Recombinant mouse hepatitis viruses (MHV) differing only in the spike gene, containing A59, MHV-4, and MHV-2 spike genes in the background of the A59 genome, were compared for their ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice infected with 500 PFU of each virus by intrahepatic injection. Penn98-1, expressing the MHV-2 spike gene, replicated to high titer in the liver, similar to MHV-2, and induced severe hepatitis with extensive hepatocellular necrosis. S A59 R13, expressing the A59 spike gene, replicated to a somewhat lower titer and induced moderate to severe hepatitis with zonal necrosis, similar to MHV-A59. S 4 R21, expressing the MHV-4 spike gene, replicated to a minimal extent and induced few if any pathological changes, similar to MHV-4. Thus, the extent of replication and the degree of hepatitis in the liver induced by these recombinant viruses were determined largely by the spike protein.Various strains of mouse hepatitis virus (MHV) induce different patterns of pathogenesis, including enteritis, hepatitis, encephalitis, and demyelination in the mouse (20,21). We are considering three strains here, MHV-A59, MHV-2, and MHV-4 (an isolate of MHV-JHM). The MHV-A59 strain is dualtropic, producing moderate to severe hepatitis as well as mild to moderate acute meningoencephalitis and chronic demyelination in C57BL/6 weanling mice (29, 30). The MHV-4 strain causes severe acute encephalitis, chronic demyelination, and only minimal levels of hepatitis (6, 23). The MHV-2 strain causes severe hepatitis and meningitis but is unable to cause encephalitis (7,20,42). There are previous studies demonstrating a relationship between attenuation of neurovirulence (6, 10, 13, 42) or hepatitis (14, 28) and the presence of mutations and variations in the spike (S) gene. The S protein, found on the virion envelope and on the plasma membrane of infected cells, is responsible for attachment to viral receptor and viruscell fusion during viral entry and for cell-to-cell fusion later during infection. S is a 180-kDa glycoprotein, which (in the case of most MHV spike proteins) is cleaved into two noncovalently associated 90-kDa subunits, the amino-terminal S1 and carboxy-terminal S2 subunits (14,33). It is speculated that the S1 subunit forms the globular head of the spike and the S2 subunit forms the membrane-bound stalk (8). Recently, a receptor-binding activity has been demonstrated using a recombinant protein containing the amino-terminal 330 residues of the S1 subunit of MHV-JHM (25, 41). S2 is believed to contain the domain that mediates fusion of viral and cell membranes (5, 8). The MHV-2 spike, while highly homologous in sequence to the spike proteins of other MHV strains, remains uncleaved and does not mediate fusion (44,45).Using targeted recombination technology (11,12,35), we have directly demonstrated that the spike protein is a major determinant of the neuropathogenic properties of MHV (39). When the S gene of MHV-4 was introduced into the background of MHV-A59, the resulting recombi...

Recombinant murine coronaviruses, differing only in the spike gene and containing the strain A59 (moderately hepatotropic) and JHM (neurotropic) spike genes in the background of the JHM genome, were compared for the ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice. Interestingly, expression of the A59 spike glycoprotein within the background of the neurotropic JHM strain does not reproduce the A59 hepatotropic phenotype. Thus, the JHM genetic background plays a dominant role over the spike in the determination of hepatotropism.The murine coronavirus mouse hepatitis virus (MHV) has a single-stranded, positive-sense RNA genome of approximately 31 kb (35). MHV infection of the central nervous system (CNS) has been used as a model for the study of chronic demyelinating diseases (3,6,9,12,19,23,26,32). Some MHV strains cause a wide range of liver injuries that range from minimal changes to fulminant hepatitis (5,11,13,20). The development of targeted RNA recombination (14,24,25,38) has allowed the generation of chimeric recombinant viruses differing only in the spike gene (22). Targeted RNA recombination was developed by using the MHV-A59 strain (24, 25), and thus, until recently, only the A59 genetic background has been used for generation of recombinant viruses. Using chimeric A59 recombinant viruses, we have previously demonstrated that the spike protein of MHV is a major determinant of neurovirulence (33, 34), demyelination (4), and hepatotropism (27). We further demonstrated that the ability to induce hepatitis is largely determined by the spike gene when the rest of the viral genes are derived from A59 (27). We compared three isogenic recombinant viruses differing only in the spike gene and expressing the spike protein of A59 (RA59; formerly named S A59 R13), MHV-2 (Penn 98-1), or MHV-JHM (SJHM-RA59; formerly named S 4 R21), all in the background of the A59 genome. We found that after intrahepatic inoculation with 500 PFU per mouse, the spike gene determined the viral load in the liver and that the amounts of antigen staining and necrosis in the liver correlated with the viral load (27). Our results were not surprising, since the spike glycoprotein is responsible for viral receptor attachment, entry, and cell-to-cell fusion (7,8,39). Thus, the spike would be expected to play a crucial role in initiation of infection, as well as in virus spreading (8). Our goal in this study was to explore the role of the viral genetic background in the development of murine coronavirus-induced hepatitis. Our data demonstrate that the presence of background genes from the neurotropic JHM strain of MHV eliminates A59 spike hepatotropism even after inoculation with 10 6 PFU directly into the liver.We have previously described the targeted recombination technology used to select recombinant viruses (in the A59 background) differing only in the spike gene, including the wild-type A59 recombinant (RA59) and the A59 recombinant virus expressing the JHM spike (SJHM-RA59) (27,33). In this study, we have co...

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