Ten-year long monitoring of laboratory mouse and rat colonies in French facilities: a retrospective study

From 1988 to 1997, a total of 69 mouse colonies and 36 rat colonies were examined for the presence of antibodies to 14 indigenous viruses of mice and rats. Among mouse viruses, high positivity rates were observed with mouse hepatitis virus (MHV), Theiler's encephalomyelitis virus (THEMV), minute virus of mice (MVM), Sendai virus and pneumonia virus of mice (PVM); the prevalence rates were high in rats with Khilam's rat virus (KRV), THEMV, Toolan's H-1 virus, Sendai virus, Parker's rat coronavirus (RCV/SDA) and PVM. During the last decade, the prevalence of some agents such as MHV, Sendai virus, THEMV, PVM and MVM has apparently decreased although they were still present in 1997 (except for PVM). Another point is the constant increase of colonies found free of viruses through this decade, demonstrating the efforts of the French research community to increase the quality of hygiene in laboratory animals.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Serological examinations on natural infections with mouse pathogens in inbred mouse strains: difference in antibody detection among the strains (author's transl)

Serological surveys on several infections were performed on the inbred mouse strains maintained at the Central Institute for Experimental Animals. In the first survey, 11 strains of mouse, which were 8 weeks of age or older and were kept in separate cages in the same animal room, were tested for antibodies to Salmonella enteritidis, Corynebacterium kutscheri, Tyzzer's organisms, Mycoplasma pulmonis, mouse hepatitis virus (MHV), Sendai virus (HVJ), pneumonia virus of mice (PVM) and minute virus of mice (MVM). Positive results were obtained in MHV, HVJ, PVM and MVM. Positive rates for these viruses except for MVM were different among mouse strains. In the second survey, 5 strains of mouse kept together in the same cage for 4 weeks after weaning were examined for MHV and HVJ antibodies. Positive rates to MHV were different among mouse strains as observed in the first survey. For HVJ antibody, no difference was demonstrated in positive rates unlike in the first survey, but the titers varied between the strains. These results suggest the difference in antibody response to natural infections dependent on mouse strains.     

Mouse hepatitis virus S in weanling Swiss mice following intranasal inoculation

Three-week-old outbred mice were inoculated intranasally with a mildly pathogenic strain of mouse hepatitis virus (MHV-S). Tissues were analyzed for distribution of infectious virus, lesions, and viral antigen at intervals up to 49 days after inoculation. Sera were tested for neutralizing antibody to MHV-S. Within the first week of infection, virus was isolated from lung and brain of most mice and liver of one mouse, but not from blood, spleen, or intestine. Microscopic lesions consisted of mild olfactory mucosal necrosis, neuronal necrosis of olfactory bulbs and tracts, lymphoplasmacytic infiltrates and vacuolation in the brain, mild nonsuppurative pulmonary perivascular lymphocyte infiltration, focal interstitial pneumonia, and focal necrotizing hepatitis. The presence and distribution of MHV antigen, as determined by indirect immunofluorescence, correlated with virus recovery and acute lesions. No virus or antigen was demonstrable beyond day 7. Serum antibody was first detected on day 10, and titers peaked on day 28 after infection.     

The effectiveness of a microisolator cage system and sentinel mice for controlling and detecting MHV and Sendai virus infections

Experiments were conducted to determine (a) whether BALB/c mice housed on soiled bedding can be used as sentinels for the detection of Sendai virus and MHV from infected mice housed in microisolators, and (b) whether the microisolator caging system protects mice against Sendai virus and MHV infections. Sentinel mice were housed in microisolator cages, exposed continuously to soiled bedding and bled at 21 and 42 days for serology. All sentinel mice were seropositive for MHV by 42 days; however, sentinel mice exposed to soiled bedding were seronegative for Sendai virus at 21 and 42 days. These results suggest that sentinels housed on soiled bedding may not detect all infectious murine viruses. This study also showed that the microisolator caging system provided an effective barrier against MHV infection at the cage level and suggests that the microisolators should protect mice against other infectious agents.     

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.     

Reactivities of 4 murine coronavirus antigens with immunized or naturally infected rat sera by enzyme linked immunosorbent assay

Four murine coronavirus antigens, sialodacryoadenitis virus (SDAV) strain TG, Parker's rat coronavirus (PCV) strain 8190, mouse hepatitis virus (MHV) strains S and NuU, were examined for their reactivities to hyperimmunized and naturally infected rat sera by ELISA. With the immunized sera, SDAV and PCV antigens reacted best with respective homologous sera. MHV antigens reacted with all antisera, anti-SDAV, anti-PCV, and anti-MHV-S at approximately the same level, and MHV-S showed a slightly higher reactivity than MHV-NuU. The reactivities of the sera from various colonies to these antigens were in the order--from high to low--of SDAV, MHV-S, MHV-NuU, and PCV. None of sera negative for SDAV antigen reacted positively to the other antigens. Within the sera positive for SDAV, the positivities were in the order of MHV-S, MHV-NuU, and PCV. These results suggested that, although homologous antigens are best to detect SDAV or PCV infection by ELISA, MHV antigen can be used if highly cross-reactive viral strain is selected.     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

Murine gammaherpesvirus 68 cyclin D homologue is required for efficient reactivation from latency

Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses beta-galactosidase as a marker (MHV68(cy-)). MHV68(cy-) grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68(cy-) show deficient growth of the MHV68(cy-) in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68(cy-) virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68(cy-) had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.     

Susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species

The susceptibility of laboratory mice to intranasal and contact infection with mouse hepatitis virus (MHV)-related coronaviruses was tested in infant CD1 mice. One day old mouse pups were inoculated intranasally with respiratory MHV-S, enteric MHV-Y, rat sialodacryoadenitis virus (SDAV), human coronavirus OC43 (HCV-OC43) or bovine coronavirus (BCV). Twenty-four hours later, they were placed in direct contact with age matched sham inoculated pups. Indices of infection in virus inoculated mice included lesions by histopathology and viral antigen by immunoperoxidase histochemistry in brain, lung, liver and intestine at 3 days after inoculation. Indices of infection in contact mice included mortality or seroconversion by 21 days after exposure. Infant mice were susceptible to infection with all five viruses. Transmission by direct contact exposure occurred with MHV and SDAV, but not HCV or BCV. Furthermore, adult mice were not susceptible to infection with HCV. Tissue distribution of lesions and antigen varied markedly among viruses, indicating that they do not induce the same disease as MHV. This study demonstrates that although these coronaviruses are antigenically closely related, they are biologically different viruses and disease patterns in susceptible infant mice can be used to differentiate viruses.     

Genetic resistance to lethal Sendai virus pneumonia: virus replication and interferon production in C57BL/6J and DBA/2J mice

Resistant C57BL/6J and susceptible DBA/2J mice were exposed to aerosols of Sendai virus and killed at intervals to 12 days. Lungs were removed and assayed for infectious virus and interferon. Mean virus titers were 6 to 400 times higher in DBA/2J mice than in C57BL/6J mice 3 to 10 days after exposure. Mean interferon titers were 10 to 140 times higher in DBA/2J mice than in C57BL/6J mice 4 to 7 days after exposure. These results suggest that genetic resistance to the lethal effects of Sendai virus is expressed through control of viral replication within the first 72 hours of infection and that early expression of inherited resistance is not regulated by interferon.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

Duration and strain-specificity of immunity to enterotropic mouse hepatitis virus.

We examined the duration and strain-specificity of immunity to enterotropic mouse hepatitis virus (MHV). Two strains of enterotropic MHV (MHV-Y and MHV-RI) were determined to be distinct virus strains by serum neutralization and by enzyme immunoassay. BALB/cByJ mice immunized by oral infection with either MHV-Y or MHV-RI developed serum MHV IgG titers that remained stable for more than 6 months. The animals were protected from reinfection with the homologous virus strain at 1 and 6 months after an initial immunizing infection, based on intestinal histology and polymerase chain reaction for a 375-base-pair segment of the membrane glycoprotein gene. Immunity was also fully protective against challenge with the heterologous strain 1 month after initial immunization and partly protective after 6 months. Maternally-derived passive immunity prevented MHV infection in 1-week-old pups challenged with the homologous strain of MHV, and pups challenged with the heterologous virus strain were partially protected.     

The chemokine macrophage-inflammatory protein-1 alpha and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1 alpha (MIP-1 alpha). Here we examine the role of MIP-1 alpha in the pathogenesis of this disease using mice deficient in MIP-1 alpha or its receptor, CCR1. The inflammatory response to PVM in MIP-1 alpha-deficient mice was minimal, with approximately 10-60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1 alpha-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1 alpha/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.     

Pneumocystis carinii pneumonia in the rat model.

Groups of barrier-raised but not certified virus-free Sprague-Dawley rats, obtained from the same source over the course of several years, were placed on an identical immunosuppressive regimen. This caused reactivation of latent Pneumocystis carinii infection, manifest as P. carinii pneumonia (PCP) of varying severity. Rats were euthanized after 9-12 wk of immunosuppression. An assessment of the severity of the induced PCP was made, based on the total number of organisms extracted from the lungs and their ability to proliferate in short-term cell culture. Serum samples obtained at sacrifice were tested by indirect immunofluorescence for antibodies to coronavirus, parvovirus, Sendai virus, pneumonia virus of mice (PVM) and Mycoplasma pulmonis. A total of 60 rats were examined. Thirty-four of these (57%) developed moderate or severe PCP. No antibodies were detected to either coronavirus or Mycoplasma pulmonis in any of the rats. Although antibodies were detected to parvovirus in 13/60 (22%), to PVM in 29/60 (48%), and to Sendai virus in 47/60 (78%), there was no apparent correlation between the presence or absence of antibodies to these agents and the severity of PCP. Sequential observations during the course of immunosuppression are needed to clarify the role of concomitant infections in the development of PCP.     

Influence of viral infections on body weight, survival, and tumor prevalence in Fischer 344/NCr rats on two-year studies

Sendai virus (SV), pneumonia virus of mice (PVM), and rat coronavirus/sialodacryoadenitis virus (RCV/SDAV) were common viral infections of rats in the National Cancer Institute-National Toxicology Program (NCI-NTP) studies from 1977 to 1983. Influence of these viral infections on body weight, survival, and prevalences of spontaneous tumors in the F344/NCr rats of 28 diet control groups at five different laboratories were evaluated. Tumor prevalences evaluated in this investigation included the following: leukemia and tumors of the anterior pituitary, lungs, salivary glands and Harderian glands in both sexes; adrenal pheochromocytomas in male rats; and mammary tumors in female rats. SV and PVM but not RCV/SDAV infections were associated with significant (P less than 0.05) decreases in body weights of male and female rats. Male rat groups with PVM infection had a lower prevalence of leukemia and male rat groups with RCV/SDAV infection had a higher prevalence of anterior pituitary tumors than the corresponding uninfected groups. Female rat groups with SV infection had greater survival and a higher prevalence of lung tumors than groups without SV infection. However, none of the tumor prevalence and survival differences were statistically significant when interlaboratory variability and time-related effects were taken into account.     

Adverse effects of mouse hepatitis virus on ascites myeloma passage in the BALB/eJ mouse.

During experimental serial passage of ascites myelomas through BALB/cJ mice, unexpected illness and premature deaths occurred. Postmortem examination of affected mice revealed focal or diffuse discolored depressed areas in the liver and, in some cases, splenomegaly. Histopathologic findings consisted of focal to diffuse areas of necrosis with minimal leukocytic infiltration. Aerobic and anaerobic bacterial cultures of livers and spleens from affected mice were negative. Mouse hepatitis virus (MHV) was isolated from livers of clinically ill mice and from the ascites myeloma lines. An MHV contaminated ascites myeloma line, when passed into nude (nu/nu) mice, killed the animals in 6 days; the virus was isolated from livers of inoculated mice. Attempts to determine the source of the infection were unsuccessful. Serologic survey of newly acquired mice indicated no evidence of antibodies to MHV while mice in holding rooms had titers that ranged from 1:10 to 1:40. Two solid myeloma lines (being maintained by subcutaneous passage) were negative for MHV when tested by virus isolation techniques, and nine lines were negative to 11 murine viruses when tested by mouse antibody production assay. Attempts to demonstrate Eperythrozoon coccoides in control BALB/cJ mice were unsuccessful. Because of the outbreak, changes were made in animal handling procedures. A colony of BALB/cAn mice negative to MHV antibodies was established to provide animals for experimental passage of tumors, and animals in both the breeding and transfer room were placed under filter tops. The results were encouraging. In the four newly established tumor lines, one having been passed 46 times, no illness or unexplained deaths were observed.