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
Abstract
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.
Subject
MHV-1lung pathogenesis, SARS-CoV
mouse hepatitis virus strain 1
reverse genetic
whole genome
Citation
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 https : / /hdl .handle .net /1969 .1 /152630.