Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice.

Mouse hepatitis virus A59 (MHV-A59) infection of adult BALB/c mice induced a severe, transient atrophy of the thymus. The effect was maximal at 1 week after infection, and thymuses returned to normal size by 2 weeks after infection. There was no effect of glucocorticoids, since thymus atrophy was also found in adrenalectomized, infected mice. In infected thymus, immature CD4+ CD8+ lymphocytes were selectively depleted, and apoptosis of lymphocytes was increased. The MHV receptor glycoprotein MHVR was detected on thymus epithelial cells but not on T lymphocytes. In a small number of stromal epithelial cells, but in very few lymphocytes, the viral genome was detectable by in situ hybridization. These observations suggested that MHV-A59-induced thymic atrophy results not from a generalized lytic infection of T lymphocytes but rather from apoptosis of immature double-positive T cells that might be caused by infection of a small proportion of thymus epithelial cells or from inappropriate secretion of some factor, such as a cytokine.     

Altered splenic T cell function of BALB/cByJ mice infected with mouse hepatitis virus or Sendai virus

Mouse hepatitis virus and Sendai virus are among the most common viruses naturally infecting laboratory mice. Concanavalin A-stimulated in vitro proliferative responses of splenocytes were examined after infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus (MHV-JHM) or Sendai virus. Mice were exposed to these viruses by presumed natural routes (per os or intranasally). Immunodepression was marked but transient among BALB/cByJ mice exposed to MHV-JHM. Among mice exposed to Sendai virus and examined over a 21-day period, spleen cells from only one mouse, sacrificed 10 days postinoculation, exhibited a severely impaired ability to respond to concanavalin A. Lymphokine production by spleen cells from control and infected mice was then assessed. IL 2 was either absent or present at very low levels in culture supernates of concanavalin A-unresponsive spleen cells from MHV-JHM-infected mice. Spleen cells from the single Sendai virus-infected mouse also produced very low levels of IL 2. In contrast, IL 1 was detected in supernatants of all spleen cell cultures derived from control, MHV-JHM-infected, or Sendai virus-infected mice. There was not a clear correlation between concanavalin A responsiveness and the ability of spleen cells to produce interferon-gamma. These results stress the importance of using laboratory mice of known microbiological status for immunologic experiments.     

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.     

RNA of mouse hepatitis virus.

The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.     

Naturally occurring mouse hepatitis virus infection in the nude mouse.

A group of athymic nude mice developed an unusual chronic wasting disease within 1-3 months after their arrival into the laboratory. Affected nude mice had severe, acute-to-chronic, active hepatitis with multinucleated giant hepatocytes and fibrosis. Vascular and central nervous system lesions were frequently present, giant cell peritonitis, ascites, and multinucleated giant cells in the intestinal epithelial villi were less frequently observed. Mouse hepatitis virus was isolated from the livers of three mice with lesions. The virus, when inoculated into nude mice, produced lesions similar to those observed in the natural outbreak.     

Origin and biological properties of a new BALB/c mouse sarcoma virus.

A focus-forming virus previously isolated from a BALB/c mouse hemangiosarcoma has been shown to be replication defective. Analysis of individual BALB/c mouse sarcoma virus (BALB-MSV) nonproducer transformants for expression of helper virus-coded proteins revealed genetically stable variants that expressed two, three, or all four gag gene products in the absence of detectable helper viral env gene expression. The type-specific antigenic determinants of helper viral proteins encoded by the BALB-MSV genome and by the B-tropic virus isolated from the BALB-MSV stock were demonstrated to be indistinguishable from those of BALB:virus-1, a known endogenous virus of BALB/c cells. These findings imply that a BALB/c endogenous virus was involved in the generation of BALB-MSV. By the same immunological approach, the presence of at least a portion of the Moloney-MuLV gag gene has been identified in two other transforming viruses--Moloney-MSV and Abelson lymphosarcoma virus--previously isolated from the BALB/c strain. The tissue culture properties of cells transformed by these defective viruses were also shown to be distinguishable. These findings indicate that transforming virus isolates of the same inbred strain differ in their transforming activities as well as in the helper viral sequences stably associated with their genomes.     

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Polyadenylate in the virion RNA of mouse hepatitis virus.

Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.     

Host age and genotypic effects on enterotropic mouse hepatitis virus infection

Intestinal lesions due to infection with an enterotropic strain of mouse hepatitis virus (MHV-Y) were found to be more severe and wide-spread in BALB/cByJ and Cr1:CD-1(ICR) mice than in SJL mice inoculated at 1 week of age, using nonparametric ranking analysis. Lesions and viral antigen were limited largely to the bowel, but also occurred in the liver and brain of some mice. BALB/cByJ mice developed a particularly high prevalence of brain infection, resulting in mortality after the enteric phase of infection had ceased. MHV-Y antigen was present in neurons, glia and vascular endothelium in a vascular distribution. Cr1:CD-1(ICR) pups inoculated with MHV-Y at 4 or 7 days of age developed severe typhlocolitis, enteritis and encephalitis with moderate mortality. Pups infected at 2 or 3 weeks of age had mild intestinal lesions with minimal alteration of mucosal architecture, no encephalitis and no mortality. These results demonstrate that host age and genotype influence the course of enterotropic mouse hepatitis virus, as has been shown previously with non-enterotropic, respiratory-type strains of mouse hepatitis virus.     

Characterization of accessory cell function during acute infection of BALB/cByJ mice with mouse hepatitis virus (MHV), strain JHM.

Earlier studies revealed defective concanavalin A-stimulated proliferation and cytokine production by spleen cells derived from BALB/cByJ mice acutely infected with mouse hepatitis virus (MHV), strain JHM. Based on those observations, assays of in vitro antigen-presenting cell (APC) function were undertaken. APC function of unfractionated spleen cells from individual MHV-infected mice was highly variable. Experiments using pooled spleen cells derived from MHV-infected mice revealed that adherent spleen cell APC function was impaired to a much greater degree than B cell APC function. Adherent cells derived from peritoneal exudates of infected mice exhibited an APC defect that was similar in magnitude to that observed for splenic adherent cells. Splenic B cells derived from acutely infected BALB/cByJ mice harbored infectious MHV. In contrast, lysates of adherent spleen cells from acutely infected mice did not kill intracerebrally inoculated neonatal mice, but did induce seroconversion among all survivors. Despite impairment of APC function of cells derived from MHV-infected donors, neither indomethacin nor accessory cells from uninfected control mice restored concanavalin A-induced proliferative responses of spleen cells collected from acutely infected mice. These results and those of earlier studies suggest that, although APC function is impaired, in vitro T cell dysfunction exhibited by spleen cells from MHV-JHM-infected donors is probably related to an inherent proliferative defect subsequent to T cell activation. Defective concanavalin A-stimulated proliferation does not appear to be secondary to accessory cell function suppression or to inhibitory factors secreted by accessory cells.     

Isolation of a Sindbis virus variant by passage on mouse plasmacytoma cells.

A variant of Sindbis virus has been isolated by growing a stock of virus, previously passaged on chicken embryo cells, in mouse plasmacytoma (MOPC 315) cells in suspension culture. An indirect immunofluorescence test and infective center assay showed that only a small fraction of cells could be infected by the stock wild-type virus, but that the population of virus accumulating after a few passages on the mouse cells had host-range properties distinct from the stock virus. The mouse-passaged virus retained its virulence for the original host and back-passaging of this virus on chicken cells did not change its newly acquired properties. Thus, this variant appears to be a genetically distinct form of Sindbis that adsorbs to and grows much better than the stock virus on several types of mouse cells including cultures of mouse macrophages.     

BALB/c myeloma retroviruses have mink cell focus-inducing activity.

We have determined the in vitro host range of the cloned MO-21 and FL-1 murine myeloma retroviruses grown in SC-1 cells that were originally isolated from cloned MOPC-21 and FLOPC-1 BALB/c plasmacytoma cell lines. These viruses are able to replicate in murine (BALB/3T3, NIH/3T3) as well as numerous heterologous cell lines. These myeloma retroviruses also exhibit mink cell focus-inducing activity. MO-21 and FL-1 shared interference patterns with each other, but their replication was not interfered with by ecotropic, xenotropic, or amphotropic viruses. The lack of cross-interference with ecotropic or xenotropic viruses distinguishes these isolates from other mink cell focus-inducing viruses.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

Changes in growth characteristics of mouse ascites tumor AISM during the process of passage in vivo

The growth characteristic of mouse ascites tumour AISM was observed on two distant passages (35th and 117th) in vivo. The following growth differences were established; the duration of life time of tumour bearing mice is less at the 35th passage (8 days) in comparison to the 117th passage (12 days); the common tumour cell mass at the terminal stage of life of tumour is more than 10 times less at 35th passage (10(8) cells) than at 117th passage (1.2.10(9) cells). The growth rate at 35th passage increases to the 4th day and at 117th passage to the 6th day. It is suggested that the tumour growth rate and the final size of tumor cell mass depend on the cell ploidity and chalone growth control.     

Effects of a bacteriocin from Mycobacterium smegmatis on BALB/3T3 and simian virus 40-transformed BALB/c mouse cells

The effects of a bacteriocin from Mycobacterium smegmatis ATCC 14468 on simian virus 40-transformed BALB/c mouse cells (mKS-A TU-7 cells) and nontransformed BALB/3T3 cells originating from the BALB/c mouse strain were studied. The percentage of nigrosin-unstained (viable) cells in the bacteriocin-treated mKS-A TU-7 cells decreased time-dependently with an increase in the bacteriocin activity. There was a time-dependent decrease in the bacteriocin activity after treatment with the cell membrane preparation from mKS-A TU-7 cells. There was no apparent effect of the bacteriocin on the viability of nontransformed BALB/3T3 cells. Wheat germ agglutinin blocked the toxic effect of bacteriocin on mKS-A TU-7 cells. These results indicate that the higher sensitivity and binding capacity of the tumor cells to the bacteriocin is probably due to the presence of a large amount of N-acetyl-glucosamine or closely related sugar residues with a high affinity for bacteriocin, as compared with normal cells. The bacteriocin produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the nontransformed cells.     

Two genetically transmitted BALB/c mouse mammary tumor virus genomes located on chromosomes 12 and 16.

We have examined EcoRI-restricted cellular DNA from BALB/c mouse-hamster somatic cell hybrids by blot hybridization for the presence of mouse mammary tumor virus-related sequences. Results of this analysis show that mouse mammary tumor virus-related proviral copies are located on chromosomes 16 (16-kilobase-pair fragment) and 12 (10.5- and 7.7-kilobase-pair fragments).     

Difference in response to mouse hepatitis virus among susceptible mouse strains.

After intraperitoneal inoculation with a high-virulent mouse hepatitis virus (MHV) a significant difference was seen in survival time between DDD and CDF1 (BALB/c X DDD) mice, while 50% lethal doses were not significantly different. With 3 X 10(3) PFU of the virus CDF1 and DDD mice died in 45 and 120 hr, respectively, on the average. This difference of susceptibility between DDD and CDF1 mice was first demonstrable at the age of 1 week and was more pronounced at the age of 4 weeks but showed no dependence of the sex. Virus titers ran 2 to 3 log higher in the liver and blood of CDF1 than in those of DDD mice, while being only 1 log higher in the spleen. At an early stage of infection viral antigen was demonstrable by immunofluorescence in sinusoidal lining cells of the liver more prominently in VDF1 than in DDD mice. Interferon production occurring in parallel with virus growth was significantly higher in CDF1 than in DDD mice. In DDD mice, liver lesions were rather focal with some accumulation of round cells, while they were confluent with poor cellular response in CDF1 mice. Viral growth in cultured peritoneal macrophages from CDF1 mice was 1 log higher than in those from DDD mice. The results suggest that the divergence in response to MHV among susceptible mice greatly depends upon the susceptibility of macrophages and reticuloendothelial cells which constitute primary targets of the virus.