Strategy for systematic assembly of large RNA and DNA genomes: Transmissible gastroenteritis virus model

A systematic method was developed to assemble functional full-length genome s of large RNA and DNA viruses. Coronaviruses contain the largest single-st randed positive-polarity RNA genome in nature. The similar to 30 kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an imp ortant pathogen in swine. Using a novel approach, six adjoining cDNA subclo nes that span the entire TGEV genome were isolated. Each clone was engineer ed with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of similar to 28.5 kb in length. Transcripts de rived from the full-length TGEV construct were infectious, and progeny viri ons were serially passaged in permissive host cells. Viral antigen producti on and subgenomic mRNA synthesis were evident during infection and througho ut passage. Plaque-purified virus derived from the infectious construct rep licated efficiently and displayed similar plaque morphology in permissive h ost cells. Host range phenotypes of the molecularly cloned and wild-type vi ruses were similar in cells of swine and feline origin. The recombinant vir uses were sequenced across the unique interconnecting junctions, conclusive ly demonstrating the marker mutations and restriction sites that were engin eered into the component clones. Pull-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of T GEV could theoretically be used to precisely reconstruct microbial or eukar yotic genomes approaching several million base pairs in length.