Severe Fever With Thrombocytopenia Syndrome Virus Genome And Expression Of Its Glycoprotein Gn-Gc

ObjectiveIsolated and identified severe fever with thrombocytopenia syndrome(SFTS)virus strains.Sequencing the whole genome and classified the genotypes.Constructed recombinant plasmids to achieve the soluble expression of envelope protein Gn-Gc.Methods1.Collected 14 patients serum of SFTS samples from Zhejiang Province.Virus RNA was extracted and detected from the patient serum inoculated by Vero cells.2.The random primer sequencing and oligo(d T)beads selection sequencing were used for library construction of 3 samples.Whole genome sequencing was performed on NGS platform.Established a SFTS virus genome detection based on next generation sequencing.3.The 14 samples were sequenced by NGS platform.The S,M,and L genomic segments of these SFTS virus strains were phylogenetically analyzed together with those of 40 SFTS virus strains available from Gen Bank.4.According to the SFTS virus Gn-Gc protein gene sequence,3126 bases were cloned to p ET-His.Transformed and expressed by BL21(DE3).5.Measured the Gn-Gc protein by SDS-PAGE and Western-blot.Results1.The 14 patients serum inoculated Vero cells and showed obvious CPE on the 5th day.Virus RNA was detected by Tag Man real-time quantitative RT-PCR and showed positive.2.The oligo(dT)beads selection sequencing has a greater advantage than the random primer sequencing.The depth and coverage of the random primer sequencing were lower than that of the oligo(d T)beads selection sequencing.Three samples were sequenced by oligo(d T)beads selection showed coverage was over 99% and depth was over 900.The average alignment rate of database from NCBI was about 94.5%.3.14 strains of SFTS virus complete genomes were sequenced.phylogenetic analysis showed that the 14 SFTS virus strains clustered into two independent genotypes(D and E).4.Successfully constructed the expression vector p ET-His-Gn-Gc.The recombinant plasmid and fragment were consistent with the sequencing results.5.The recombinant protein Gn-Gc was about 100 k Da.SDS-PAGE and Western-blot results showed the same electrophoresis strip about 100 k Da.ConclusionsEstablished an efficient and specific genome detection method based on next-generation sequencing for SFTS virus.The SFTS virus strains of Zhejiang Province in recent years were sequenced and classified.Constructed the vector for soluble expression of SFTS virus glycoprotein Gn-Gc.Established a stable system of expression and purification.