Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Protective effects of losartan in mice with chronic viral myocarditis induced by coxsackievirus B3

AimTo investigate whether losartan has protective effects in mice with chronic viral myocarditis induced by coxsackievirus B3 (CVB3).Main methodsThirty two male Balb/c mice were intraperitoneally injected with CVB3 (10 × TCID₅₀) to induce chronic viral myocarditis (CVM). Losartan at 12.5 mg/kg (n = 16) or normal saline (n = 16) were orally administered daily for 28 days to these mice. Uninfected mice (n = 6) were used as controls. On day 29, all mice underwent anesthesia and echocardiography prior to sacrifice. Serum IL-17, IL-4, IFN-γ and TNF-α levels were measured by enzyme-linked immunosorbent assay, and cardiac tissues were histologically examined after hematoxylin & eosin staining. In addition, the effect of losartan on the virus titers in primary cultured neonatal rat cardiomyocytes infected with CVB3 was measured on Hep-2 cells at 72 h post infection.Key findingsMice infected with CBV3 had significantly increased mortality, heart/body weight ratios, necrosis and inflammatory scores and decreased cardiac ejection fractions, compared with the controls (all P < 0.05). Losartan significantly decreased mortality from 40.0% to 12.5%, heart/body weight ratios from 7.08 ± 2.17 to 4.15 ± 0.99, and necrosis and inflammatory scores from 3.33 ± 0.50 to 2.50 ± 0.65 (all P < 0.05), and increased ejection fractions from 55.80 ± 9.25 to 72.31 ± 12.15 (P < 0.05). Losartan significantly enhanced IL-4, and decreased IFN-γ, TNF-α and IL-17 (all P < 0.05). In the in vitro experiment, losartan had no influence on virus titers.SignificanceLosartan protects mice against CVB3-induced CVM, most likely through upregulating Th2 responses, and down-regulating Th1 and Th17 responses.     

Divergent effects of leptin in mice susceptible or resistant to obesity.

Consumption of a high-fat diet decreases hypothalamic neuropeptide Y (NPY) and increases proopiomelanocortin (POMC) and brown adipose uncoupling protein (UCP)-1 mRNA in obesity-resistant SWR/J but not obesity-prone C57Bl/6J mice. Although leptin was elevated in both strains in response to a high-fat diet, its role in the development of diet-induced obesity has remained unclear since insulin and other factors that affect similar tissue targets are altered. Thus, we administered recombinant leptin by subcutaneous infusion to chow-fed mice to mimic the changes in plasma leptin across its broad physiologic range. We observed strain differences in responsiveness to reduced and elevated leptin levels. A reduction in leptin during fasting evoked a greater response in C57Bl/6J mice by decreasing energy expenditure and thyroxin, increasing corticosterone and stimulating food intake and weight gain during refeeding. However, C57Bl/6J mice were less responsive to an increase in leptin in the fed state. Conversely, the leptin-mediated response to fasting was blunted in SWR/J mice, whereas an increase in leptin profoundly reduced food intake and body weight in SWR/J mice fed ad libitum. Sensitivity to fasting in C57Bl/6J mice was associated with higher hypothalamic NPY mRNA and reduced POMC and UCP-1 mRNA expression, while the robust response to high leptin levels in SWR/J mice was associated with suppression of NPY mRNA. These results indicate that differences in leptin responsiveness between strains might occur centrally or peripherally, leading to alteration in the patterns of food intake, thermogenesis and energy storage.     

T cells expressing the gamma delta T-cell receptor potentiate coxsackievirus B3-induced myocarditis.

Initial studies determined whether intraperitoneal (i.p.) injection of BALB/c mice with 0.1, 1.0, and 10 mg of adriamycin (a cardiotoxic anthracycline antibiotic) for times ranging between 1 and 9 weeks prior to i.p. injection of 10(5) PFU of coxsackievirus B3 (CVB3) would alter the severity of virus-induced myocarditis. Prior adriamycin exposure enhanced pathogenicity of a poorly pathogenic CVB3 variant (H310A1) but had no effect on myocarditis produced by the pathogenic variant (H3). Cardiac virus concentrations were equivalent in H3- and H310A1-infected mice irrespective of adriamycin treatment. BALB/c mice treated with either 0.1 ml of complete Freund's adjuvant (CFA), 10 mg of adriamycin, or 10(5) PFU of H3 and H310A1 i.p. developed cytolytic Thy 1.2+ lymphocytes (CTL) to H3-infected myocytes 7 days later. CFA-, adriamycin-, and H3-treated mice developed CTL expressing the gamma delta+ T-cell receptors, while H310A1-infected animals did not. Only H3- and H310A1-infected mice developed alpha beta+ CTL. Treatment of BALB/c mice with 0.1 ml of CFA 5 days prior to H310A1 infection dramatically increased myocarditis. Selective depletion of gamma delta+ T cells abrogated this effect. The ability of gamma delta+ T cells to augment the pathogenicity of H310A1 infection was confirmed by adoptive transfer of CFA-stimulated T cells depleted of either gamma delta- or gamma delta+ cells into H310A1-infected recipients.     

Variation in susceptibility of Balb/c mice to coxsackievirus group B type 3-induced myocarditis with age

The severity of cardiac lesions in coxsackievirus group B, type 3 (CVB3)-infected Balb/c mice depends upon both the age and the sex of the animal at the time of viral inoculation. Suckling animals (1-3 weeks old) of either sex develop few cardiac lesions. Thereafter, males rapidly demonstrate increasing disease susceptibility peaking at 16-18 weeks old and then decreasing susceptibility from 20 to 40 weeks of age. Female susceptibility increases much more gradually and myocarditis in this sex never reaches maximal levels as seen in males. Increased susceptibility correlates with virus concentrations in the heart and anti-CVB3 titers in the serum. Cardiac injury is dependent on functional T lymphocytes since treatment of the animals with rabbit anti-mouse thymocyte serum abrogates inflammation and myocyte necrosis. Sex-associated steroid hormones influence both virus concentrations and immune responses in mice and are probably responsible for variations in disease susceptibility throughout the animal's life.     

LPS promotes CB3-induced myocarditis in resistant B10.A mice.

Coxsackie virus B3 (CB3) infection of A/J or A.SW mice results in autoimmune myocarditis characterized by a diffuse mononuclear cell infiltrate and heart-specific autoantibodies. C57BL/10 congenic mice that are identically treated are resistant to this disease. CB3-infected resistant B10.A mice were treated with LPS to determine if this immunomodulator alters disease susceptibility. In contrast to mice infected only with CB3 or treated only with LPS. CB3-infected/LPS-treated (CB3/LPS) B10.A mice developed autoimmune myocarditis similar to that observed in susceptible A/J or A.SW mice. By Day 14, CB3/LPS-induced disease was characterized by significant mortality, myocardial immunoglobulin deposition, and mononuclear cell infiltration of the heart. Immunohistochemical examination revealed deposits of IgG in the heart tissue and serum IgG autoantibodies reactive with sarcolemmal and fibrillary antigens in normal heart tissue. This serum IgG reacted with normal mouse cardiac antigens of a wide range of molecular weights by Western immunoblotting. Because LPS treatment is capable of increasing cytokine levels as well as MHC Class I and Class II expression in heart tissue, it suggests that these factors may contribute to susceptibility to autoimmune myocarditis in CB3-infected mice.     

Interferon and neutralizing antibody in sera of exercised mice with coxsackievirus B-3 myocarditis.

Weanling ICR albino Swiss mice were inoculated ip with 1.9 x 10(4) PFU of coxsackievirus B-3 (Nancy) and subsequently forced to swim vigorously daily in a preheated pool (33 degrees). Viremias and virus in hearts of exercised mice were respectively 75 x 1000 x greater than in infected, but not exercised mice. At 24 hr after inoculation, pooled serum from mice that had been swum had no circulating interferon, while infected but not swum mice had interferon activity at a dilution of 1:10. At 72 hr after infection, circulating interferon disappeared from infected (not swum) mice, but continued to be present in high titers through the sixth day in sucklings forced to swim. Interferon was first detected in the hearts of both groups at 48 hr. Quantities in both infected groups were generally similar. Neutralizing antibodies were found in these baby mice on the 13th day of infection and were 16 x greater in nurslings that were not exercised. Measures of corticosterone taken at 4 PM daily were similar in infected, infected-swum, and uninfected mice.     

Hormonal regulation of CD4(+) T-cell responses in coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infection causes significant cardiac inflammation in male, but not female, B1.Tg.Ealpha mice. This gender difference in disease susceptibility correlates with selective induction of CD4(+) Th1 (gamma interferon-positive) cell responses in animals with testosterone, whereas estradiol promotes preferential CD4(+) Th2 (interleukin-4 positive [IL-4(+)]) cell responses. Differences in immune deviation of CD4(+) T cells cannot be explained by variation in B7-1 or B7-2 expression. Infection significantly upregulated both molecules, but no differences were detected between estradiol- and testosterone-treated groups. Significantly increased numbers of activated (CD69(+)) T cells expressing the gammadelta T-cell receptor were found in male and testosterone-treated male and female mice. In vivo depletion of gammadelta+ cells by using monoclonal antibodies inhibited myocarditis and resulted in a shift from a Th1 to Th2 response phenotype. Taken together, our results indicate that testosterone promotes a CD4(+) Th1 cell response and myocarditis by promoting increased gammadelta+ cell activation.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis

Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.     

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.     

Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis

Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory.     

Colony-forming cells and colony-stimulating activity during listeriosis in genetically resistant or susceptible mice

Serum colony-forming activity (CSA) and colony-forming cells (CFC) of resistant (C57BL/10 ScSn) and susceptible (BALB/cJ) mice were studied during Listeria monocytogenes infection. Key findings were also checked in susceptible CBA/H mice. Prompt, bacterial dose-dependent increases in serum CSA were observed in all mice following infection. In response to the same challenge dose, serum CSA increased more in susceptible mice, possibly because rapid bacterial proliferation lead to high bacterial numbers. Thus CSA is not a limiting factor which accounts for the differences in Listeria resistance, but is produced in response to bacterial load. In uninfected mice, there were higher numbers of colony-forming cells in the bone marrow and spleen of resistant mice than in susceptible mice. By 24 hr postinfection there was a sharp drop in total cell numbers including CFC, in the bone marrow of resistant C57BL/10 ScSn mice. This coincides with the time when monocytes have been first observed in the blood of infected mice and when differences in bacterial growth between the mouse strains were first observable. Since the superior resistance of C57BL/10 mice has been shown to be radiosensitive, it is probable that this larger, readily mobilized reserve of monocyte/granulocyte precursors in the resistant mice plays an important role in early control of infection. The significance of this is discussed.     

Role of CD1d in coxsackievirus B3-induced myocarditis

The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant J alpha 281 gene, but minimal disease in BALB/c CD1d(-/-) animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. V gamma 4(+) T cells show increased activation in both H3-infected BALB/c and J alpha 281(-/-) mice compared with CD1d(-/-) animals. The activated BALB/c V gamma 4(+) T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (K(d)/D(d)) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d(-/-) myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d(+) mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that V gamma 4(+) T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. II. Pathogenesis

The pathogenesis of mousepox due to infection with ectromelia virus strain NIH-79 was characterized in genetically susceptible (BALB/cAnNCr) and genetically resistant (C57BL/6NCr) mice. BALB/c mice inoculated subcutaneous (s.c.) or intranasally (i.n.) had high mortality. Most mice died within 7 days from severe necrosis of the spleen and liver. Necrotic foci in livers of BALB/c mice that survived beyond 7 days often were accompanied by mononuclear cell infiltrates and by hyperplasia of lymphoid tissues. C57BL/6 mice inoculated by either route remained asymptomatic and necrotic lesions were mild or absent, whereas focal non-suppurative hepatitis and lymphoid hyperplasia were prominent. Infectious virus and viral antigen were distributed widely in tissues of BALB/c mice, but had limited distribution in C57BL/6 mice. Both mouse strains had infection of the respiratory tract, genital tract, oral tissues and bone marrow, and BALB/c mice also had infection of the intestines. Both strains also developed serum antibody to vaccinia virus antigen after infection. The results show that ectromelia virus occurs in tissues conducive to mouse to mouse transmission and that the severity and character of mousepox lesions correlate directly with resistance and susceptibility to infection. They also support the concept that cellular immunity contributes to survival from infection.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. I. Clinical responses

Clinical responses to infection with ectromelia virus strain NIH-79 were determined in several strains of inbred mice. All mice were equally susceptible to infection, but mortality was strain dependent. BALB/c AnNCr, A/JNCr, DBA/2NCr and C3H/He/NCr MTV- mice were highly susceptible to lethal infection whereas AKR/NCr and SJL/NCr mice were moderately susceptible and C57BL/6NCr mice were highly resistant. Death rates were influenced strongly by virus dose and by route of inoculation. High doses were associated with early and high mortality. For a given dose, intraperitoneal inoculation resulted in the highest mortality and death rates were progressively reduced in mice inoculated by the footpad, subcutaneous and intranasal routes. Footpad swelling was prominent in resistant mice and in survivors among susceptible strains. Deaths among AKR and SJL mice were sporadic and often occurred late irrespective of virus dose. It is suggested that this pattern could be influenced by secondary contact infections or by immunologic injury associated with host responses to ectromelia virus.     

Trace element changes in the myocardium during coxsackievirus B3 myocarditis in the mouse

During most infections plasma, concentrations of trace elements change, but it is unclear if this reflects changes in infected target tissues. In coxsackievirus B3 (CB3) infection, the myocardium is a target in both humans and mice. The concentrations of 12 trace elements were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) in the myocardium of sham-inoculated controls and infected A/J mice 4 and 7 d postinoculation. The size of the inflammatory lesion was positively correlated to the virus content of the heart, as estimated by histopathology and in situ hybridization, respectively. Iron, cobalt, vanadium, and selenium showed transient changes, whereas for the other elements, tendencies on d 4 were manifest on d 7. A threefold increase in calcium on d 7 suggests prestages of calcification, whereas increases in zinc, selenium, and copper may be the result of the accumulation of immune cells. The magnesium decrease may contribute to the increased sensitivity to cardiac arrhythmias in myocarditis.     

The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis.

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.     

Fractionation and immunologic assessment of KCl-extracted cardiac antigens in coxsackievirus B3 virus-induced myocarditis.

Hypertonic salt extracts prepared from the heart tissues of adolescent CD-1 mice were fractionated on Sephadex G-100 columns. Two separate fractions were obtained. Fraction I, containing the antigenic immunoreactive activity, was able to inhibit the migration of CVB3-PPD immune mouse peritoneal exudate cells (IMPEC) as well as PEC from mice infected with CVB3 virus alone. Fraction II did not have antigenic activity as assessed by the agarose droplet cell migration inhibition assay. As controls, Fraction I prepared from the livers of spleens of CVB3-infected CD-1 mice was unable to inhibit the migration of CVB3 IMPEC. Unimmunized or "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited by Fraction I. Antibodies prepared against Fractions I and II were unable to neutralize CVB3m virus in the plaque reduction test, and polyacrylamide gel analysis revealed multiple bands in 10% SDS gels.     

Infection of primary cultures of murine splenic and thymic cells with coxsackievirus B4

Infection of primary cultures of total splenic and thymic cells from BALB/c and C3H/HeN mice with CVB4 E2 and JVB strains has been investigated. The presence of positive-strand viral RNA within cells was determined by semi-nested RT-PCR, and viral replication was attested by detection of intracellular negative-strand viral RNA and by release of infectious particles in culture supernatants. Viral replication occurred with both CVB4 strains to an extent dependent on the genetic background of the host. No interferon-alpha production was detected in the supernatants of CVB4-infected cultures using biological titration. Together these results suggest that infection of splenic and thymic cells can play a role in virus dissemination, and therefore in the pathophysiology of CVB4 infections.