Generation and Genetic Characterization of Avian Reovirus Temperature-Sensitive Mutants

Abstract

There currently is little known about the genetic and biological functions of avian reovirus (ARV), an atypical member of the family Reoviridae and the prototype of all nonenveloped viruses that induce syncytia formation. In this study, we created ARV temperature-sensitive (ts) mutants by chemical mutagenesis of ARV strain 138. We developed a novel efficiency of lysis (EOL) screening technique and used it and the classical efficiency of plating (EOP) assay to identify 17 ARV ts mutants. Pairwise mixed infections of these mutants and evaluation of recombinant progeny ts status led to their organization into seven recombination groups. This indicates that these new groups of mutants represent the majority of the ARV genome. To phenotypically characterize the ts mutants, progeny double-stranded RNA (dsRNA) produced at permissive and nonpermissive temperature was measured. Some mutants were capable of dsRNA synthesis at the restrictive temperature (RNA(+)), which indicates the effects of their ts lesions occur after RNA replication. Most mutants were RNA(-), which suggests their mutations affect stages in viral replication that precede progeny genome synthesis.

Avian Reovirus Temperature-Sensitive Mutant tsA12 Has a Lesion in Major Core Protein σA and Is Defective in Assembly

“…Quail QM5 fibrosarcoma cells were maintained in medium 199 supplemented with 10% fetal calf serum, 10% tryptose phosphate broth, and 2 mM glutamine. Viral infections were carried out on QM5 cell monolayers and incubated in the above medium supplemented with 100 U of penicillin/ml, 100 g of streptomycin sulfate/ml, and 1 g of amphotericin B/ml, as described previously, and viral titers were determined by a plaque assay (33).…”

“…At various times postinfection, cells were harvested and disrupted by freeze-thawing twice and sonication. The yield of infectious progeny virions was determined by a plaque assay on QM5 cells as described previously (33).…”

“…Reassortants were generated essentially as described previously (33). Briefly, subconfluent QM5 monolayers were coinfected with the mutant clone tsA12 and wild-type ARV176 at various MOI ratios (5/5, 2/8, and 8/2 PFU per cell).…”

“…The ARV efficiency of plating (EOP) assay was described previously (33). Briefly, QM5 cell monolayers in six-well tissue culture plates were infected with serial 10-fold dilutions of each viral stock in duplicate and then incubated at 33.5°C (permissive temperature) and 39.5°C (nonpermissive temperature).…”

“…Recently, members of our laboratory chemically generated a set of 17 ARV ts mutants by the mutagenesis of ARV138. We assigned 11 of these mutants to 7 of the 10 expected recombination groups, named A through G, and began to characterize them (33). Recombination group A contains two mutant clones (tsA12 and tsA146).…”