Potential Genetic Contributions to a Pneumopathogenic MHV-1 Virus: Analysis by Targeted Recombination and Whole Genome Sequencing in the MHV-1 Model of SARS-COV Lung Disease
MetadataShow full item record
A targeted recombination system was developed for MHV-1 by generation of a Donor plasmid that consisted of sequences consisting of the first 448 nucleotides of the MHV-1 genome fused to sequences from codon 28 in the HE gene through the 3’UTR to the poly(A) tail. The Donor plasmid was transcribed in vitro and transfected into FCWF cells that had been infected with a felinized MHV-1 recombinant virus. Recombinant viruses were selected by overlaying infected/transfected FCWF cells onto murine DBT cells and monitoring for syncytia formation and cell death. Recombinant viruses were plaque purified and expanded in murine cells. Several recombinant viruses that were not significantly different from MHV-1 in tissue culture were isolated, but none were pneumopathogenic in the A/J mouse. During the generation of multiple wild type MHV-1 stocks for mouse studies we discovered that MHV-1 rapidly lost pnuemopathogenicity during passage in DBT cells. This finding demonstrated that targeted recombination may not be a viable method of genetic manipulation of MHV-1 because the multiple passages in cell culture required to generate viruses by targeted recombination may cause loss of virulence. Using Next-Generation sequencing technology a virulent and non-virulent MHV-1 passage were sequenced, and two potential mutations that are present in the subpopulation of virulent viruses were identified that may contribute to pneumopathogenicity: nsp13 C17015A and ns4 G28454A. We are developing an infectious cDNA clone using in vitro assembly of MHV-1 cDNA fragments. The fragments are flanked by type II restriction enzymes which, when digested liberate cDNA fragments that only contain MHV-1 genetic sequence and can be ligated together. By housing portions of the MHV-1 genome in low-copy plasmids we were able to create a system that is easily maintained in bacteria and easily manipulated by restriction digestion or site-directed mutagenesis. This infectious clone will be used to determine if the mutations that were discovered during the sequencing of the MHV-1 pneumovirulent virus are sufficient and able to generate a pnueumopathogenic virus. The infectious clone will also be used to determine the role, if any, of ns2 in an MHV-1 infection of lungs.
lung pathogenesis, SARS-CoV
mouse hepatitis virus strain 1
McGruder, Brenna Mariechen (2014). Potential Genetic Contributions to a Pneumopathogenic MHV-1 Virus: Analysis by Targeted Recombination and Whole Genome Sequencing in the MHV-1 Model of SARS-COV Lung Disease. Doctoral dissertation, Texas A & M University. Available electronically from