key: cord-0010227-ea3gwnw7 authors: Hirano, Norio; Goto, Naoaki; Ogawa, Tetsuo; Ono, Katsuhiko; Murakami, Toshiaki; Fujiwara, Kosaku title: Hydrocephalus in Suckling Rats Infected Intracerebrally with Mouse Hepatitis Virus, MHV‐A59 date: 2013-11-14 journal: Microbiol Immunol DOI: 10.1111/j.1348-0421.1980.tb02887.x sha: 4a0ab0f14f7953e300169d389be454cf40c1036f doc_id: 10227 cord_uid: ea3gwnw7 After intracerebral inoculation of mouse hepatitis virus, MHV‐A59 strain, into 3‐ to 5‐day‐old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of a considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus‐specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection. Mouse hepatitis virus (MHV), which is a coronavirus (26, 35) prevalent in mouse colonies without apparent illness (10, 32) , is known to cause fatal hepatitis (3, 12, 25, 29) , encephalitis (1, 6, 12, 19, 25, 29) , or enteritis (5) in mice. Recently, two coronaviruses, rat coronavirus (RCV) (31) and sialodacryoadenitis virus (SDAV) (4) from rats were shown to share a common antigen with MHV (4, 8) , and Bhatt et al (3) demonstrated that SDAV is also capable of causing respiratory infection in mice. While Bhatt and Jacoby (2) observed some differences in pathogenicity between SDAV and RCV, the speculation has arisen that MHV, and SDAV might be only variations within the murine coronaviruses which are transmissible between mice and rats. Recently, our routine serological surveillance revealed a high incidence of antibody to MHV in rat sera in Japan (11) . The present study was carried out to determine the response of suckling rats to intracerebral inoculation with some MHV strains, and reveals that strain MHV-A59 has the ability to cause hydrocephalus at high frequency. MATERIALS Virus. Laboratory and wild strains of MHV, MHV-1 (12), MHV-2 (29), MHV-3 (8) , JHM (6) , MHV-S (34), MHV-A59 (25) , NuA, NuU, and Nu66 (18), were used. All these virus strains were propagated in DBT cells (14) (15) (16) and their infectivity was assayed on the same cells as described previously (14) (15) (16) . Virus titer was expressed in plaque-forming units (PFU). Animals. Wistar rats in a late stage of pregnancy were obtained from a commercial barriered breeding colony, which had been serologically checked for the absence of antibody to MHV (8) . Suckling rats were nursed by their dams throughout the experiments. Inoculation. Inoculation was by the intracerebral (i.c.), intranasal (i.n.), or intraperitoneal (i.p.) route, with 0.02 ml of virus for i.e. and i.n. and 0.2 ml for i.p. inoculation, respectively. In some cases, 2.5 mg of cortisone acetate (Cortone, Merck-Banyu, Tokyo) per rat was administered subcutaneously shortly after virus inoculatio n. Virus content of the organs. Animals were killed at intervals and the brain, liver, and spleen were stored at -20 C. Blood was aseptically collected from the heart, diluted 10-fold in phosphate buffered saline (PBS, pH 7.2), and stored at -20 C until virus assay. Immunofluorescence and histopathology. Samples of brain, liver, and spleen were frozen in n-hexane chilled with acetone and dry ice, and 6-,um sections were made by cryostat. After being fixed with cold acetone for 5 min, the sections were stained by the direct method with fluorescein isothiocyanate-conjugated rabbit IgG against MHV-2 at 37 C for 60 min, as described previously (13) . Ten 3-to 5-day-old rats were inoculated i.e. with 105 to 106 PFU of nine strains of MHV. As shown in Table 1 , typical central nervous system (CNS) symptoms were observed in the JHM-and MHV-A59-inoculated groups, and with cortisone treatment, and neither brain lesions nor virus were detected at autopsy. The propagation of MHV in the brain of rats and hamsters has been reported for JHM (1, 3, 6, 28) and MHV-A59 (25) strains. The present study demonstrated the propagation of MHV-A59 in suckling rats, and cerebral lesions were established, with brain virus titers of 107-108 PFU/0.2 g at maximum levels. The infected rats developed CNS symptoms such as trembling, spasms, ataxia, and convulsions. Virus-specific immunofluorescence was detected in neurons of the cerebral cortex. Anomalies in the brain were produced only with strain MHV-A59 of the nine strains tested. After i.e. inoculation of suckling rats with MHV-2, MHV-3, JHM, NuA, and NuU, 103 (or more) PFU of virus per 0.2 g were recovered from the brain but no brain lesions were produced. No virus was detectable after i.e. inoculation with MHV-1, MHV-S, and Nu66. Brain lesions were found in all survivors inoculated i.e. with MHV-A59 at ages less than 7 days, and their development and severity were closely associated with the inoculum dose. Such lesions were produced only after i.e inoculation. Furthermore, 14-and 30-day-old rats were resistant to i.e. inoculation even with the administration of cortisone which is known to enhance MHV infection in mice (17, 18) . These results indicate that the malformation is produced only when rats less than 7 days old are inoculated with MHV-A59 by the i.e. route and that the age of the host and the route of infection are important for the development of hydrocephalus in rats, as reported for MHV-2 infection in mice (17) . Virus growth in the brain was parallel with the development of clinical signs, and virus-specific antigen was demonstrated in numerous neurons of the cerebral cortex by direct immunofluorescence. In addition, histopathological changes in the cortex were correlated with the presence of virus antigen detected by immunofluorescence. As for the pathogenicity of MHV-A59 for rats, our observation was different (25) , who reported that i.c.-inoculated newborn Fisher rats showed no evidence of disease. However, brain lesions could also be induced in suckling Fisher rats as in Wistar rats (unpublished data), suggesting that the virus could produce such a lesion in other strains of rat. Concerning MHV infection of the CNS, i.c. inoculation of the JHM strain into mice caused initial damage in oligodendroglia cells followed by demyelination, as reported by Lampart et al (23) , Weiner (36) , and Powell and Lampart (33) . Goto et al (13) demonstrated that mitral cells of olfactory bulbs were initially affected in weanling mice by i.n. infection. Recently, Nagashima et al (28) observed the development of demyelinating encephalitis in weanling rats after i.c. inoculation with the JHM strain and detected virus antigen in oligodendroglia cells by immunofluorescence. In the present study virus-specific fluorescence and maximum virus titers were observed in numerous neurons of the cortex during the early stage of infection; however, hydrocephalus was produced in the late stages, when virus was not detected. These observations indicate a difference in target cells and pathogenicity between the JHM and MHV-A59 strains. The first observation of experimental hydrocephalus as a sequence of viral encephalitis in hamsters infected with non-neuroadapted mumps virus was reported by Johnson et al (21) . Influenza A in rhesus monkeys (24) , parainfluenza type 2 in hamsters (20) , reovirus type 1 in hamsters, ferrets, mice, and rats (22), Ross River virus (27) and Newcastle disease vaccine virus (7) in mice have also been shown to induce hydrocephalus. These experiments show that nonfatal infections with those viruses result in stenosis of the aqueduct leading to hydrosephalus. Recently, Norrby and Kristensson (30) reported that subacute encephalitis and hydrocephalus in hamsters were caused by measles virus released from persistently infected cell cultures, and that no definite lesion of the aqueduct was found in animals with hydrocephalus. In the present study aqueductal stenosis was not found in the affected animals, and it was thought possible that hydrocephalus was produced as a sequence to the destruction of immature developing nervous tissues by the virus. 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