Replicase genes of murine coronavirus strains A59 and JHM are interchangeable: differences in pathogenesis map to the 3' one-third of the genome

Abstract

The important roles of the spike protein and other structural proteins in murine coronavirus (MHV) pathogenesis have been demonstrated; however, the role of the replicase gene remains unexplored. We assessed the influence of the replicase genes of the highly neurovirulent MHV-JHM strain and the hepatotropic and mildly neurovirulent A59 strain in acute infection of the mouse. Analysis of chimeric A59/JHM recombinant viruses indicates that the replicase genes are interchangeable and that it is the 3' end of the genome, encoding the structural proteins, rather than the replicase gene, that determines the pathogenic properties of these chimeras.

FIG. 1.

Scheme of targeted RNA recombination. Feline cells (FCWF) were infected with fMHV-A59, a chimeric recombinant MHV-A59 virus expressing the feline infectious peritonitis virus (FIPV) spike (A), or with fMHV-JHM B3b, a chimeric recombinant MHV-JHM virus expressing the FIPV spike (B), and then electroporated with pJHM-derived (A) or pMH54-derived (B) in vitro-transcribed RNA to generate recombinant JHM viruses expressing the replicase gene of A59 (repA59-RJHM) (A) and A59 viruses expressing the replicase gene of JHM (repJHM-RA59) (B). Infected and electroporated FCWF cells were overlaid onto murine L2 cells, and recombinant viruses were selected for their ability to infect murine cells as previously described (15). Four independent chimeric viruses and two independent wild-type recombinant viruses per construct were generated and characterized in vitro and in vivo. The positions of open reading frames (ORF1, ORF2a, ORF4, and ORF5a), genes, plasmids, and 3′ untranslated region (3′UTR) are indicated.

FIG. 2.

Viral growth kinetics of recombinant viruses in murine L2 cells. Released (A) and cell-associated virus (B) of RA59 (•), RJHM (▪), repJHM-RA59 (○), and repA59-RJHM (□). No significant differences were observed between RA59 and repJHM-RA59 viruses, demonstrating that a chimeric recombinant A59 virus expressing the replicase gene of JHM replicates similarly to the wild-type recombinant A59. In contrast, a chimeric recombinant JHM virus expressing the A59 replicase gene (repA59-RJHM) replicated to lower levels than RJHM did. At the indicated times, virus titers were determined in cells and culture supernatants by plaque assay in L2 cells. The data shown represent the mean titers of triplicate samples. Four independent chimeric recombinant viruses were analyzed. Data from one independent recombinant virus per construct are shown.

FIG. 3.

Viral load in the livers of C57BL/6 mice at 1, 3, 5, and 7 days p.i. after intrahepatic inoculation with 500 PFU (A) and 10⁵ PFU (B) of recombinant wild-type viruses RA59 and RJHM and chimeric recombinants repJHM-RA59 and repA59-RJHM. Viral titers were determined by plaque assay and presented as log₁₀ PFU/gram of liver. Error bars represent logarithmic standard deviations (SDs). The limit of detection was 200 PFU/g of tissue. RA59 and repJHM-RA59 replicated to higher viral titers than RJHM and repA59-RJHM did (P < 0.05). No significant differences in viral titers were observed among viruses differing only in the replicase gene (RA59 versus repJHM-RA59 and RJHM versus repA59-RJHM). Data represent the averages plus SDs of four independent chimeric and two wild-type recombinant viruses. Ten to 20 mice were evaluated for each point.

FIG. 4.

Viral load in the brains (A) and livers (B) of C57BL/6 mice at 1, 3, 5, and 7 days p.i. after intracranial inoculation. Mice were inoculated intracranially with LD₅₀s of RA59, RHJM, repJHM-RA59, and repA59-RJHM recombinant viruses. No significant differences in viral titers were observed for viruses in the brain. In the liver, RA59 and repJHM-RA59 replicated to higher titers than RJHM and repA59-RJHM did (P < 0.05). Data represent the averages plus standard deviations (error bars) of four independent chimeric and two wild-type recombinant viruses. Ten to 20 mice were evaluated for each point.