蓝舌病病毒基因节段2与6的重配导致病毒血清型与增殖特性的改变

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

摘要:

【背景】蓝舌病病毒(Bluetongue Virus，BTV)是一种侵染反刍动物的虫媒病毒，基因重配可引起病毒的快速变异。【目的】通过我国强致病性BTV-16型毒株与弱致病性BTV-4型毒株间Seg-2与Seg-6基因节段的重配，探讨病毒基因重配与表型变异之间的关系。【方法】采用全长cDNA扩增与高通量测序获取BTV-16/V158的全基因组序列，构建病毒的真核表达质粒，通过免疫荧光与Western blot检测目的蛋白表达；通过RT-PCR、体外转录与细胞转染等方法建立BTV反向遗传体系并获取基因重配病毒；通过蚀斑分析、增殖曲线分析与血清中和试验，比较亲本毒株与基因重配病毒在生物学特性上的差异。【结果】获取的BTV-16/V158毒株基因组大小为19 186 bp，与中国和印度BTV-16型毒株具有最近的亲缘关系；将表达BTV VP1、VP3与NS2的真核表达质粒转染细胞，检测到目的蛋白的表达；将BTV的7种真核表达质粒与基因组ssRNA共转染BHK-21细胞，成功拯救出与亲本毒株生物学特性一致的病毒；将BTV-16/V158毒株的Seg-2与Seg-6替换为BTV-4/YTS4毒株的对应基因节段，拯救出基因重配病毒BTV-16/V158-RG (BTV-4/S2，S6)；与亲本病毒相比较，基因重配病毒在BHK-21细胞上形成的蚀斑变小，增殖能力减弱，血清型由BTV-16型转化为BTV-4型。【结论】建立了我国流行BTV-16型毒株的反向遗传体系，BTV Seg-2与Seg-6的基因重配可引起病毒在细胞上增殖能力的改变与血清型改变。研究结果为BTV基因重配致病毒变异与新型基因工程疫苗的研究提供了基础。

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.