Development And Stability Study Of Recombinant Duck Tembusu Virus Expressing Foreign Genes

The newly emerged duck infectious disease caused by duck Tembusu virus(DTMUV)hasbeen widedissemination in China and it has caused massive loss in duck industry in China since 2010.Based on an attenuated strain(FX2010-180P)generated through serial passages of a DTMUV isolate in the chichen embryo fibroblasts,we explored to insert the reporter genes in different locations of the attenuated strain.Fortunately,we found an insertion site that can express exogenous proteins successfully,which laid a foundation for further development of new vaccines against this disease.In order to explore whether DTMUV can stably express foreign proteins or not,fusion PCR was used to insert green fluorescent protein gene(EGFP)between 5' untranslated region(UTR)and C gene,C gene and PrM gene,PrM gene and E gene,E gene and NS1 gene and internal ribosome introducing site(IRES)and EGFP between NS5 gene and 3' UTR respectively.Using four plasmids as templates,the full-length of FX2010-180 P strain,the T7 promoter and exogenous gene sequence were generated,then it was transcriptedinto infectious RNA in vitro.After 4 days of transfection into DF-1 cells,only recombinant DTMUV with exogenous gene inserted between NS5 gene and 3'UTRcan fluoresce.The fluorescence intensity of recombinant virusdecreased gradually with the passage times increasing,and the fluorescence disappeared completely in the P5 generation.Sequencing results showed that the recombinant virus lost not only506 nucleotides of IRES 3'end and the whole EGFP,but also 67 nucleotides of FX2010-180 P strain 3' end followed by termination codon.So we constructed a plasmid that lack of these 67 nucleotides on the basis of inserting exogenous genes between NS5 gene and 3'UTR and use the same method to rescue the virus.Surprisingly,recombinant virus could proliferate and cause cytopathic effect similar to parental viruses.Recombinant viruses by successive passages showed that the fluorescence gradually weakened after passage to generation P5 and disappeared completely after passage to generation P7 which indicating that the recombinant virus still could not express exogenous proteins stably.High throughput sequencing revealed that it has at least seven kinds ofvirusesin cell supernatants of P2 recombinant viruses.It shows that the recombinant virus contain multiple missing locations,and the length of each location is different.After further passage,the types of recombinant viruses in cell supernatants are becoming fewer and fewer,and eventually they exist in one or two relatively stable states.Although this locus can express foreign genes,but it is unstable after generations.We should continue to look for insertion site that can stably express exogenous proteins in future studies.