Identification Of Tembusu Virus Noncoding Subgenomic RNA And Study On Its Functions In Viral Pathogenesis

Duck Tembusu virus(DTMUV)disease can cause severely decreased egg production and/or severe neurological disorders bringing huge economic losses in the Chinese duck industry.DTMUV,as an important member of the genus Flavivirus and the family Flaviviridae,is similar to other virus of Flavivirus and can be transmitted by mosquitoes,it has been reported that TMUV can be transmitted to humans,which brings hidden dangers to the public health safety in our country.The DTMUV is an enveloped virus,and its genome length is about 11 kb.It contains an open reading frame that encodes three structural proteins and seven non-structural proteins.The 5' and 3' end of the ORF contains a UTR(Untranslated region,UTR)respectively,the 5' UTR is composed of 94 nucleotides,and the3' UTR is composed of 618 nucleotides.Although the UTR is not involved in the viral proteins encoded by the viral genome,they have rich biological functions and play an important role in the process of viral genome replication and translation.The recent sudies indicated the flavivirus produce a unique and highly structured noncoding subgenomic RNA(sgRNA)of 0.3-0.5 kb derived from the genome during viral replication,which is essential for virus-induced cytopathicity and pathogenicity.The generation of sgRNA is a unique phenomenon of flavivirus,the productin of which is due to the conservative advanced structure of viral genomic 3'UTR which blocks the cleavage of the genome by host 5'-3' exonuclease XRN1.The terminal structure of sgRNA 5' prevented the downstream sequence from degradation,which can form advanced structure to interact with RNA or protein to perform related functions.In recent years,the sgRNA of flavivirus has been studied continuously,however,until now,there remains no report of identification and functional studies on DTMUV sgRNA,and this work is performed for the study of DTMUV sgRNA.The present study employed the high throughput deep sequencing,northern blot hybridization,exoribonuclease XRN1 treatment to identify sgRNAs sequences.By aligning the sgRNA sequences to the DTMUV3'UTR,the exactly sgRNAs targeted regions were finally obtained.First of all,the production of sgRNA can be intuitively confirmed by analyzing the results of northern blot.The results of the high throughput deep sequencing showed that a class of small RNA from genomic 3'UTR enriched in host cells.Combined with the analysis of the prediction result of 3'UTR secondary structure,it was found that the total length of DTMUV sgRNAsequence was about 528 nt,and by aligning the sgRNA sequences to the DTMUV 3' UTR,the accurate location of sgRNA in virus genome was confirmed.Additionally,according to the result of prediction of sgRNA secondary structure,we obtained the complete secondary structure information of sgRNA,and the exactly sgRNAs targeted regions were finally obtained.Importantly,the structure located in 3'UTR which can hinder the digestion of XRN1 was found.Based on this analysis,we identified the potential mutation sites in this structure.Furthermore,our previously constructed DTMUV full-length cDNA infectious clone were used for engineering the recombinant DTMUV with the deletion or point mutation of the corresponding partial 3'UTR region which cannot produce the sgRNAs or reduce ability to produce sgRNAs during the viral replication.In vitro,the viral pathogenesis and relative viral gene expression were tested which revealed the sgRNAs has a role in facilitating efficient viral genome replication and virus-induced cytopathicity in cell culture.In vivo,the pathogenicity of the mutation recombinant DTMUV were tested by inoculating the ducklings and compared with the native DTMUV which revealed the sgRNA has a role in the DTMUV pathogenesis and host antiviral mechanism.Through this study,we confirmed the production of DTMUV sgRNA.This study not only clarified the sgRNA was essential for the viral replication,virus-induced cytopathicity,pathogenicity,escape host antiviral mechanism;but also provided a basis for the further development of an sgRNA deleted recombinant DTMUV based attenuated vaccine.