Repeated Bottleneck Transfers Can Lead to Non-cytocidal Forms of a Cytopathic Virus: Implications for Viral Extinction

Several biological subclones of a biological clone of foot-and-mouth disease virus (FMDV) have been subjected to many plaque-to-plaque (serial bottleneck) transfers in cell culture. At transfer 190 to 409, clones underwent a transition towards a non-cytolytic (NC) phenotype in which the virus was unable to produce plaques, representing at least a 140-fold reduction in specific infectivity relative to the parental biological clone. NC clones, however, were competent in RNA replication and established a persistent infection in cell culture without an intervening cytolytic phase. In one clone, the transition to the NC phenotype was associated with the elongation of an internal oligodenylate tract that precedes the second functional AUG translation initiation codon. The pattern of mutations and their distribution along the FMDV genome of the clones subjected to serial bottleneck transfers were compared with the pattern of mutations in FMDV clones subjected to large population passages. Both the corrected ratios of non-synonymous to synonymous mutations and some specific mutations in coding and non-coding regions suggest participation of positive selection during large population passages and not during bottleneck transfers. Some mutations in the clones that attained the NC phenotype were located in genomic regions affecting the capacity of FMDV to kill BHK-21 cells. The resistance to extinction of clones subjected to plaque-to-plaque transfers marks a striking contrast with regard to the ease of extinction mediated by lethal mutagenesis. The results document a major phenotypic transition of a virus as a result of serial bottleneck events.