Systematic study of the genetic response of a variable virus to the introduction of deleterious mutations in a functional capsid region.

We have targeted the intersubunit interfaces in the capsid of foot-and-mouth disease virus to investigate the genetic response of a variable virus when individual deleterious mutations are systematically introduced along a functionally defined region of its genome. We had previously found that the individual truncation (by mutation to alanine) of 28 of the 42 amino acid side chains per protomer involved in interactions between capsid pentameric subunits severely impaired infectivity. We have now used viral RNAs individually containing each of those 28 deleterious mutations (or a few others) to carry out a total of 96 transfections of susceptible cells, generally followed by passage(s) of the viral progeny in cell culture.

Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines.

We have rationally engineered foot-and-mouth disease virus to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid intersubunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulfide bonds or electrostatic interactions between subunits. Two engineered viruses were normally infectious, genetically stable, and antigenically indistinguishable from the natural virus but showed substantially increased stability against irreversible dissociation.

Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies.

A severe limitation to fully realize the dramatic potential for adaptation of RNA virus quasispecies may occur if mutations in vast regions of the sequence space of virus genomes lead to significant decreases in biological fitness. In this study the detection and selection by heat of thermostable variants from different foot-and-mouth disease virus (FMDV) populations were attempted, in order to explore whether FMDV may generally accept a substantial increase in thermostability without compromising its infectivity. The results obtained with both uncloned and cloned populations of different serotypes, recovered from cytolytic or persistent infections and subjected to either very few passages or extensive passaging in cells, indicate that the presence of thermostable virus variants, even in small proportions, is not a general feature of FMDV quasispecies.