Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction.

Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node.     

Neonatal infection with mouse thymic virus: effects on cells regulating the antibody response to type III pneumococcal polysaccharide.

Mice infected neonatally with mouse thymic virus (TA) were evaluated at different ages with respect to their ability to give a plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III), as well as the degree of amplifier and suppressor thymus-derived (T) cell activity present. B cell activity matured rapidly from 2 to 4 weeks of age and was not affected by TA infection. Amplifier T cell activity matured progressively over the first 8 weeks of life and was transiently suppressed in TA-infected mice at 4 weeks of age. Suppressor T cell activity measured at 2,4, and 6 weeks of age was unaffected by TA. The findings suggest that TA is highly tropic for T cells and has selective effects on subpopulations of T cells.     

Thymic necrosis following oral inoculation of mouse thymic virus

Mouse thymic virus (MTLV;ICTV designation murid herpesvirus 3) infects developing T lymphocytes of neonatal mice, causing thymic necrosis and acute immunosuppression. Infected animals shed virus indefinitely. However, although transmission in nature is presumably by contact and is likely to involve the oral-nasal route, virtually all experimental studies with MTLV have used systemic (intraperitoneal) inoculation. In order to determine whether systemic inoculation causes artifacts in pathogenesis of the infection, effects of intraperitoneal and oral-nasal inoculation were compared in newborn mice. Thymic necrosis occurred with either route of inoculation, although rate of infection was lower with oral inoculation, varying from about 20% to 67%. There were no gross differences in pathogenesis. Orally infected animals seroconverted and shed virus. These data indicate that the apparent lymphotropism of thymic virus, and induction of thymic necrosis, are not dependent on route of inoculation.     

Murine viruses in an island population of introduced house mice and endemic short-tailed mice in Western Australia

House mice (Mus domesticus) were recently introduced to Thevenard Island, off the northwest coast of Western Australia. This island is also habitat for an endangered native rodent, the short-tailed mouse (Leggadina lakedownensis). Concerns have been raised that house mice may pose a threat to L. lakedownensis both through competition and as a source of infection. To assess the threat to L. lakedownensis posed by viral pathogens from M. domesticus, a serological survey was conducted from 1994 to 1996 of both species for evidence of infection with 14 common murine viruses (mouse hepatitis virus, murine cytomegalovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus strains FL and K87, minute virus of mice, mouse parvovirus, reovirus type 3, Sendai virus, Theiler's mouse encephalomyelitis virus, polyoma virus, pneumonia virus of mice, and encephalomyocarditis virus) and Mycoplasma pulmonis. Despite previous evidence that populations of free-living M. domesticus from various locations on the Australian mainland were infected with up to eight viruses, M. domesticus on Thevenard Island were seropositive only to murine cytomegalovirus (MCMV). Antibodies to MCMV were detected in this species at all times of sampling, although seroprevalence varied. Infectious MCMV could be isolated in culture of salivary gland homogenates from seropositive mice. In contrast, L. lakedownensis on Thevenard Island showed no serological evidence of infection with MCMV, any of the other murine viruses, or M. Pulmonis, and no virus could be isolated in culture from salivary gland homogenates. Although MCMV replicated to high titers in experimentally infected inbred BALB/c laboratory mice as expected, it did not replicate in the target organs of experimentally inoculated L. lakedownensis, indicating that the strict host specificity of MCMV may prevent its infection of L. lakedownensis. These results suggest that native mice on Thevenard Island are not at risk of MCMV infection from introduced house mice, and raise interesting questions about the possible selective survival of MCMV in small isolated populations of M. domesticus.     

The effect of biological response modifiers on chronic and latent murine cytomegalovirus infections

Host-mediated antiviral effect of 2 biological response modifiers (BRM), OK-432, and PS-K, against murine cytomegalovirus (MCMV) was evaluated in chronically or latently infected mice. In the early stage of chronic MCMV infection, the BRM-induced resistance was evidenced by decrease in infectious viruses replicated in the salivary glands and by augmented cytotoxic activity of the spleen cells against YAC-1 cells and MCMV-infected mouse embryonic fibroblasts (MEF). In the late stage of chronic MCMV infection, the BRM treatment did not eliminate MCMV from the mice, but did prevent exacerbation of MCMV infection in the salivary glands induced by administration of cyclophosphamide (CY). In mice latently infected by MCMV, BRM treatment suppressed CY-induced reactivation of MCMV in the salivary glands. It was suggested that the antiviral effect of BRM against MCMV in chronically or latently infected mice was based on activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTL).     

Immunologic effects of neonatal infection with mouse thymic virus.

Mouse thymic virus is a herpesvirus that causes extensive thymic necrosis when given to newborn mice. During the time of acute infection spleen cells have markedly diminished reactivity to T cell phytomitogens and to allogeneic cells and are incapable of effecting a primary in vitro response to a "T-dependent" antigen; responses to B cell mitogens and to a T-independent antigen are unimpaired. Spleens from acutely infected mice have low theta antigen normal numbers of immunoglobulin-bearing cells. Surprisingly, despite widespread necrosis and cellular depletion, thymic cell reactivity to mitogens is unimpaired. However, the ability to thymocytes to proliferate and to generate cytotoxic killer cells in response to allogeneic cells is diminished.     

Studies of latent cytomegalovirus infection: the macrophage as a virus-harboring cell

During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV. Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

Antigen-specific T cells maintain an effector memory phenotype during persistent Trypanosoma cruzi infection

Infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Central and South America. Control of acute experimental infection with T. cruzi is dependent on a robust T cell and type 1 cytokine response. However, little evidence exists demonstrating the development and persistence of a potent antiparasite T cell memory response, and there has been much speculation that the majority of the immune response to T. cruzi infection is not directed against the parasite. In this study, we used an experimental mouse model of T. cruzi infection to test both the Ag specificity and the functional and phenotypic characteristics of the responding T cell population. We observed a vigorous antiparasite response from both CD4(+) and CD8(+) T cells that was maintained in the face of persistent infection. T cells from infected mice also proliferated in response to re-exposure to Ag, and CD8(+) T cells underwent spontaneous proliferation when transferred to naive congenic mice, both characteristic of central memory T cells. Interestingly, T cells from infected mice showed significant down-regulation of CD62L, a characteristic associated with an effector memory phenotype. These results suggest that T cells maintained in mice with chronic T. cruzi infection are fully functional memory cells that cannot be easily categorized in the current central/effector memory paradigm.     

In vivo selection of lymphocyte-tropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection.

This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.     

Transplantable polyoma virus-induced epitheliomas harbor immature T lymphocytes

Some salivary epitheliomas induced in C3H/Bittner (C3H/Bi) mice by neonatal injection of the fifth in vitro passage of parotid tumor agent isolate of mouse polyoma virus are infiltrated with T lymphocytes having an immature phenotype. Serially transplantable tumor lines have been obtained from primary salivary epitheliomas, and some of these secondary tumors are also infiltrated with variable proportions of immature T cells. A subline has been selected which, in syngeneic hosts, becomes richly infiltrated by immature lymphocytes, as defined by bearing both CD4 and CD8 surface markers. While some sublines produced this infiltrate, others did not. There is no relationship between the extent or nature of the T-cell infiltrate and the passage number (generation) of the tumors or the residence period of the transplanted tumors in the host. The results strongly suggest that subsets of salivary epitheliomas resemble the thymic microenvironment sufficiently that T-lymphocyte maturation, at least in part, proceeds readily.     

Central neuropathogenesis of vesicular stomatitis virus infection of immunodeficient mice.

To determine whether central neuropathogenesis associated with vesicular stomatitis virus (VSV) infection is regulated by T cells, we have examined the effects of intranasal infection of mice lacking T cells. The mice examined were of two kinds: (i) thymus-deficient BALB/c nu/nu nice and (ii) BALB/c mice experimentally depleted of T cells by systemic infusions of a monoclonal antibody to the CD4 or CD8 cell surface molecules. These mice were infected intranasally with a single dose of replication-competent VSV. Brain tissue homogenates were analyzed for the presence of infectious virus. For each population of mice, infection-related mortality was assessed. In histological sections of brain, the distribution of viral antigens (Ags) was examined by immunocytochemistry. We found that recovery of infectious virus from homogenates of tissues obtained from athymic nu/nu animals was more than 10 times greater than that from samples from their euthymic littermates. With a single exception in a BALB/c nu/nu mouse, virus was not isolated from the spleen when it was administered intranasally. In these experimental infections, athymic mice succumbed 1 to 2 days before their euthymic littermates. A dose of virus that resulted in half of the nu/+ survival rate was uniformly lethal to nu/nu mice. In experiments with BALB/c mice depleted of either CD4+ or CD8+ T cells by in vivo antibody treatment, histological analysis revealed an increase in viral Ag distribution in comparison with control (medium-infused) infected mice. Necrosis and inflammation paralleled the extent of viral Ag expression. Viral Ags were detected in discrete areas that usually remain uninfected in immunocompetent mice. These areas include the neocortex and caudate putamen nuclei, the piriform cortex, and the lateral olfactory tract. Neuronal loss and necrosis were consistently found in the olfactory bulb and the horizontal/vertical band of Broca. In some of the T-cell depleted mice, necrosis was also evident in the hippocampus, fimbria, mammillary bodies, and hypothalamic nuclei. In the brain stem, perivascular cuffing was evident, but with little necrosis. Collectively, these data suggest that CD4+ and CD8+ T cells make only a minor contribution to the development of histopathology but rather function together to limit viral replication and transsynaptic or ventricular spread of virus, thus promoting recovery. The primary effectors of histopathology appear to be related more to the cytopathologic nature of the virus infection and non-T-cell-mediated mechanisms.     

Role of thymic output in regulating CD8 T-cell homeostasis during acute and chronic viral infection

Although it is well documented that CD8 T cells play a critical role in controlling chronic viral infections, the mechanisms underlying the regulation of CD8 T-cell responses are not well understood. Using the mouse model of an acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we have examined the relative importance of peripheral T cells and thymic emigrants in the elicitation and maintenance of CD8 T-cell responses. Virus-specific CD8 T-cell responses were compared between mice that were either sham thymectomized or thymectomized (Thx) at approximately 6 weeks of age. In an acute LCMV infection, thymic deficiency did not affect either the primary expansion of CD8 T cells or the proliferative renewal and maintenance of virus-specific lymphoid and nonlymphoid memory CD8 T cells. Following a chronic LCMV infection, in Thx mice, although the initial expansion of CD8 T cells was normal, the contraction phase of the CD8 T-cell response was exaggerated, which led to a transient but striking CD8 T-cell deficit on day 30 postinfection. However, the virus-specific CD8 T-cell response in Thx mice rebounded quickly and was maintained at normal levels thereafter, which indicated that the peripheral T-cell repertoire is quite robust and capable of sustaining an effective CD8 T-cell response in the absence of thymic output during a chronic LCMV infection. Taken together, these findings should further our understanding of the regulation of CD8 T-cell homeostasis in acute and chronic viral infections and might have implications in the development of immunotherapy.     

Murine lymphocytic choriomeningitis: the history of a natural cross-infection from wild to laboratory mice.

A new locus of wild house mice tolerantly infected with the virus of lymphocytic choriomeningitis (LCM) has been identified in the United Kingdom. Evidence is presented which indicates that these mice were the source of infection in a laboratory mouse breeding colony, the mode of transmission probably being bites on tails and limbs exposed through wire-grid flooring. The results of an experiment which simulated indirect exposure of SPF mice to tolerantly infected wild mice supported earlier observations that without injury to the epidermis the risk of spread of infection from the infective urine, saliva or faeces is low.     

Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.     

Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.     

Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.