Data_Sheet_1_The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.zip

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

神经坏死病毒（Nervous necrosis virus, NNV）是一种嗜神经性病毒，可感染包括欧洲鲈（*Dicentrarchus labrax*）在内的多种鱼类，引发病毒性神经坏死病。NNV拥有双节段正链单股RNA（(+) ssRNA）基因组，分为编码RNA聚合酶的RNA1以及编码衣壳蛋白的RNA2。欧洲鲈中流行最广的NNV种类为红点带石斑鱼神经坏死病毒（red-spotted grouper nervous necrosis virus, RGNNV），该病毒会导致鱼苗和幼鱼出现高死亡率。反向遗传学（reverse genetics）研究已证实，RGNNV衣壳蛋白的270位氨基酸与该病毒在欧洲鲈体内的毒力相关。NNV感染会产生可适应多种选择压力（如宿主免疫应答或宿主物种切换）的准种（quasispecies）与重配体。
为更深入地解析RGNNV种群的变异特性及其与毒力的关联，研究人员以两种RGNNV重组病毒感染欧洲鲈标本：一种是对欧洲鲈具有高毒力的野生型毒株rDl956，另一种为单突变毒株Mut270Dl965，其对该宿主的毒力较弱。研究团队通过逆转录实时定量PCR（RT-qPCR）对脑组织中的两个病毒基因组节段进行定量，并通过下一代测序（Next Generation Sequencing, NGS）分析全基因组准种的遗传变异特性。结果显示，感染低毒力毒株的鱼类脑组织中，RNA1与RNA2的拷贝数较感染高毒力毒株的样本低1000倍。此外，两组实验对象在Ts/Tv比值、重组频率以及RNA2节段突变谱的遗传异质性方面均存在显著差异。上述结果表明，双节段RNA病毒的完整准种可因其中一个节段的共有序列发生单个点突变而发生改变。
金头鲷（*Sparus aurata*）是RGNNV的无症状携带者，因此rDl965在该物种中被视为低毒力分离株。为评估rDl965的准种特性是否在另一种易感性不同的宿主中保持保守，研究人员用rDl965感染幼年金头鲷，并按照前述方法开展分析。值得注意的是，金头鲷体内rDl965的病毒载量与遗传变异特性均与欧洲鲈体内的Mut270Dl965相似。这一结果提示，RGNNV突变谱的遗传变异与进化可能与其毒力密切相关。