Ortega-Del Campo Sergio, Díaz-Martínez Luis, Moreno Patricia, García-Rosado Esther, Alonso M Carmen, Béjar Julia, Grande-Pérez Ana
Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.
Centro de Supercomputación y Bioinnovación (SCBI), Universidad de Málaga, Málaga, Spain.
Front Microbiol. 2023 Jun 15;14:1182695. doi: 10.3389/fmicb.2023.1182695. eCollection 2023.
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 (). 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 () 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（NNV）是一种嗜神经病毒，可在包括欧洲海鲈在内的多种鱼类中引发病毒性神经坏死病。NNV具有双节段（+）单链RNA基因组，由编码RNA聚合酶的RNA1和编码衣壳蛋白的RNA2组成。海鲈中最常见的NNV毒株是红斑石斑鱼神经坏死病毒（RGNNV），可导致幼鱼和稚鱼的高死亡率。反向遗传学研究表明，RGNNV衣壳蛋白的第270位氨基酸与海鲈中的RGNNV毒力相关。NNV感染会产生准种和重配体，它们能够适应各种选择压力，如宿主免疫反应或宿主物种间的转换。为了更好地理解RGNNV群体的变异性及其与RGNNV毒力的关联，用两种RGNNV重组病毒感染海鲈样本，一种是对海鲈具有高毒力的野生型rDl956，另一种是对该宿主毒力较低的单突变病毒Mut270Dl965。通过RT-qPCR对脑中的两个病毒基因组片段进行定量，并通过下一代测序（NGS）研究全基因组准种的遗传变异性。感染低毒力病毒的鱼脑中RNA1和RNA2的拷贝数比感染高毒力病毒的鱼脑中的拷贝数低1000倍。此外，在RNA2片段的Ts/Tv比值、重组频率和突变谱的遗传异质性方面，发现了两个实验组之间的差异。这些结果表明，双节段RNA病毒的整个准种会因其一个片段的共有序列中的单点突变而发生变化。黑鲷是RGNNV的无症状携带者，因此rDl965在该物种中被认为是低毒力分离株。为了评估rDl965的准种特征在另一种表现出不同易感性的宿主中是否保守，用rDl965感染黑鲷幼鱼，并按上述方法进行分析。有趣的是，rDl965在黑鲷中的病毒载量和遗传变异性与Mut270Dl965在海鲈中的相似。这一结果表明，RGNNV突变谱的遗传变异性和进化可能与其毒力相关。
