MHV strains are routinely used as models for various diseases. MHV-1, which has been known for some time to be highly pneumotropic, has been shown to reproduce the
clinical and pathological symptoms seen in humans infected with SARS in A/J mice, providing a convenient animal model that can be investigated without the restrictions necessary to work with the SARS-coronavirus. A reverse genetic cDNA assembly system was developed for the betacoronavirus mouse hepatitis virus strain A59 (MHVA59), in 2002. A similar approach was used to assemble a full-length infectious cDNA of MHV-1. Our system was designed so that it would be compatible with the previouslydeveloped MHV-A59 system in order to allow for the creation of chimeric viruses to
identify virulence factors in MHV-1. Seven cDNA fragments representing the entire MHV-1 genome will be generated by RT-PCR, long-accurate PCR, and cloning. These fragments are designed such that the restriction site recognition sequences are removed during restriction enzyme digestion, which creates unique 4 base overhangs to allow for sequential ligation of the cDNAs. Mutations that will not affect the coding sequence were introduced into these plasmid cDNA overhangs to make them compatible with
MHV-A59 fragments, which had been previously generated by the Baric lab. Once all seven genomic fragments are cloned, this work will allow for the genetic modification of the entire genome and will make it possible to identify and study the genes that have been hypothesized to be responsible for pneumovirulence in MHV-1 and may lead to more insight to the pathogenesis of SARS-CoV
