J. Vet. Med. B 37, 721-727 (1990) 0 1990 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1 - 1793 Department of Veterinary Microbiology, Iwate University, Morioka, Japan Natural Infection of Nude Mice with Low-virulent Mouse Coronavirus N. HIRANO and K. ONO Address of author: Dr. NORIO HIRANO, Department of Veterinary Microbiology, Iwate University, Morioka 020, Japan With 2 figures und li tubles (Received for publication March 22, 1990) Summary The outbreak of wasting disease of nude mice occurred in the laboratory colony of a Phar- maceutical Company. The viruses producing cytopathic effect with syncytium formation were isolated from the wasted nude mice by DBT cells, and were identified as mouse coronavirus by direct immunofluorescence. The nude mouse colony was closed and all the nude mice (about 500) were killed by the reason of disease control. At autopsy about 60 % of nude mice showed necrotic hepatitis. By the virus isolation to see the source of contamination, viruses were isolated from the feces of apparently healthy mice of ICR, CDFl, DBA/2 and C3H, and from human cancer cell line stocked in liquid nitrogen. In experimental infection, the isolates produced only mild hepatitis in ICR mice treated with cortisone. By cross-neutralization test, the nude isolate reacted closely with the virus from C3H mice but not with the virus from cancer cell line. The isolates from nude and C3H mice produced experimentally wasting disease with necrotic hepatitis in nude mice. These findings suggest that wasting disease in nude mice might be caused by low-virulent mouse coronavirus shed in feces from C3H mice introduced before the outbreak of disease. Introduction Athymic and immunodeficient nude mice have been widely used for immunologic as well as cancer researches. The mutant mice are highly susceptible for common infectious agents such as Sendai virus and mouse coronavirus (murine hepatitis virus; MHV), suffering from “wasting disease” characterized by depression and body weight loss (1, 3, PANTELOURIS (15) described that wasted and dead nude mice frequently had multiple necrotized foci in the liver. From wasted nude mice with hepatitis, SEBESTENY and HILL (16) isolated a low-virulent MHV, and many authors isolated low-virulent MHV strains (1, 11, 12, 13, 19). Mouse coronavirus has been recognized as a major pathogen causing wasting syndrome in nude mice (3, 17). During November and December 1986, acute deaths occurred in nude mice at a laboratory colony in Pharmaceutical Company near Tokyo. At autopsy necrotized lesions were detected in the liver of some wasted and dead nude mice. This report deals with isolation of a low-virulent MHV from the cases and epizootiology. 11, 16, 18-20). U.S. Copyright Clearance Center Code Statement: 0931 - 793/90/3710-0721$02.50/0 722 HIIUNO and ONO Material and Methods Animal room: Three hundreds to 600 athymic nude mice aged 6- to 15-week old had been usually kept for using various studies, since 1983, and about 200 nude mice were monthly introduced from a commercial breeder (Japan SLC, Hamamatsu). Ten mice each were kept in plastic cages (215x320 130mm) with a filter cap within a unit with HEPA-filter. They were given gamma irradiated commercial pellet (Clea Japan) and filtrated aseptic water by automatic system. Animal rooms were supplied with filtrated fresh air. In respected rooms near to this colony (room 8), inbred and hybrid mice of various strains were kept, as illustrated in Fig. 1. The nude mouse colony was closed in late December 1986 and about 300 of 500 nude mice were revealed to have liver necrosis at autopsy. Immunofluorescence and virus isolation: Direct immunofluorescence was performed on frozen sections of the liver by using anti-MHV-2 rabbit antibody conjugated with fluorescein isothiocyanate (4-5). Ten % homogenates of liver and spleen tissues were prepared in Eagles minimum essential (MEM) and after centrifugation at 3,000 rpm for 10 min, the supernatant was inoculated into DBT cell cultures (4,5). For isolation from feces, a spoonful of freshly collected feces was homogenized in 20 ml of MEM containing penicillin G (2,000 IU/ml) and streptomycin (2,000 pg/ml) and the supernatant was inoculated into DBT cell cultures after centrifugation at 3,000 rpm for 10 min. The DBT cells were grown at 37C in MEM containing 10 /o newborn calf serum (NCS), 10% tryptose phosphate broth (TPB) and kanamycin (0.06 mg/ml). For maintaining the cells and harvesting the virus, NCS concentration was reduced to 5 %. Infected DBT cells grown on coverslips were fixed with cold acetone for 10 min, and the coverslips were subjected to immunofluorescence (4-5). Plaque assay: Isolates were assayed for infectivity by plaque method described previously on DBT cell system (4, 5). As reference viruses, MHV-2 (14), MHV-NuA (11), feline herpesvirus (FHV) strain 406 and feline calicivirus (FCV) strain 410 (10) were used. FHV and FCV were also assayed by plaque m