Establishment Of Minireplicon System For Severe Fever With Thrombocytopenia Syndrome Bunyavirus

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease newly identified in China, which caused by a novel bunyavirus named severe fever with thrombocytopenia syndrome virus (SFTSV). Since2010, SFTS has been reported in China’s Hubei, Anhui, Shandong, Henan, Jiangsu and Liaoning. Some of the critically patients died due to multiple organ damages. The viruses were isolated from patients’blood and ticks. The function of non-coding regions is still unclear.Minireplicon derived by reverse genetic technology, and has been applied to study the functions of non-coding sequences with a variety of viruses in the bunyaviridae. In the minireplicon system, viral coding sequence is replaced by an assayable reporter, the non-coding regions acts as regulatory elements, mimic viral replication. Minireplicon provides a powerful tool to study the regulatory function of non-coding sequences of viruses.SFTSV contains a tripartite, single-stranded RNA genome of negative polarity. The large (L) RNA segment contains6368nucleotides with one open reading frame from nucleotide17to6271. The M segment contains3378nucleotides, and the ORF is from nucleotide19to3240. The S segment contains1744nucleotides of ambisense RNA with two open reading frames. The virus RNA from nucleotide43to780encodes nucleocapsid protein, and complement RNA segment from nucleotide835to1716encodes nonstructural protein. This study intends to use the non-coding sequences of the L, M, S segments to establish the minireplicon system of SFTSV, and investigate the regulation capacity of NCR of the three segments.In this study RNA polymerase I system was used to construct SFTSV minireplicon. GFP and luciferase genes were chosen as reporter genes. GFP and luciferase genes were amplified and flanked by non-coding regions of L, M, S segments of SFTSV strain HB29respectively, and then cloned into plasmid pHH21under the control of pol I promoter.The ribonucleoprotein (RNP) consists of the genomic RNA, L protein and N protein, which is the smallest functional unit of transcription and replication for many negative strand RNA viruses. This study utilized both the L protein and N protein of SFTSV in minireplicon system, which are supplied either by superinfection with SFTS helper virus or by L protein and NP expressing plasmids. The Vero cells are superinfected with SFTSV at a multiplicity of infection of1PFU/cell. Seven days later, indirect immunofluorescence assay (IFA) and Real-time PCR were used to detect the virus in infected cells. Then the plasmids of L, M, S segments non-coding regions controlled GFP were transfected into virus infected Vero cells. Twenty four hours posttransfection, no GFP expressions were observed.The reporter contained plasmid cotransfects with L and N proteins expressing plasmids, which has successfully used to the virus of bunyaviridae. The L and N protein eukaryotic expression plasmids are constructed by Dr. Wang Tao. We used those plasmids of VR1012-L and VR-1012-NP cotransfect with GFP reporter plasmid in293T cells. Twenty four hours posttransfection, green fluorescence could be observed in the three segments of L, M, and S.The reporter plasmids integrated with luciferase gene cotransfected with L and N proteins expressing plasmids by the same way in293T cell line. Twenty four hours posttransfection, luciferase activity was tested. The NCR controlled luciferase gene was expressed, and expression level of luciferase was different between L, M and S minireplicons.In summary, the minireplicon system for SFTSV was established, the NCR of each L, M and S segment was sufficient to play an important role in transcription and replication of a minigenome.