Development Of The Reverse Genetics System For An Attenuated Duck Tembusu Virus Strain

The newly emerged duck infectious disease caused by duck Tembusu virus (DTMUV) has resulted in massive loss in duck-raising industry in China since2010. The disease causes duck death, retarded growth, high fever, loss of appetite, and egg production decline. Based on an attenuated strain (FX2010-180P) generated through serial passages of a DTMUV isolate in the chicken embryo fibroblasts (CEF), the reverse genetics system for the attenuated strain was developed in this study. The establishment of a reliable reverse genetics system of DTMUV would lay the foundation for the studies on the molecular basis of DTMUV pathogenicity.In order to develop the reverse genetics system for FX2010-180P strain, viral RNA was extracted and reverse transcribed into cDNA firstly. Then the whole genome was amplified segmentedly by polymerase chain reaction (PCR) to produce6fragments whose terminal sequence overlapped with each adjacent fragment. The PCR products were inserted into pSIMPLE18plasmid separately. The larger segment containing the sequence of3656to10991nucleotides of viral genome was obtained by restriction-enzyme digestion and ligation of the segments cut from3plasmids constructed previously, and then inserted into pSIMPLE18plasmid. Finally,4recombinant plasmids containing4DNA segments covering whole genome sequence were prepared. Using these4plasmids as the templates, the PCR product of full-length viral genome was generated, together with the T7promoter in the5' terminate was introduced by PCR. To rescue the virus, PCR products containing full-length viral genome was transcribed in vitro to produce the viral RNA, and then viral RNAs were purified and transfected into DF-1cells. Comparing with the control cells, cytopathic effect (CPE) on the cells transfected with the viral RNA appeared at48hours post-transfection and severe CPE was detectable at72hours post-transfection. After identified by RT-PCR and indirect immunofluorescence (IFA), it was proved that DTMUV (R-FX2010-180P) was rescued successfully. Sequence analysis confirmed no unexpected mutation in rescued virus.To explore the possibility to express exogenous proteins in DTMUV, inside ribosomal entry site (IRES) and the gene of green fluorescent protein (EGFP) were introduced between the NS5gene and the3'non-coding sequence of FX2010-180P by the way of PCR, restriction-enzyme treatment and ligation. The long DNA product containing T7promoter in the5'terminate, and full-length genome of FX2010-180P with the insertion of IRES and EGFP was generated by PCR. After the transcription of PCR product in vitro, the RNA product was purified and transfected into the DF-1cells, a recombinant DTMUV expressing EGFP was rescued. The recombinant virus was passed3times in DF-1cells, and EGFP was expressed stably in each passage.