE2 strain was 10,724 bases,with an open reading frame encoding for 3,391 amino acids,flanked by 96 nts and 452 nts at the 5' UTR and 3' UTR,respectively.The nucleotides identity and the amino acid identity between YNPE1 strain and two closely strains,Sri Lanka DK87 strain?KP398852?and Thailand TH/BID-V2270 strain?FJ687427?,both were 98%.The phylogenetic analysis revealed that YNPE1 belongs to genotype I.The YNPE2 strain had 99.0%nucleotide identity and 99.8%amino acid identity with two closely related strains,ThD2₀078₀1 strain?DQ181797?and DENV-2/TH/BID-V2157/200 strain?FJ639832?.The phylogenetic analysis suggested that the YNPE2 strain belonged to Asian I genotype and was likely derived from Thailand strain?DQ181797?.Moreover,selection pressure analysis revealed two amino acid sites of the NS4B and NS5 proteins,with important evidence of positive selection.Conclusion:This study firstly revealed the complete genome sequence and molecular characterization of the new DENV-1 and DENV-2 strains isolated from two imported case from Myanmar,thus providing a valuable reference genome source for future surveillance,epidemiology and vaccine development of DENY virus in Yunnan,China.Background:circRNA is a ubiquitously expressed endogenous RNA with many biological functions,such as miRNA sponge,regulatory factors,and translation templates.The circRNAs are also closely associated with tumors and neurodegenerative diseases.circRNA has been found in a variety of circular double-stranded DNA tumor viruses such as EBV and KSHV.As a DNA tumor virus research model,the circRNA expression profile of SV40 has not been reported so far.Method:High throughput sequencing was performed for SV40 infected Vero cells and uninfected cells after removing the rRNA or RNase R-digesting.The data of the RNA-seq were analyzed by CIRI software to identify virus-derived circRNAs.The candidate circRNAs were verified by various experimental methods,including divergent PCR,Sanger sequencing,RNase R digestion and Northern blot.Subcellular localization analysis was performed after separating the cytoplasm and nucleus of the infected cells.Overexpression of the circRNA was used to initially explore its function.Result:In total of 10 and 8 circRNAs were obtained in the rRNA-removed or RNase R-treated RNA-seq data.Four of them are obtained by the two different sequencing methods.However,only a 147 nt circRNA was identified as a new circRNA molecule derived from SV40 by divergent PCR,Sanger sequencing,RNase R digestion and Northern blot.Since this circRNA was produced by back-splicing from the second exon of SV40 17kT,it was named circ-17kT.circ-17kT is continuously expressed in Vero cells with the trend of firstly increasing and then decreasing.Subcellular localization results showed that circ-17kT was mainly localized in the nucleus in the early stage of infection.Overexpression of circ-17kT analysis revealed that circ-17kT can promote the expression of SV40 LT mRNA.Conclusion:This study firstly revealed the circRNA expression profile of SV40.It was also found that circ-17kT was located in the nucleus in the early stage of infection.And circ-17kT can promote the expression of SV40 LT mRNA,suggesting that it may be involved in SV40-induced cell transformation.This will provide a favorable reference for exploring the pathogenicity of SV40,virus-host interactions,and function of circ-17kT.