key: cord-336416-vas0b6dt authors: Wege, H.; Koga, M.; Stephenson, J. R.; Siddell, S.; ter Meulen, V. title: NEUROVIRULENCE AND PERSISTENCY OF MOUSE HEPATITIS VIRUSES IN RATS 1 1 Supported by the Deutsche Forschungsgemeinschaft and Hertie-Stiftung date: 1980-12-31 journal: Animal Virus Genetics DOI: 10.1016/b978-0-12-255850-4.50067-6 sha: doc_id: 336416 cord_uid: vas0b6dt ABSTRACT The murine coronavirus JHM induces in weanling rats different types of central nervous diseases ranging from an acute panencephalitis to a late demyelinating encephalomyelitis. The occurrence and rate of these different disease types is associated with the virus variant used for inoculation. Except for MHV 2, neurovirulence was not observed in four other murine coronavirus strains. The relationship of these coronaviruses to JHM was investigated by cross neutralization and oligonucleotide maps of their genomic RNA. Murine coronaviruses induce in mice a variety of diseases, ranging from acute hepatitis, enteritis and encephalomyelitis to inapparent infections. Of particular interest are the central nervous system (CNS) diseases associated with this virus group, especially the mouse hepatitis virus strain JHM reveals a distinct neurovirulence for mice and rats (1, 2, 3, 4) . In rats different disease processes accompanied by demyelination are observed (5, 6, 7) , providing the opportunity to investigate the virus host relationships which lead to myelin destruction. In the present communication the neurovirulence of four murine coronavirus strains (MHV 1, MHV 2, MHV 3 and MHV A59) for rats is compared to the JHM virus. Neurovirulence of MHV-JHM. Infection of suckling rats with JHM-virus regardless of virus history resulted in an acute encephalomyelitis characterized by necrotic lesions in all parts of the CNS followed by hepatitis and rapid death within eight days after infection. Intracerebral inoculation of weanling rats however, led to three different courses of a central nervous disease (5,6,7) as summarized in table 1. The Acute Panencephalitis (APE) is characterized by a rapid onset within 12 days post infection (p.i.), severe necrosis and acute inflammations in most parts of the brain. The Subacute Demyelinating Encephalomyelitis (SDE) does not occur before 12 days p.i. and shows a more protracted clinical course. Neuropathologically, inflammatory changes are present consisting mainly of mononuclear lymphocytes, plasmacells and macrophages without lesions of necrosis. In contrast to the APE neuronal involvement is minimal or absent. Moreover, a primary demyelination in restricted areas of the brain and spinal cord is noticed which can easily be detected by histological staining procedures. The Late Demyelinating Encephalomyelitis (LDE) develops after an incubation period of 2 -8 months. The main histological lesions of the CNS consist of demyelinating areas present in the spinal cord and cerebrum. Demyelination is accompanied by remyelination of the CNS and PNS type. Inflammatory infiltrations can be detected in the neighborhood of demyelinating plaques. The attempts to isolate infectious virus from diseased brain material were successful in all animals tested, regardless of the different CNS disease process. It is noteworthy, that even in the animals with LDE infectious virus was present in brain, indicating JHM virus persistency during the incubation period. The occurrence and rate of the different types of CNS disease induced by JHM virus is associated with the properties of the virus preparation used as inoculum as summarized in table 2. Uncloned wild type JHM virus induced in weanling rats mainly APE and SDE but only occasionally LDE. This finding suggests that uncloned JHM consists of a heterologous virus population with different biological properties. This is supported by the observation that cloned tissue culture adapted JHM virus always caused in weanling rats an APE, whereas certain temperature sensitive mutants derived from that JHM virus clone after mutagenization with fluoruracil caused SDE or LDE. No acute disease process could be induced with these ts-mutants. Reisolated mutants from diseased animals with LDE did not always maintain their temperature sensitivity but were different from revertants by the type of neurovirulence. Moreover, some characteristics in tissue culture were similar to the ts-mutants originally inoculated. The reisolate ts 42-1027 is an example for this phenomenon. This variant was reisolated from a diseased animal 3 months after infection with ts 42. The reisolate is much less temperature sensitive than the original mutant. It had an efficiency of plating of 1,6 x 10~1 compared to 1,4 x 10~4 for ts 42. In animals, this reisolate caused predominantely a high rate of SDE in contrast to its parental virus, which induced only few cases of LDE. Neurovirulence of other MHV-strains. The virulence of cloned MHV-strains (MHV 1, MHV 2, MHV 3 and MHV A59) was compared to JHM virus after intracerebral inoculation of defined virus doses into weanling rats (Table 3 ) . With the highest virus dose used, only MHV 2 induced one case of an acute encephalomyelitis. No clinical disease was observed in the other animals injected with MHV 1, MHV 3 and MHV A59. Pathological investigations carried out 24 days after virus inoculation revealed only mild inflammations in liver tissue without detectable neuropathological changes. No infectious virus was recovered from CNS tissue 4 -8 days p.i. All the inoculated animals developed a humoral immune response against the different murine coronaviruses except for MHV 1. This virus strain grows in tissue culture lines of murine origin but appears not to be infectious for rats. Cross Neutralization among MHV-strains. Antigenic comparisons among the different cloned murine coronavirus strains were carried out by cross neutralization. The results are summarized in table 4 and indicate that each virus strain is antigenically distinct in the structural protein(s) which induced neutralizing antibodies. However, some cross neutralization among the murine coronaviruses can be noted. MHV JHM and MHV 2 demonstrated a significant bilateral cross reactivity. MHV A59 and MHV 1 cross reacted only in one direction, since anti MHV 1 serum hardly neutralized MHV A59 virus. Relative oligonucleotide homology between strains of murine coronaviruses. The genomic RNA of the cloned MHV strains was labeled by growth in presence of ^2pj 10S p] lorus# «p^ genome from the purified viruses was extracted and further purified as described previously (8, 9, 10) . After RNAse digestion oligonucleotides were analysed in a two dimensional gel electrophoresis (11) . The position of the large oligonucleotides within the fingerprint was compared. The result of this study (manuscript in preparation) is summarized in table 5 and indicates distinct differences between JHM virus and the other murine coronavirus strains. MHV 2, MHV 3 and MHV A59 are more closely related to each other than with MHV JHM, underlining the unique properties of this virus. COMMENTS From the five coronaviruses tested, only JHM revealed significant neurovirulence for weanling rats. The other murine coronaviruses induce beside liver inflammations no CNS lesion in general even after intracerebral inoculation. JHM virus infection resulted in three different CNS processes depending on the property of the inoculated virus preparation. This association provides a basis to investigate the mechanisms by which the different pattern of diseases are induced. It is conceivable that biochemical analysis of different mutants might unravel a specific marker which contributes to neurovirulence. The observation that JHM virus persists in weanling rats for months before LDE is recognized suggests that specific events, either host or virus derived, prevent a rapid spread of virus infection. The temperature sensitivity of clones provides only a marker for a genetic lesion, because this lesion might be independent from the regions influencing the type of neurotropism which develops after inoculation in rats. This is suggested by the reisolation of mutants with reduced temperature sensitivity which nevertheless retain a specific neurovirulence. At present the selective neurotropism of different JHM mutants cannot be directly correlated to biological or biochemical properties of this virus. However, the preliminary data obtained from the comparison of oligonucleotide patterns indicates that JHM contains unique RNA sequences which are not found among the other murine coronaviruses. Further studies will define the genetic basis which contributes to neurovirulence, virus persistency and virus host interactions which lead to a demyelinating CNS disease. Proc.Natl. Acad.Sci. USA We thank Hanna Wege and Margarete Sturm for excellent technical assistance and Helga Schneider for typing the manuscript.