VIROLOGY 170,556-560 (1969) SHORT COMMUNICATIONS Spread of a Neurotropic Murine Coronavirus into the CNS via the Trigeminal and Olfactory Nerves STANLEY PERLMAN,*+ GARY JACOBSEN,* AND ADEL AFIFI*+ Departments of Pediatrics, tMicrobiology, *Anatomy, and Neurology, University of Iowa, Iowa City, Iowa 52242 Received January 17, 1989; accepted March 2. 1989 The route of entry into the central nervous system (CNS) of most neurotropic viruses has not been established. The coronavirus, mouse hepatitis virus strain JHM (MHV-JHM). causes acute encephalomyelitis and acute and chronic demyelinating diseases and is an important model system for virus-induced neurological disease. Suckling C57BU6 mice infected intranasally with MHV-JHM develop either the acute encephalomyelitis or a late onset, symptomatic demyelinating encephalomyelitis, depending on whether they are nursed by unimmunized or immunized dams. Analysis by in situ hybridization was used to determine the route of entry of MHV-JHM into the CNS in these mice. At early times, viral RNA was detected only in the trigeminal and olfactory nerves and in their immediate connections in all mice. A few days later, MHV-JHM RNA was found throughout the brain in mice dying of the acute encephalomyelitis, but remained confined to the entry sites in mice which did not develop acute disease. These results suggest that MHV- JHM enters the CNS via an interneuronal route in all mice, but that the presence of maternal antibody prevents the dissemination of virus via extracellular fluid. In addition, MHV-JHM may establish low-level persistence in the trigeminal or olfactory nerve or in one of its connections in mice that do not develop acute encephalomyelitis. o 18s Academic Press. Inc. Coronaviruses, positive-stranded RNA viruses, are able to infect persistently many types of animals (1, 2). Neurotropic strains of the murine coronavirus, mouse hepatitis virus (MHV), cause acute encephalomyelitis and acute and chronic demyelinating diseases in sus- ceptible rodents (2-70). For this reason, mice or rats infected with these viruses are useful for determining the factors important for both acute and persistent CNS infections caused by RNA viruses. One such fac- tor is the relationship between the initial site of viral en- try and the sites of preferential replication in animals that become clinically ill either acutely or after a latent period of several weeks. Young mice develop an invariably fatal acute en- cephalomyelitis if inoculated with the appropriate dose of MHV-JHM. Acute disease can be prevented by the use of mutant virus (either temperature sensitive or neutralization resistant), by administration of neutraliz- ing monoclonal antibody prior to inoculation, or by suckling by immunized dams (1 I- 76). Suckling C57BU6 mice infected intranasally with the JHM strain of MHV and nursed by immunized dams re- main asymptomatic for several weeks post inoculation (p.i.), although 40-90% eventually develop a neurologi- cal disease characterized by hindlimb paralysis. Signs of encephalitis (hunching, ruffled fur, irritability) are not present at this time. MHV-JHM RNA can be detected To whom requests for reprints should be addressed. in the brainstem and spinal cord of all mice with the late onset disease (16-l 7). In this report, we used the method of in situ hybrid- ization to localize virus-specific RNA in the CNS at early times after intranasal infection. At a few days p.i., MHV- JHM RNA was detected in the trigeminal nerve and some of its associated brainstem nuclei or tracts and in the olfactory system in all mice, whether nursed by immunized or unimmunized dams. Suckling C57BU6 mice inoculated intranasally with 6 X lo4 PFU MHV-JHM and nursed by unimmunized dams develop encephalitic symptoms at approximately 4 days p.i. and die by 5 days p.i. Few histological changes are present at 3 days p.i., but over the next 2 days, extensive perivascular, parenchymal, and lepto- meningeal inflammatory cell infiltrates and widespread areas of necrosis become apparent (16). The brains from acutely infected mice were removed at 3, 4. and 5 days p.i. and analyzed for the presence of MHV-JHM RNA by in situ hybridization (17, 78) using a 35S-labeled antisense RNA probe synthesized as de- scribed previously (17, 19). This probe was comple- mentary to all of MHV genes 5 and 6 and portions of genes 4 and 7 (17,20). No annealing occurs with brains or spinal cords from uninfected mice using this method of in situ hybridization (77). At 3 days p.i., MHV-JHM RNA was invariably present in the olfactory bulb (Fig. 1 B). After an additional 24 hr, virus-specific RNA could also be detected in more distal portions of the olfactory 0042-6822/89 $3.00 CopyrIght 0 1999 by Academic Press. Inc. All rights of reproductton in any form reserved. 556 SHORT COMMUNICATIONS 557 FIG. 1. Temporal appearance of MHV-JHM RNA in mice with acute encephalomyelitis. The offspring of unimmunized C57BU6 dams (Jac