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.     

长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法的建立及初步应用

目的建立长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物MHV的检测。方法根据已发表的小鼠肝炎病毒（MHV）S基因序列,设计合成引物。提取MHV细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、稳定性、重复性试验。并对65只长爪沙鼠及12只小鼠进行检测。结果建立的MHVRT-PCR检测方法特异、敏感、稳定。以MHVRNA逆转录产物为模板,所能检测RNA最小模板浓度为3．1pg／μL,可检测病毒最小滴度为10^-3／mL。65只沙鼠经RT-PCR检测,均为阴性,12只小鼠经RT．PCR检测,有3只MHV阳性,测序结果与Genbank中MHV核酸序列同源性均为97％。结论建立的长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法可用于长爪沙鼠、小鼠等实验动物MHV的检测。     

Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-processing function of the Kupffer cells of both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

Abstract The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-proceesing function of the Kupffer cellsof both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

Murine cytomegalovirus infection of mouse testes.

With the aim of illustrating a mechanism of cytomegalovirus (CMV) venereal transmission, we induced murine CMV infection in the mouse testes of immunologically competent mice. Using in situ cytohybridization, we were able to show that murine CMV-specific DNA was associated with spermatocytes and mature sperm. Electron microscopy studies also supported sperm infection. The virus could be reisolated from infected epididymal sperm by cocultivation with mouse embryo fibroblasts. We found no difference in either the sexual performance or the fertilization efficiency of the sperm between infected and uninfected males.     

Transfer of Borrelia burgdorferi s.s. infection via blood transfusion in a murine model

Without antibiotic treatment, the Lyme-disease-causing bacterium, Borrelia burgdorferi can be cultured from the peripheral blood of human patients nearly 6 wk post-tick bite. To determine if Lyme disease spirochetes can be transmitted from a spirochetemic donor mouse to a naive recipient during blood transfusion, blood taken from immunocompetent infected mice was transfused into either immunodeficient (SCID) mice, inbred immunocompetent animals (C3H/HeJ), or outbred mice. Nine of 19 (47.7%) immunodeficient mice, 7 of 15 (46.8%) inbred immunocompetent mice, and 6 of 10 (60.0%) outbred mice became infected with B. burgdorferi after transfusion. Our results indicate that it is possible to acquire B. burgdoferi infection via transfused blood in a mouse model of Lyme borreliosis.     

Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically

Helicobacter typhlonius, one of the most recently identified members of this rapidly expanding genus of bacteria, was detected by polymerase chain reaction (PCR) in an animal facility and studied for tissue distribution in sentinel mice (Helicobacter-free, barrier maintained). Immunodeficient athymic nude-nu (nu/nu), Helicobacter-sensitive C3H/HeJ and Helicobacter-resistant C57BL/6J mouse strains were used to study whether Helicobacter species could spread to the sex organs and be transmitted vertically. Sentinel mice of these three strains became infected at different times after first exposure and Helicobacter was detected by PCR for a brief period in the sex organs. The potential for PCR-positive tissues from the ovary, uterus, testis and epididymis to transmit infection was investigated via in vitro fertilization (IVF), embryo transfer (ET) or ovary transplantation in immunodeficient athymic nude-nu mice and did not result in contamination of the recipient females.     

Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction.

Sequence analysis of the nucleocapsid protein genes of five strains of mouse hepatitis virus (MHV) disclosed that the 3' region of the nucleocapsid protein gene contains highly conserved sequences unique to MHV. We designed a pair of primers to amplify cDNA from such sequences of MHV by using the polymerase chain reaction (PCR). Six isolates of wild-type MHV, as well as prototype viruses, were amplified successfully and detected in ethidium bromide-stained agarose gels. The sequence identity of PCR products was readily verified by confirming target size and a MflI site within the target. The sensitivity of our PCR assay was estimated to be sufficient to detect a single cell infected with MHV. This new approach may permit more sensitive and rapid detection of MHV in biologic materials than current methods such as virus isolation, the infant mouse bioassay, and the mouse antibody production test.     

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection.

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.     

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.     

Alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus

Pre-infection with mouse hepatitis virus (MHV) strains S, 3, or JHM reduced the ability of mice to seroconvert to PVM. Geometric mean antibody titers to PVM among MHV pre-infected mice were lower than those for control mice given only PVM, and dually infected mice seroconverted to PVM later than mice given PVM alone. PVM was not recovered from normally permissive respiratory tract tissues of MHV-S pre-infected mice. Pre-infection of DBA/2 mice with MHV-S compromised the susceptibility of these mice to lethal Sendai virus infection but did not substantially reduce the titers of infectious Sendai virus recovered from the lungs. Serologic responses to Sendai virus and lung Sendai virus titers were similar in Sendai virus-resistant C57BL/6 mice pre-infected or not with MHV-S.