Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5' nontranslated region

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5' nontranslated region (5'NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5'NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5'NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5'NTR alone or the 5'NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5'NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5'NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.     

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.     

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.     

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.     

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.     

Effect of gold(I) compounds on the virulence of an amyocarditic strain of coxsackievirus B3

Coxsackieviruses, especially B strains (CVB), are known etiological agents of myocarditis. Both amyocardititc and myocarditic strains exist and at least one amyocarditic strain, CVB3/0, can convert to virulence when passaged through selenium or vitamin E-deficient mice. Gold(I)-containing compounds, such as aurothiomalate (ATM) and aurothioglucose (ATG), can act as selenium antagonists. In this study, we examined the effect of intraperitoneal administration of equal doses of ATM or ATG on the virulence of CVB3/0. ATM but not ATG increased mortality in CVB3/0-infected mice. CVB3/0-infected mice treated with ATM had total necrosis of the pancreatic exocrine tissue. Heart damage also occurred in ATM-treated mice but did not correlate with mortality. Increased viral titers and persistence were observed in ATM-treated mice and, to a lesser extent, in ATG-treated mice. Thus, under our conditions, only ATM increased the virulence of CVB3/0, whereas ATG did not. On the other hand, both ATG and ATM inhibited thioredoxin reductase activity in heart and pancreas, but neither affected glutathione peroxidase activity. In contrast, dietary selenium deficiency reduces both enzyme activities. Thus, it is unlikely that these compounds affect virulence by acting as selenium antagonists.     

Murine natural killer cells limit coxsackievirus B3 replication

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.     

Augmentation of pathogenesis of coxsackievirus B3 infections in mice by exogenous administration of interleukin-1 and interleukin-2.

Two variants of coxsackievirus B3 (CVB3) which differ dramatically in the ability to induce myocarditis in BALB/c mice were studied. H3 virus infection of murine monocytes in vitro resulted in release of concentrations of interleukin 1 (IL-1) and alpha/beta interferon that were high compared with those of cells infected with the H310A1 virus variant. In vivo, H3 virus infection caused substantial inflammatory cell infiltration of the myocardium, and lymphocytes from these animals gave predominantly Th1-cell responses to either whole H3 virus or overlapping peptides of the CVB3 vp1 capsid protein, as determined by IL-2 production. In contrast, H310A1 virus infection produced minimal myocarditis and Th1-cell responses, but Th2-cell activation was more pronounced than in H3 virus-infected mice (as determined by IL-4 concentrations). Exogenous treatment of H310A1 virus-infected mice with either IL-1 or IL-2 restored both myocarditis susceptibility and Th1-cell responses to whole virus and vp1 peptides. Furthermore, H310A1 virus-infected mice given exogenous IL-1 showed substantial in situ IL-2 deposition in the myocardium. These results indicate that CVB3-induced myocarditis may depend upon release of specific cytokines during infection and that activation of Th1 cells may be an important factor in pathogenesis.     

Host selenium status selectively influences susceptibility to experimental viral myocarditis

The purpose of the present work was to determine whether dietary selenium (Se) deficiency could influence the injurious effect of human viruses other than Coxsackie virus B3 (CVB3) on mouse heart. Weanling C3H/HeN mice were fed a Se-deficient or Se-adequate diet for 4 wk and then were inoculated intraperitoneally with one of the following viruses: Coxsackie virus B1 (CVB1), echovirus 9 (EV9), Coxsackie virus A9 (CVA9), or herpes simplex 1 (HSV1). Polio virus 1 (PV1) was employed as a negative control. Prior to inoculation, mean serum Se levels were 430 versus 61 ng/mL in adequate versus deficient mice, respectively. Ten days later, hearts were removed and processed by routine histological procedures. Cardiac lesions were scored according to the number and size of myocarditic foci. Significantly greater heart damage resulting from CVB1 and EV9 was observed in Se-deficient than in Se-adequate mice, and the Se status had no influence on CVA9-induced myocarditis. In contrast, heart damage caused by HSV1 was significantly milder in Se-deficient than in Se-adequate mice. Therefore, it may be concluded that the Se status of the murine host selectively influences the degree of viral-induced myocarditic lesions.     

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid‐protein‐specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti‐VP2 and anti‐VP1 sera detected the viral proteins extracted from CVB3‐infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti‐CVB3 antibodies in virus‐infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3‐induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti‐CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus‐induced myocarditis.     

Lymphocytes protect against and are not required for reovirus-induced myocarditis.

Many studies suggest that host lymphocytes are damaging, rather than protective, in virally induced myocarditis. We have investigated the role of lymphocyte-based immunity in murine myocarditis by using a myocarditic reovirus (reovirus serotype 3 8B), nonmyocarditic reoviruses, adoptive transfer experiments, and mice with severe combined immunodeficiency (SCID mice). Prior to infection, passive transfer of monoclonal antibodies specific for 8B capsid proteins protected neonatal mice against 8B-induced myocarditis, indicating that humoral immunity can protect against myocarditis. Some monoclonal antibodies acted by blocking viral spread to and/or replication in the heart. Passive transfer of reovirus-immune, but not naive, spleen cells prior to infection protected neonatal mice from 8B-induced myocarditis. Depletion of either CD4 or CD8 T cells resulted in increased viral titer in the heart but did not abrogate immune cell-mediated protection against myocardial injury. This shows that both CD4 and CD8 T cells can act independently to protect myocardial tissue from reovirus infection. In addition, reovirus 8B caused extensive myocarditis in SCID mice. This confirms a prior report (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989) that T cells are not required for reovirus-induced myocarditis and demonstrates for the first time that B cells are not required for reovirus-induced myocarditis. We used SCID mice and a panel of reoviruses to assess (i) the relationship between growth in the heart and myocardial damage and (ii) the possibility that nonmyocarditic reoviruses exhibit a myocarditic phenotype in the absence of functional lymphocytes. Growth in the heart was not the sole determinant of myocarditic potential in SCID mice. Although 8B induced myocarditis in SCID mice, no or minimal myocarditis was found in SCID mice infected with four reovirus strains previously shown (B. Sherry and B. N. Fields, J. Virol. 63:4850-4856, 1989) to be nonmyocarditic or poorly myocarditic in normal neonatal mice. We conclude that (i) humoral immunity and cellular immunity are protective against, and not required for, reovirus-induced myocarditis and (ii) the potential to induce cardiac damage is a property of the virus independent of lymphocyte-based immunity.     

Coxsackievirus-B3-induced myocarditis: virus receptor antibodies modulate myocarditis

Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.     

柯萨奇病毒B3型诱导的免疫偏离与心肌炎发病关系的研究

目的 研究2种近交系小鼠在柯萨奇病毒B3型(CVB3)感染后辅助性T细胞(Th)免疫偏离对心肌炎发病的影响。方法 用CVB3腹腔感染BALB/c和C57BL/62种近交系小鼠,感染后7d通过检测小鼠血清肌酸激酶(CK)活性,观察心脏外观变化以及心脏石蜡切片H.E染色观察心脏病理改变,比较2种小鼠心肌炎的发病情况;通过体外感染心肌细胞观察病毒复制情况以及体内心脏组织病毒载量的分析,比较2种小鼠对病毒感染和复制的差异;通过检测感染小鼠细胞因子白细胞介素-4(IL-4)、IL-12和γ干扰素(IFN-γ)的表达,抗CVB3VP1抗体的亚型以及T-bet和Gata-3的表达,比较2种小鼠Th免疫偏离的情况。结果 CVB3在体外和体内都可以感染BALB/c和C57BL/6小鼠心肌细胞,但仅BALB/c小鼠感染后可发生明显的病毒性心肌炎,C57BL/6小鼠则不能;BALB/c小鼠感染后表现为Th1型免疫反应而C57BL/6小鼠则偏向于Th2型免疫反应。结论 CVB3感染2种品系小鼠表现为不同的心肌炎发生率,与其诱导了不同类型的免疫偏离密切相关。     

An attenuated variant of Coxsackievirus B3 preferentially induces immunoregulatory T cells in vivo.

BALB/c mice infected with the Woodruff variant of coxsackievirus group B type 3 (CVB3W) develop myocarditis mediated by autoimmune cytolytic T lymphocytes. A variant of CVB3W (designated H3-10A1) which infects the myocardium but induces minimal mortality of myocarditis compared to the parental virus was selected. Although H3-10A1 infections stimulate normal CTL responses to CVB3-infected myocytes, the autoimmune response to myocardial antigens is absent. Treatment of H3-10A1-infected mice with 50 mg of cyclophosphamide per kg of body weight, a treatment which preferentially eliminates suppressor cells, allows both the development of the autoimmune cytotoxic T-lymphocyte response and the expression of myocarditis. Similar treatment of CVB3W-infected mice had no effect on the disease. The presence of the immunoregulatory cells was confirmed by adoptive transfer of T lymphocytes from either H3-10A1 or CVB3W-infected donor mice into syngeneic CVB3W-infected recipients. Animals given H3-10A1-immune cells had minimal myocardial inflammation, while animals given CVB3W-immune lymphocytes developed enhanced cardiac disease. Elimination of the T-lymphocyte population from the donor cells prior to transfer abrogated suppression with the H3-10A1-immune population, showing that immunoregulation depended upon T lymphocytes. Both H3-10A1 and CVB3W have cross-reactive epitopes between the adenine translocator protein and the virion which are indicative of antigenic mimicry and may be the basis for the autoimmunity to cardiac antigens. These results suggest that immunoregulatory T cells may be primarily responsible for the nonpathogenicity of the H3-10A1 variant.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune mechanisms.     

Inhibition of coxsackie B virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.     

Aggravation of coxsackievirus, group B, type 3-induced myocarditis and increase in cellular immunity to myocyte antigens in pregnant Balb/c mice and animals treated with progesterone

Male Balb/c mice inoculated with a heart-adapted variant of coxsackievirus, group B, type 3 (CVB3M) develop severe myocarditis characterized by extensive focal lesions of inflammatory cells and necrosis of the myocardium. Females generally develop minimal myocarditis except when infected during the first and third trimesters of pregnancy. Enhanced myocarditis is usually accompanied by elevations in virus concentrations in the heart, virus-specific antibody titers, and lymphocyte mediated cytolytic activity to both uninfected and CVB3M-infected myocytes in vitro. As previously shown in males, T-lymphocyte-depleted pregnant female mice inoculated with the virus do not develop significant myocarditis indicating that immune rather than virus-mediated myocyte damage is important in myocarditis. Progesterone increases during gestation reaching maximum concentrations during the third week when heart disease is most severe. Administration of progesterone to castrated male and female mice prior to virus inoculation resulted in increased virus concentrations, cellular and humoral CVB3M-specific immunity, and myocarditis. Two hypotheses for exacerbation of the disease with elevated progesterone concentrations have been postulated: the hormone either indirectly increases cellular immune responses by enhancing virus replication, or independently enhances both T-cell responses and virus replication.     

gamma delta+ T cells regulate major histocompatibility complex class II(IA and IE)-dependent susceptibility to coxsackievirus B3-induced autoimmune myocarditis.

Coxsackievirus B3 (CVB3) infection induces myocardial inflammation and myocyte necrosis in some, but not all, strains of mice. C57BL/6 mice, which inherently lack major histocompatibility complex (MHC) class II IE antigen, develop minimal cardiac lesions despite high levels of virus in the heart. The present experiments evaluate the relative roles of class II IA and IE expression on myocarditis susceptibility in four transgenic C57BL/6 mouse strains differing in MHC class II antigen expression. Animals lacking MHC class II IE antigen (C57BL/6 [IA+ IE-] and ABo [IA- IE-]) developed minimal cardiac lesions subsequent to infection despite high concentrations of virus in the heart. In contrast, strains expressing IE (ABo Ealpha [IA- IE+] and Bl.Tg.Ealpha [IA+ IE+]) had substantial cardiac injury. Myocarditis susceptibility correlated to a Th1 (gamma interferon-positive) cell response in the spleen, while disease resistance correlated to a preferential Th2 (interleukin-4-positive) phenotype. Vgamma/Vdelta analysis indicates that distinct subpopulations of gamma delta+ T cells are activated after CVB3 infection of C57BL/6 and Bl.Tg.Ealpha mice. Depletion of gamma delta+ T cells abrogated myocarditis susceptibility in IE+ animals and resulted in a Th1-->Th2 phenotype shift. These studies indicate that the MHC class II antigen haplotype controls myocarditis susceptibility, that this control is most likely mediated through the type of gamma delta T cells activated during CVB3 infection, and finally that different subpopulations of gamma delta+ T cells may either promote or inhibit Th1 cell responses.