Sevilla N, Domingo E
Centro de Biología Molecular "Severo Ochoa", Universidad Autonoma de Madrid, Spain.
J Virol. 1996 Oct;70(10):6617-24. doi: 10.1128/JVI.70.10.6617-6624.1996.
Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.
口蹄疫病毒（FMDV）在细胞培养中显示出细胞溶解或建立持续感染的双重潜力。FMDV R100是在持续感染FMDV克隆C-S8c1的载体BHK-21细胞传代100次后拯救出的病毒，相对于亲本克隆C-S8c1，它表现出多种遗传和表型改变。几个FMDV R100群体在BHK-21细胞中经历了100次连续的细胞溶解感染，并分析了表型和遗传改变的回复情况。R100的极端温度敏感性在某些传代系列中完全或部分回复，但在其他传代系列中未回复。小斑块形态在所有情况下都回复到正常大小。对BHK-21细胞的高毒力在细胞溶解传代后未回复，甚至有所增加。在持续感染期间在R100基因组中固定的大多数突变在细胞溶解传代过程中未回复，但R100的延伸聚核糖胞苷酸序列（约460个残基，而C-S8c1中为۲۹۰个）长度显著缩短，在所检查的所有传代系列中降至۲۲۰至۲۶۰个残基的范围。在涉及非常大病毒群体的传代中，衣壳蛋白VP1的G-H环内高度保守的Arg-Gly-Asp（RGD基序；位置141至143）旁边有两个氨基酸取代（L-144→V和A-145→P）的变体占主导地位。克隆分析允许分离出具有单一取代A-145→P的突变体。病毒产生和生长竞争实验表明这两个变体的适应性与亲本病毒非常接近。结果提供了证据，表明在病毒准种中可能成为优势的变体库可能受到进化病毒群体大小的影响。一系列持续感染传代后接着一系列细胞溶解传代的最终结果是尽管表型特征回复或停滞，但基因组仍逐渐多样化。讨论了对RNA病毒进化的影响。
