Murine cytomegalovirus adrenalitis in athymic nude mice

Summary During studies of the pathogenesis of murine cytomegalovirus (MCMV) infection in athymic nude mice, we noted striking virus involvement of the adrenal glands. Because patients with the acquired immunodeficiency syndrome (AIDS) have recently been reported to have adrenal necrosis and evidence of infection of the adrenal gland with human cytomegalovirus (HCMV), we have further evaluated adrenal involvement during MCMV infection. Following virus inoculation, MCMV replicated to high titer in the adrenal glands of T-cell deficient, homozygous nude mice, but not heterozygous littermates with intact T-cell function. Concomitant with the high titers of virus, there appeared overt histological evidence of herpesvirus infection accompanied by patchy necrosis of adrenal cortical and medullary tissues. Acyclovir, which inhibits growth of MCMV, reduced virus replication in the adrenal gland. Similarly, virus replication was diminished in homozygous nude mice immunologically reconstituted by infusion of normal spleen cells three weeks prior to infection. Thus, in the absence of functioning T lymphocytes, MCMV can infect and replicate in adrenal tissues causing a progressive destructive adrenalitis.With 1 Figure     

Role of specific cytotoxic lymphocytes in cellular immunity against murine cytomegalovirus.

Cytotoxic lymphocytes were generated in vitro against murine cytomegalovirus (MCMV)-infected cells by incubation with ultraviolet light-irradiated, infected fibroblasts. When passively transferred, they reduced virus titers in spleens of mice 1 day after infection with MCMV. Protection was abrogated by anti-theta serum and complement. Spleen cells from mice infected for 6 to 14 days protected mice better than cells from mice after infection for 1, 3, or 30 days. Protection by in vitro- and in vivo-generated cells was H-2K or H-2D restricted. Specific cytotoxic T lymphocytes are therefore present and operative during acute MCMV infection. However, MCMV infection inhibited the development of primary cytotoxic response against ectromelia virus. It also suppressed the ability of lymphocytes from mice with established memory for ectromelia to develop secondary cytotoxic cells in vitro, and it inhibited the development of memory cells for the cytotoxic response to ectromelia virus. In view of these data and the inability of animals recovering from MCMV infection to eliminate all infected cells, the cytotoxic response to MCMV may be qualitatively or quantitatively deficient.     

Acute cytomegalovirus infections in leukemic mice.

Mice infected with 2 x 10(3) plaque-forming units of mouse cytomegalovirus (MCMV) 3 days after receiving 300 to 400 spleen focus-forming units of Friend leukemia virus developed a more severe MCMV infection than did normal animals. Increased severity was demonstrated by the increased amounts of MCMV recoverable from the salivary glands of leukemic mice 1 to 5 weeks postinfection. In addition, the difference in the number of virus isolations from the kidneys, spleens, livers, and lungs of animals (74 to 120) coinfected with MCMV and Friend leukemia virus compared with animals (49 of 120) infected with MCMV alone was significant (P less than 0.01). Both the 50% lethal dose and 50% infectious dose of MCMV in leukemic mice were lower than in normal animals. MCMV and Friend leukemia virus appear to interact by suppressing the ability of infected spleen cells to respond to mitogen-induced stimulation. The observations of increased severity of MCMV infections in leukemic mice closely parallel the situation observed in human leukemia patients who are at an increased risk of disease due to human cytomegalovirus infections. This mouse model may be useful in assessing the effect of antiviral (cytomegalovirus) therapy.     

Developmental disorders of the mouse brain induced by murine cytomegalovirus: Animal models for congenital cytomegalovirus infection

Developmental disorders induced by congenital cytomegalovirus (CMV) infection mainly involve the central nervous system. The type and degree of the brain disorders seems to depend on infection time during gestation, virulence, route of infection and viral susceptible cells in each embryonal stage. Since transplacental transmission has been reported not to occur with murine CMV (MCMV), we developed mouse models for congenital CMV infection by surgical injection of MCMV into the mouse conceptus or embryo at different gestational stages. For the early stage, the mouse embryos were not infected with MCMV even after injecting the virus into the blastocysts, which were developed in the pseudo-pregnant mothers or cultured in vitro. Isolated whole mouse embryos of day 7.5 of gestation (E7.5), adsorbed with a high titer of MCMV and cultured for 3 days, were susceptible to MCMV infection. Therefore, the mouse embryo acquires the susceptibility around this period. Microphthalmia and cerebral atrophy were induced in mouse embryos after injection of MCMV into the conceptus on E8.5. Viral antigen-positive cells were widely distributed in the mesenchyme around the oral and nasal cavities and in the mesenchyme around the brain, especially the endothelial cells of vessels and the perivascular mesodermal cells, then infection extends to the eyes, brain or choroid plexus. This finding suggests that mesenchymal infection may be the critical step in disrupting organogenesis, resulting in brain disorders. For the late stage, mouse embryos were infected with MCMV by injecting the virus into the cerebral ventricles on E15.5. Brains of the offspring showed massive necrosis with gliomesodermal proliferation in the cerebral cortex. Viral antigen-positive cells were observed in laminar array in the lesion-free cortex and the hippocampus, suggesting that the infected cells migrate in association with the lamina formation. Imrnuno-histochemical double-staining showed that brain cells susceptible to MCMV infection may be mainly neuronal and endothelial cells, resulting in cerebral atrophy with reduction of neuronal cells and cystic lesions, presumably due to ischemic vascular changes.     

Pathogenesis of reactivated latent murine cytomegalovirus infection.

Sixteen weeks after inoculation, murine cytomegalovirus (MCMV) can no longer be detected in the tissues of mice. However, a 2-week course of immunosuppression with antilymphocyte serum and cortisone acetate results in reactivation and dissemination of the latent virus in all animals. In this study of reactivation, MCMV was first detected in the liver, usually during the first week of immunosuppression, and virus replication was shown to be restricted to hepatocytes. Subsequently, a viremia occurred, with spread of infection to other organs. The highest titers of virus were reached in salivary glands in which replication occurred in serous acinar cells. In the lung, virus-specific abnormalities were difficult to detect because of superimposed bacterial and fungal infections. However, interstitial pneumonitis could be produced when cortisone acetate was deleted from the immunosuppressive regimen. Although the site of virus latency has not been defined, this model system will be useful for study of reactivation of latent cytomegalovirus infection.     

Pathogenesis of murine cytomegalovirus infection: the macrophage as a permissive cell for cytomegalovirus infection, replication and latency.

Macrophages harvested from the peritoneal cavities of mice of several strains were permissive to infection with murine cytomegalovirus (MCMV). Macrophages from six mouse strains released equivalent amounts of plaque-forming virus into the culture fluids and cells from mouse strains scored similarly in numbers of infectious centres. Twenty to 50% of the infected macrophages obtained after thioglycollate activation formed infectious centres. When studied by in situ hybridization, more than 82% of infected macrophages (with or without thioglycollate activation) contained MCMV DNA. Macrophages obtained from latently infected mice were examined for their content of MCMV. Using co-cultivation assays, MCMV was frequently recovered from thioglycollate activated macrophages harvested from latently infected mice but only rarely recovered from non-activated macrophages. MCMV DNA--mouse DNA hybridization assays revealed four to seven virus genome DNA copies per 100 cells. These studies indicate that macrophages harvested from mice susceptible (BALB/cSt) or resistant (C3H) to MCMV infection replicated virus equivalently and that macrophages are a reservoir of MCMV during latent and chronic infections. Activation of macrophages may be one of the important steps leading to the exacerbation of in vivo latent infections.     

Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR

Acute virus replication in murine (M) CMV infected C57BL/6 (Cmv1(r)) mice is severely limited by Ly49H+ NK cells, but not in MCMV infected BALB/c or BXD8 (Cmv1(s)) mice that lack Ly49H+ NK cells. Interestingly, other NK cell receptors may also play a role in MCMV immunity, as CMV encoded gp40 protein can diminish expression of protein ligands recognized by the NK cell receptor NKG2D. To determine whether other additional gene products might influence MCMV immunity, we designed an efficient, sensitive and reliable method for screening resistance or susceptibility phenotypes in mice. Although multiple methods are frequently used to detect and quantify infectious MCMV in mouse tissue samples collected during acute viral infection, these are not readily adaptable to high-throughput screening strategies. Hence, we utilized real-time PCR for detection and quantitative measurement of infectious MCMV genomes present in various tissues of infected mice. MCMV genomic sequence was accurately and reproducibly detected over the range 10(2)-10(8) molecules in mouse genomic DNA samples using this methodology. Importantly, it was found that quantitative real-time PCR and viral plaque assay measurements of MCMV in tissues collected from infected mice, including resistant and susceptible strains, were directly correlated. Moreover, quantitative real-time PCR results obtained during a 3-week time-course study of virus replication in spleens, livers and salivary glands of infected mice demonstrated sensitive, accurate and reproducible detection and measurement of infectious MCMV.     

Immunohistochemical localization of components of the immune barrier in the olfactory mucosae of salamanders and rats.

Immunohistochemical techniques were used to investigate the cellular distribution of components of the secretory immune system, including secretory immunoglobulin, secretory piece, and J chain, as well as other immunoglobulins and nonspecific defense factors in the olfactory mucosae of salamanders and rats. In the salamander, secretory immunoglobulin M, and J chain were localized in duct and acinar cells of Bowman's glands, in B lymphocytes, and in sustentacular cells in immature regions of the olfactory mucosa. Lactoferrin and lysozyme were also present in Bowman's glands, in sustentacular cells in immature regions of the olfactory mucosa, and in blood cells in the lamina propria. Olfactory nerve section resulted in the presence of increased numbers of secretory immunoglobulin-immunoreactive B lymphocytes and in an altered distribution of IgM, secretory piece, and lactoferrin. In the rat, secretory immunoglobulin A and J chain were localized in duct and acinar cells of Bowman's glands and in B lymphocytes in the lamina propria. Secretory piece could be demonstrated in Bowman's glands only in rats that had a prior viral infection. Other defense factors, localized in the lamina propria, included IgG in the connective tissue stroma and in B lymphocytes, IgD-immunoreactive B lymphocytes, and IgE-immunoreactive cells that were identified as mucosal mast cells. Lactoferrin and lysozyme were present in serous acinar cells of Bowman's glands and in blood cells. These results demonstrate that the olfactory mucosa is protected from pathogenic invasion by the secretory immune system as well as other immunoglobulins, lactoferrin, and lysozyme.     

Distribution of mouse cytomegalovirus in organs of white mice experimentally infected by natural route

One, 10, 21-day-old and adult mice were inoculated by peroral and/or intranasal routes with mouse cytomegalovirus (MCMV). In animals surviving generalized infection, the virus could be demonstrated in salivary glands up to 123 days postinfection (p.i.). In mouse females which had eaten their infected and diseased offspring, the virus was detectable in salivary glands up to day 121, p.i. On day 16 p.i., the virus was present in salivary glands, lungs and kidneys of mice of different age groups, but no virus was recovered from their Gasserian ganglia. These results were compared with those obtained after infection with murine alpha herpesvirus.     

The effects of protein malnutrition on the pathogenesis of murine cytomegalovirus disease.

BALB/c and CBA mice maintained on low (4%) or normal (18%) protein diets for 2 weeks after weaning were infected with a sublethal dose of murine cytomegalovirus, adjusted in proportion to body weight. Viral replication, histopathological changes and humoral responses to the virus were compared between the dietary groups 2-42 days post infection (p.i.). Higher numbers of viral antigen-positive cells and/or more prominent tissue necrosis were noted in the livers, spleens, hearts, adrenal glands, kidneys and bone marrows of infected protein-deficient mice. These mice also showed a delayed onset of leucocytic exudation in their livers and salivary glands, relative to infected mice on the normal diet. Second peaks of viral replication were detected by plaque assays in livers and spleens from protein-deficient mice and in livers from normal mice 12-18 days p.i., but few antigen-positive cells and no tissue necrosis were observed. Virus also persisted at higher titres in the salivary glands from protein-deficient mice. Although cellular immunity may be defective in these mice, humoral IgG and IgM responses to the virus were not inhibited. The influence of genetic factors on the pathogenesis of murine cytomegalovirus disease in protein-deficient mice is discussed.     

Immunohistochemical localization of components of the immune barrier in the olfactory mucosae of salamanders and rats

Immunohistochemical techniques were used to investigate the cellular distribution of components of the secretory immune system, including secretory immunoglobulin, secretory piece, and J chain, as well as other immunoglobulins and nonspecific defense factors in the olfactory mucosae of salamanders and rats. In the salamander, secretory immunoglobulin M, and J chain were localized in duct and acinar cells of Bowman's glands, in B lymphocytes, and in sustentacular cells in immature regions of the olfactory mucosa. Lactoferrin and lysozyme were also present in Bowman's glands, in sustentacular cells in immature regions of the olfactory mucosa, and in blood cells in the lamina propria. Olfactory nerve section resulted in the presence of increased numbers of secretory immunoglobulin-immunoreactive B lymphocytes and in an altered distribution of IgM, secretory piece, and lactoferrin. In the rat, secretory immunoglobulin A and J chain were localized in duct and acinar cells of Bowman's glands and in B lymphocytes in the lamina propria. Secretory piece could be demonstrated in Bowman's glands only in rats that had a prior viral infection. Other defense factors, localized in the lamina propria, included IgG in the connective tissue stroma and in B lymphocytes, IgD-immunore-active B lymphocytes, and IgE-immunoreactive cells that were identified as mucosal mast cells. Lactoferrin and lysozyme were present in serous acinar cells of Bowman's glands and in blood cells. These results demonstrate that the olfactory mucosa is protected from pathogenic invasion by the secretory immune system as well as other immunoglobulins, lactoferrin, and lysozyme.     

A new mutation involving the sublingual gland in NFS/N mice. Partially arrested mucous cell differentiation.

A new mutation in mice affecting the mucous cell differentiation of the sublingual glands is described. The normal mouse sublingual glands are mucus-secreting and virtually all the acinar cells differentiate to mucus-rich cells by the day of birth. In contrast, all endpieces of newborn mutant mice consisted of acini of immature cuboidal cells. However, normal mucous cells, staining intensively with mucin-specific stains such as Alcian blue at pH 2.5 or mucicarmine, appeared in the mutant mice from an early age singly or in groups in a small number of acini, and their number apparently increased with age to occupy over 30% of the total acinar cells. Ultrastructurally, irregular secretion granules of varying electron-density, distinct from ordinary sublingual mucin granules, were frequently observed in the cytoplasm of the immature acinar cells in the mutant phenotype. The genetic analysis showed that a single autosomal recessive gene determined the observed abnormality. This is the first salivary gland mutation and will provide a critical model for the study of salivary mucous cell differentiation.     

Adenovirus-mediated gene expression in isolated rat pancreatic acini and individual pancreatic acinar cells

 In this study we have examined the feasibility of using replication-deficient recombinant adenoviral vectors to transfer and express genes in pancreatic acinar cellsin vitro. We infected primary cultures of both isolated pancreatic acini and individual acinar cells with a recombinant adenovirus containing the coding sequence for β-galactosidase. Our data demonstrate that recombinant adenoviruses readily infect pancreatic acinar cellsin vitro. Close to 100% infection and maximal β-galactosidase expression were obtained, when acini or acinar cells were infected with 5×10⁶ or 10⁶ plaque-forming units (pfu) of virus per millitre of acini or acinar cell suspension, respectively. Examination of the time-course of β-galactosidase expression showed that there was a lag of approximately 6 h before β-galactosidase levels increased. Thereafter β-galactosidase expression increased rapidly. By 20 h post-infection β-galactosidase activity had increased from undetectable levels to 2.5–3.0 units/mg of cellular protein. Acini/acinar cells maintained a robust secretory response after adenoviral infection. The cholecystokinin-octapeptide (CCK8) dose/response curves for amylase secretion for acini and acinar cells infected with 5×10⁵ and 1×10⁵ pfu/ml of virus, respectively, were biphasic, with maximal amylase secretion being stimulated by 1 nM CCK8. In addition, the dose/response curves were identical to those obtained from control, sham-infected, acini/acinar cells. Our findings indicate that replication-deficient recombinant adenoviral vectors will be excellent tools to transfer and express genes in isolated pancreatic acini or acinar cells. Received: 27 March 1998 / Received after remission: 6 May 1998 / Accepted: 7 May 1998     

Induction of virus specific and H-2 restricted cytotoxic T cells by UV inactivated murine cytomegalovirus

Summary Infection of C3H mice with live or UV-inactivated murine cytomegalovirus (MCMV) was able to generate population(s) of lymphocytes in the spleen (CTL) which could exert a lytic effect against L cells infected with MCMV but not against uninfected or those infected with HSV-1. The effector cells proved to be theta-bearing T cells and the lysis of target cells was H-2 restricted. Data presented show that early viral protein synthesis but not viral DNA synthesis was necessary for the appearance of relevant antigenic determinant(s) on target cells. The results of co-capping experiments suggest that H-2 molecules may have close association with MCMV induced product(s) as also with murine leukemia virus glycoprotein (gp 70) which is carried by normal L cells. Despite this observation, anti-H-2 serum effectively blocked the cytolysis whereas anti-gp 70 and anti-MCMV sera failed. Anti-MCMV serum was effective in blocking cytolysis, only if the L cells were infected for 24 hours and then used as targets. MCMV infected L cells which were coated externally with inactivated Sendai virus could be effectively recognised by MCMV as also by sendai specific CTL. That the cytotoxicity exerted on such targets was of specific nature was revealed by the results of competitive blocking experiments with unlabelled targets.     

Cadaveric skin allograft-associated cytomegalovirus transmission in a mouse model of thermal injury.

As a routine procedure to provide temporary coverage for burn wounds, cadaveric skin allografts have been used in patients with massive thermal injuries. In this study, CMV infection associated with skin grafting was investigated. Graft-associated CMV transmission was shown in a mouse model of thermal injury. Skins from mice 100 days after a nonlethal dose of murine CMV (MCMV) infection contained MCMV DNA and mRNA, although the virus was not isolated from these murine skins. When these skins were grafted to burned mice, the marked growth of MCMV was demonstrated in salivary glands. No viral growth was shown in the salivary glands of unburned mice or CMV sero(+) mice after grafting with these skins. When severe combined immunodeficient beige (SCID-beige) mice were used as recipients for CMV sero(+) skins, all mice died within 30 days after the grafting. Only 1 PFU/mouse of MCMV was shown to be 1 LD(50) in SCID-beige mice, while a 50% mortality rate was shown in normal unburned mice infected with 5 x 10(5) PFU/mouse of MCMV. This indicates that a very small amount of CMV contained in skins is sufficient to induce CMV infection in immunocompromised hosts. On the other hand, human CMV (HCMV) DNA and mRNA were detected by PCR analysis in 55% (DNA) and 33% (mRNA) of cadaveric skins, although the isolation of HCMV from cadaveric skin homogenates was not achieved in tissue cultures. CMV sero(-) patients with severe burn injuries may have a high risk for CMV infection associated with allografts of cadaveric skins.     

Fine structure of the 7 postnatal days Calomys callosus palatine salivary glands.

The aim of this study has been to determine the ultrastructural characteristics of the minor palatine salivary glands on the seventh day of development and to demonstrate wether their secretion is mucous, serous or seromucous by light, transmission and scanning electron microscopy. This study has shown that the palatine gland acinar cells are predominantly mucous with some serous units. These cells contain electron dense (serous) and low electron dense (mucous) granules in the apical portions. The cytoplasmatic organelles like mitochondria, Golgi apparatus and rough endoplasmic reticulum are localized in a supranuclear portion. We could also observe the flattened myoepithelial cells surrounding the basal part of the acini with myofilaments, Golgi apparatus and mitochondria. Desmosomal junctions and membrane interdigitations are present between the acinar and the myoepithelial cells. A basal lamina, divided in two layers, an electron dense and an electron lucent is present between the glandular stroma which is composed of dense connective tissue and the endpieces.     

In vivo administration of monoclonal antibody to the NK 1.1 antigen of natural killer cells: effect on acute murine cytomegalovirus infection

Monoclonal antibody to the NK 1.1 antigen, found on the natural killer cells of a number of strains of mice, specifically suppresses NK cell function when given in vivo. Using this monoclonal antibody, we have examined the effects of specific suppression of natural killer (NK) cells in vivo on acute murine cytomegalovirus (MCMV) infection in C57BL/10ScN mice. Administration of antibody to NK 1.1 substantially lowered the resistance of C57BL/10ScN mice to lethal virus challenge. In addition, antibody administration prior to intraperitoneal infection significantly increased MCMV replication in salivary glands, lungs, and spleens. In C3H/HeN mice, a strain that lacks the NK 1.1 antigen, antibody to NK 1.1 had no effect on virus replication or lethal infection. Thus, in vivo administration of monoclonal antibody to NK 1.1 alters the course of acute MCMV infection. These findings further substantiate the role of NK cells in defense against acute MCMV infection.     

Prolonged infection of mouse brain neurons with murine cytomegalovirus after pre- and perinatal infection

Summary The susceptibility of mice at different developmental stages to a relatively low titer of cell culture-passaged murine cytomegalovirus (MCMV) infection was compared in terms of the urinary excretion of MCMV examined by plaque assay and in terms of the distribution of viral infection, determined by immunohistochemistry, using antibodies specific to the early nuclear antigen of MCMV. Viral infection on day 8.5 of gestation (E8.5) into the conceptus and intraperitoneal infection on day 15.5 of gestation (E15.5), postnatal day 2 (P2), postnatal day 11 (P11), and 30 days after birth (P30), respectively, were performed. Embryonal and perinatal mice were more susceptible to MCMV in terms of urinary excretion of the virus and the presence of viral antigen-positive cells in the brain, lungs, and kidneys. In the embryonal and perinatal infection, the viral antigen-positive cells in the neurons of the cerebral cortex and hippocampus were retained late after birth, even though the positive cells in the lungs and kidneys had disappeared. In the mice infected on E8.5, small clusters of viral antigen-positive cells were detected only in the cortex and hippocampus late after birth, without the urinary excretion of virus. These results suggest that when mice are infected with MCMV at the embryonal and perinatal stages, elimination of the infected neurons is delayed compared with that of the other cells in the lungs and kidneys. These findings provide a model for the analysis of pathogenesis of the subclinical congenital CMV infection that manifested clinically late after birth in humans as brain disorders.