Reverse genetics of rotavirus.

The genetics of viruses are determined by mutations of their nucleic acid. Mutations can occur spontaneously or be produced by physical or chemical means: for example, the application of different temperatures or mutagens (such as hydroxylamine, nitrous acid, or alkylating agents) that alter the nucleic acid. The classic way to study virus mutants is to identify a change in phenotype compared with the wild-type and to correlate this with the mutant genotype (“forward genetics”). Mutants can be studied by complementation, recombination, or reassortment analyses. These approaches, although very useful, are cumbersome and prone to problems by often finding several mutations in a genome that are difficult to correlate with a change in phenotype. With the availability of the nucleotide sequences of most viral genomes and of the tools of genetic engineering, this stratagem has drastically changed. One can now start with rationally engineering particular mutations in individual viral genes, followed by production of infectious viral particles and exploration of the phenotype (“reverse genetics”). Whereas forward genetics investigates the genetics underlying a phenotype, reverse genetics observes the phenotypic changes arising from genetic changes that were “made to order.” Reverse genetics is a relatively straightforward task with DNA viruses because virtually all viral DNA genomes, which can be mutated in vitro, are infectious upon transfection. Reverse genetics of RNA viruses involves the manipulation of their genomes at the cDNA level, followed by procedures to produce live infectious progeny virus (wild-type or mutated) after transfection of cDNAs into cells. To achieve this end, coor superinfection with a helper virus has been used in initial attempts. However, because virus particles carrying the engineered genome may be very difficult to separate from helper virus, the final aim is to create helper virus-free, plasmid-only– or RNA-only–based systems. A tractable, helper virus-free reverse genetics system is a powerful tool, because it allows precise assignment of phenotypic changes to engineered mutations in comparison with the wild-type phenotype and genome. Reverse genetics techniques can help clarify structure/function relationships of viral genes and their protein products and also elucidate complex phenotypes, such as host restriction, pathogenicity, and immunogenicity. Kanai et al. (1) have now developed a reverse genetics system for rotaviruses (RVs), which are a major cause of acute gastroenteritis in infants and young children and in many mammalian and avian species. Worldwide RV-associated disease still leads to the death of over 200,000 children of <5 y of age per annum (2) and thus represents a major public health problem. The work by Kanai et al. (1) is the most recent addition to a long list of plasmid-only–based reverse genetics systems of RNA viruses, a selection of which is presented in Table 1 (3–13). A potent reverse genetics system for RVs (1) represents a long-awaited breakthrough and is a major technological advance over the most-sophisticated helper virus-dependent reverse genetics procedures recently developed for RVs (14, 15). Using a previously validated approach (12), Kanai et al. (1) constructed plasmids, each containing the cDNA of 1 of the 11 RV RNA segments (SA11 strain), inserted between a T7 RNA polymerase (T7Pol) promoter (5′end) and the antigenomic hepatitis δ-virus ribozyme (3′end), from which, upon cotransfection into BHK cells constitutively expressing T7Pol, authentic full-length viral ss(+)RNA transcripts would be synthesized. Although this procedure or a modification using ss(+)RNAs transcribed from cDNA clones in vitro was successful in “rescuing” infectious reovirus (12) or bluetongue virus (13) particles, it did not lead to the recovery of infectious RV progeny (1, 16). Based on previous findings that fusion-associated small transmembrane (FAST) proteins (encoded by Aquareovirus and some Orthoreovirus species) increased the yield of heterologous mammalian orthoreovirus substantially (1, 17), RV replication was found to be significantly increased in infected cells when a reovirus FAST protein was expressed from a transfected plasmid (1). Furthermore, the overexpression of the vaccinia virus-capping enzyme increased translatability of reovirus (+)ssRNAs, and coexpression of both greatly