The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV-JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV-JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.

The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM ("receptor-independent spread"). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.Mouse hepatitis virus (MHV) strain JHM is an enveloped, nonsegmented positive-strand RNA virus belonging to the Coronaviridae family within the Nidovirales order (7). JHM infection of experimental animals has been extensively used to study virus pathogenesis in the central nervous system (CNS). Susceptible mice develop an acute, fatal viral encephalomyelitis after intranasal or intracranial inoculation, with clinical signs that include hunching, ruffled fur, irritability, and lethargy. Viral antigen is detected in gray-and white-matter structures throughout the brain. Rodents chronically infected with MHV develop demyelinating diseases with many similarities to multiple sclerosis in humans (21,55,56). Chronic infection results if animals are infected with an attenuated virus or with a virulent virus in the presence of anti-JHM antibodies or T cells (5,13,26,31,39,48,57,69). In one model of chronic infection, suckling mice were infected with JHM and nursed by dams previously immunized against the virus (48). Under these conditions, sufficient antibody was transmitted to suckling mice to prevent acute encephalitis but not to mediate virus clearance. Consequently, 40 to 90% of mice developed hindlimb paralysis and histological evidence of a demyelinating encephalomyelitis at 3 to 8 weeks postinfection (p.i.) (48). Since the inception of this maternal antibody model, we have used a specific strain of JHM, JHM.Iowa (JHM.IA), for all of our experiments.

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