Gene reassortment of segment 2 and 6 altered serotype and proliferation of bluetongue virus

Abstract:

[Background] Bluetongue virus (BTV) belongs to arbovirus that infects ruminants and varies frequently due to gene reassortment. [Objective] To explore the correlation of gene reassortment and phenotypic variation of BTV caused by reassortment of the Seg-2 and Seg-6 between virulent BTV-16 strain and the weakly pathogenic BTV-4 strain. [Methods] The full genome sequence of BTV-16/V158 was obtained by full-length cDNA amplification (FLAC) and next generation sequencing (NGS). Eukaryotic expression plasmids of BTV were constructed and their expressed proteins were verified through immunofluorescence assay (IFA) and Western Blot (WB). Reverse genetic system of BTV was established through RT-PCR, in vitro transcription and cell transfection, and reassortant BTVs were rescued using the established system. The differences in biological characteristics between parental and reassortment BTV were compared through viral plaque formation assay, proliferation curve analysis, and serum neutralization test. [Results] The genome of BTV-16/V158 strain was 19 186 bp in length, which shared the closest relationship with the Chinese and Indian BTV-16 strains. The expressions of target proteins were confirmed by IFA and WB in the cells which were transfected with eukaryotic plasmids expressed VP1, VP3 and NS2 of BTV. BTV strain, named as BTV-16/V158-RG, was successfully recovered by transfecting BHK-21 cells with BTV eukaryotic expression plasmids and genomic ssRNA, which showed consistent biological characteristics with its parental virus BTV-16/V158. Furthermore, reassortment virus, named as BTV-16/V158-RG (BTV-4/S2, S6), which carried the Seg-2 and Seg-6 genes of BTV-4 strain in a backbone of BTV-16/V158 was successfully rescued. Compared with its parent virus, BTV-16/V158-RG (BTV-4/S2, S6) showed smaller viral plaques and weak proliferation on BHK-21 cells, with its serotype converted from BTV-16 to BTV-4. [Conclusion] the reverse genetic system of Chinese BTV-16 strain was successfully established. Reassortment of the Seg-2 and Seg-6 altered the proliferation and serotype of the viruses.

Key words:bluetongue virus, reverse genetics, reassortment, VP2 and VP5 gene, biological characteristics