key: cord-0004916-8qfgke03 authors: Hirano, N.; Murakami, T.; Taguchi, F.; Fujiwara, K.; Matumoto, M. title: Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes date: 1981 journal: Arch Virol DOI: 10.1007/bf01320795 sha: f9527a8cab01c24ea95dcf1ec3e21f1342f57dde doc_id: 4916 cord_uid: 8qfgke03 The pathogenicity for mice of nine strains of mouse hepatitis virus was studied in mice free from the virus by the intracerebral, intraperitoneal, intravenous and intranasal routes of inoculation. The pathogenicity for mice of nine strains of mouse hepatitis virus was studied in mice free from the virus by the intracerebral, intraperitoneal, intravenous and intranasal routes of inoculation. I t has been known that there is a wide variation in virulence and organ tropism for mice among many established strains and fresh isolates of mouse hepatitis virus (MHV) (17) . However, studies on the pathogenesis of I~[I-IV infection have been greatly hampered by difficulty to obtain MHV-ffee mice. Since we devised a very sensitive checking method for MHV infection (4, 5) , MttV-free mice have become available from commercial breeder colonies or laboratory colonies which are routinely checked by this method to ascertain absence of MHV. Using these MHV-free mice we have made some studies on the pathogenicity of MHV strains, i.e., 11, 21) , MHV-S (22, 23, 24) , J H M (9) and fresh isolates from wasting nude mice (6, 12) . The present study was undertaken to further compare several MHV strains for the pathogenicity in MIIV-ffee mice by different routes of inoculation. The MHV-strains used were MHV-1 (7), MItV-2 (18), MHV-3 (3), MHV-S (19), MHV-A59 (16), J H M (2) and 3 strains, NuA, NuU and Nu66, which were isolated from nude mice with wasting syndrome and chronic hepatitis in our laboratory (6, 12) . These strains were maintained by serial passage in cultures of mouse cell line DBT (10, 13, 14) . Virus was purified by plaque cloning (10, 13, 14) and propagated in DBT cells. Culture fluid harvested from infected cell cultures after incubation at 37°C for 24 hours was clarified by centrifugation and stored at 5b* 0304-8608/81/0070/0069/$ 01.00 iNL H~RANO et aI. : --70 ° C until use in the present study. These materials were assayed for infectivity by the plaque count method in DBT cells (10, 13, 14) . ICR mice, 4 weeks of age, were obtained from a commercial breeder colony that was proven by anamnestic serology to have no MHV infection (5) . Mice were inoculated by intracerebral (i.c.), intraperitoneal (i.p.), intravenous (i.v.) and intranasal (i.n.) routes with 0.02, 0.2, 0.2 and 0.02 ml amounts of virus, respectively. Serial decimal dilutions of each virus material were inoculated by these routes using 5 mice per virus dilution. Inoculated mice were observed for any clinical manifestations for 2 weeks. The 50 per cent lethal dose (LDs0) in plaqueforming units (PFU) was calculated by the method of Karber. Dead and surviving mice were examined for gross pathological changes. In some experiments 2.5 mg of cortisone acetate was injected subcutaneously shortly after virus inoculation, as enhancement of pathogenicity of MHV by corticosteroids has been reported (17) . The results are summarized in Table 1 . Strain MHV-2 was highly virulent for ICR mice, killing 4-week-old mice in 2 to 6 days after i.c., i.p. and i.v. inoculation. Dead mice showed marked swelling and confluent necrosis in the liver and hemorrhage in the duodenum. These results confirm previous findings (10, t 1, 21). However, large amounts of virus were required for i.n. inoculation to kill mice, in agreement with our previous results (tt). Cortisone treatment somewhat enhanced virulence by this route. Strain MHV-3 demonstrated essentially the same pathogenicity for weanhng mice as did strain MHV-2. DICK et al. (3) reported that strain MHV-3 was highly virulent, causing acute hepatitis in weanling mice. Strain JHM was highly neurotropic, killing weanling ICR mice in 5 to 9 days after i.c. inoculation even with very small amounts of virus. Infected mice produced nervous disorders resulting in limb paralysis. On the other hand, the strain caused no death by i.p. or i.v. inoculation even with an inoenlum of t0 s PFU. However, when treated with cortisone, mice infected by these routes developed acute hepatitis even with 103 PFU and died in 3 to 5 days. When inoculated i.n. with 105 or 106 PFU, some mice died of cerebral disorders. The cortisone treatment somewhat enhanced virulence by i.n. route. Neurotropism of this strain has been studied by previous workers (1, 2, 9, 26) . Strain NuA, like strain JHM, was shown to be highly neurotropie. The strain killed weanling ICR mice in 2 to 6 days even with an inoculum as small as l0 PFU by i.c. inoculation. No hepatitis was observed in dead mice. When inoculated i.v. or i.p., strain NuA caused fatal hepatitis, but death occurred erratically in relation to virus dose. These findings are somewhat different from our previous reports (6, 12) that without cortisone the strain could not produce acute fatal hepatitis in weanling ICI% mice even with 106 PFU by i.p. route. The reason for this discrepancy is obscure, but seems to be due to advanced serial passage in DBT cells. The cortisone treatment enhanced virulence by these routes of inoculation. The strain caused acute fatal hepatitis after i.n. inoculation, although relatively large amounts of virus were required. The cortisone treatment increased virulence by this route. Marked enhancement of pathogenicity by cortisone has been reported for this strain (6, 12) . Strain MHV-A59 caused fatal hepatitis by all routes of inoculation tested, although relatively large amounts of virus were required. Enhanced virulence was shown by the cortisone treatment. Similar results were obtained with strain Nu66. Previously we reported that, without cortisone, strain Nu66 could not produce acute fatal hepatitis in weanling ICR mice even with l0 s PFU by i.p. inoculation (6, 12) . The reason for this discrepancy is obscure, but seems to be due to passages in DBT cells. Strain NuU did not cause acute fatal hepatitis by all the routes tested even with 105 or 10 s PFU. The cortisone treatment elxhanced virulence, causing fatal hepatitis. Previously we reported similar results after i.p. inoculation with this strain (6, 12) . Strain MHV-S did not cause fatal hepatitis by these routes of inoculation even with l0 s PFU, confirming our previous results (22) . However, fatal hepatitis could be produced by cortisone treatment as previously reported by us (22) . Strain MHV-t showed very low pathogenicity by these routes as did strain MHV-S, in agreement with previous observations (8) . The cortisone treatment enhanced virulence of MHV-1, confirming previous results (15, 20, 25) . The data presented herein indicate that these MHV strains are widely varied in virulence and organ tropism. In the present study we used ICR mice, 4 weeks of age. Previous workers reported differences in the pathogenicity of MHV according to the strain and the age of mice (tl, 2t, 26). Another factor involved in the pathogenicity of MHV is the a~laptation through passage in vivo or in vitro as shown for/quA and Nu 66 strains in the present study. Further studies are needed to solve these problems and to elucidate the pathogenesis of MHV infections. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. II A murine virus {JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. I. Isolation and biological properties of the virus A virus related to that causing hepatitis in mice (MHV) An attempt to check five infections in breeding colonies with retired breeder mouse sera Problems in checking inapparent infections in laboratory mouse colonies: an attempt at serological checking by anamnestie response Wasting disease in nude mice infected with facultatively virulent mouse hepatitis virus A hepatitis virus of mice Mouse hepatitis virus and its pathogenic action Nasoencephalopathy of mice infected intranasally with a mouse hepatitis virus, JHM strain Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture Pathogenicity of mouse hepatitis virus for mice depending upon host age and route of infection Isolation of lowvirulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis Mouse hepatitis virus {MHV-2). Plaque assay and propagation in mouse cell line DBT cells Utility of mouse cell line DBT for propagation of mouse hepatitis virus Relationship of phagocytic activity to pathogenicity of mouse hepatitis virus as affected by triolein and cortisone A hepatitis virus complicating studies with mouse leukemia Coronaviruses : a comparative review Acute hepatitis associated with mouse leukemia. I. Pathological features and transmission of the disease Mouse hepatitis virus infection as highly courageous, prevalent, enteric infection of mice Susceptibility of cortisone-treated mice to infection with mouse hepatitis virus Difference in response to mouse hepatitis virus among susceptible mouse strains Age-dependent response of mice to a mouse hepatitis virus Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus Factors involved in the age-dependent resistance of mice infected with low-virulence mouse hepatitis virus Effect of X radiation and cortisone on mouse hepatitis virus infection in germfree mice Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus) Authors' address: Dr. M. MA~b')~OTO, The Kitasato Institute, 5-9-1 Shirokane, Minato-Ku, Tokyo, Japan 108.Received May 15, 1981