A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli.

Infectious clone methodology is a valuable tool of modern experimental virology. However, its use is often constrained by the instability of infectious clone constructs during propagation in E. coli. To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general. An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E. coli by insertion of short introns. To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture. The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines.