VIROLOGY 175,418-426 (1990) Identification of the Spinal Cord as a Major Site of Persistence during Chronic Infection with a Murine Coronavirus STANLEY PERLMAN,*+ GARY JACOBSEN,* ANN LOUISE OLSON,* AND ADEL AFIFI*+j Depanments of *Pediatrics, tMicrobiology, *Anatomy, and Neurology, University of Iowa, Iowa City, Iowa 52242 Received September 2 1, 1989; accepted December 12, 1989 After intranasal inoculation, mouse hepatitis virus (MHV) gains entry into the central nervous system (CNS) via the olfactory and trigeminal nerves. Under the appropriate conditions, some mice develop clinically apparent demyelinating encephalomyelitis several weeks later, with virus always present in the spinal cord. To determine the pathway by which virus reaches the cord, brains and spinal cords of infected, asymptomatic mice were analyzed by in situ hybridization. Viral RNA was always detected in the anterior part of the upper spinal cord. A similar analysis of mice with the recent onset of hindlimb weakness showed that viral RNA was detected in the same location. The results suggest that MHV is transported to the spinal cord via well-defined neuroanatomic pathways and that viral amplification with resultant clinical disease occurs from this site of persistence in the anterior spinal cord. This process of viral amplification may involve the generation of viral variants as has been described for MHV-infected rats. No major changes in viral RNA or protein could be detected when MHV isolated from mice with hindlimb paralysis was analyzed. The data suggest that the generation of viral variants is not important in the pathogenesis of the late onset neurological disease induced by MHG in mice. 0 1990Academic Press, inc. INTRODUCTION Mouse hepatitis virus (MHV), a single-stranded posi- tive polarity RNA virus, causes many diseases in sus- ceptible rodents, including gastroenteritis, hepatitis, and a variety of neurological diseases (Siddell et al., 1983). The JHM strain of MHV (MHV-JHM) is neuro- tropic and causes acute encephalitis and acute and subacute demyelinating diseases in mice and rats (Cheever et a/., 1949; Weiner, 1973; Lampert et a/., 1973; Nagashima et a/., 1978; Sorenson et al., 1980; Stohlman and Weiner, 1981). Suckling mice inoculated intranasally or intracerebrally develop an invariably fatal acute encephalitis; this fatal outcome can be pre- vented if mice are inoculated with attenuated virus, if they receive an infusion of protective antibody, or if they are nursed by immunized dams (Buchmeier et al., 1984; Pickel et a/., 1985; Dalziel et al., 1986; Fleming et a/., 1986, 1989; Perlman et al., 1987). Although suckling C57BU6 mice inoculated intrana- sally and nursed by immunized dams are protected from the acute fatal disease, some of them (40-90%) later develop a demyelinating encephalomyelitis char- acterized clinically by hindlimb paralysis. Previously, we have described the spread of MHV-JHM through the central nervous system (CNS) of suckling mice nursed by unimmunized and immunized dams (Perl- man et a/., 1988, 1989). After intranasal inoculation, To whom requests for reprints should be addressed. virus enters the brain via the olfactory and trigeminal nerves and spreads along known neuroanatomic path- ways. In mice dying from the acute encephalitis, virus is localized in many parts of the brain whereas it re- mains confined to the sites of entry and their associ- ated structures in asymptomatic mice protected by maternal antibody. The exact localization of MHV-JHM in asymptomatic mice at later times p.i. was not deter- mined in these experiments. In mice which subse- quently develop hindlimb paralysis, virus is always present in the spinal cord and less consistently can be detected in specific locations in the brain which are neuroanatomic connections of the initial sites of viral entry. Thus virus can be detected in distant connec- tions of the olfactory nerve such as the hippocampus and mammillary nuclei and in those of the trigeminal nerve such as the caudal thalamus and the cerebral cortex (Perlman et a/., 1988, 1989). Virus can easily be isolated from the brains and spi- nal cords of mice with hindlimb paralysis (Perlman et a/., 1987). Characterization of these mouse isolates has not been published, but MHV-JHM isolated from rats with a similar neurological disease (Nagashima et a/., 1978; Sorensen et al., 1980) causes an attenuated disease on subsequent inoculation into susceptible rats and contains a shortened spike (S) glycoprotein and mRNA (Morris et a/., 1989). In other studies, a longer glycoprotein has been associated with in- creased neurovirulence in young rats (Taguchi et al., 1985). 0042.6822/90 $3.00 CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 418 MHV PERSISTENCE IN THE SPINAL CORD 419 In this report, we describe the location of viral RNA in the CNS of asymptomatic mice at later times p.i. and compare this to the distribution of vir