Coxsackievirus B3-induced chronic myocarditis in outbred NMRI mice

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population. STUDY DESIGN/METHODS: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA. RESULTS: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection. CONCLUSIONS: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.     

Experimental CVB3-induced chronic myocarditis in two murine strains: Evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection

In order to analyse the relationships between enteroviral replication and the myocardial damage at the onset of chronic cardiac infection, 2 mouse strains with different degrees of immunological competence (NMRI nu/nu, DBA/2) were infected by a myocarditic Coxsackie virus B3 (CVB3-M1) variant. At 31 days post-inoculation, plaque-forming assay, polymerase chain reaction (RT-PCR), and immunohistochemistry were carried out for detecting viruses and viral components in the myocardium. The virological findings were related to histopathological changes in the myocardium as well to the dilatation of both cardiac ventricles. Chronic myocardial lesions characterized by large fibrosis areas and interstitial inflammatory infiltrates were detected together with cardiomegalia in 52.6% (10/19) of athymic mice and in 9% (2/22) of euthymic mice. Viral replication foci were located and were found only in myocarditic cells adjacent to myocardial inflammatory lesions by immunostaining myocardial tissue sections with anti-serum to VP1 virus capsid protein. Using PCR followed by microwell capture hybridization assay, a large excess of viral positive strand RNA over negative strand was semiquantified in heart tissue from mice with chronic myocarditis, whereas approximately equal amounts of plus and minus strand RNA were detected in cases of persistent cardiac infection without chronic myocardial injuries. These findings provide evidence of the major role of viral replication in the pathogenesis of chronic murine CVB3-induced cardiomyopathy. The results indicate that the cardiac persistence of enteroviral RNAs can be observed without chronic cardiomyopathy, which could be explained by a defective viral positive RNA replication. J. Med. Virol 52:206–214, 1997. © 1997 Wiley-Liss, Inc.     

Selenium deficiency contributes to the chronic myocarditis in coxsackievirus-infected mice

Both coxsackievirus B3 (CVB3) infection and selenium (Se) deficiency play a pivotal role in Keshan disease of the heart. The Se deficiency was known to contribute to the CVB3-induced myocarditis in acute and subacute phase of infection. However, its effect on the myocarditis in chronic phase of infection has not been examined yet. To address this question, we kept mice on a Se-replete or Se-deficient diet for 28 days, infected them intraperitoneally with CVB3 and maintaining previous diets, we examined them for next 90 days for several parameters indicative of the infection or disease. We found out that the mice on the Se-deficient diet exhibited a higher mortality, lower serum glutathione peroxidase (GPx) activity, evident histopathological changes indicative of myocarditis, and a higher level of viral RNA in the heart. Summing up, these data suggest that the Se-deficiency creates a chronic myocarditis-prone condition by fostering the active virus replication.     

Sustained nitric oxide synthesis contributes to immunopathology in ongoing myocarditis attributable to interleukin-10 disorders

Ongoing coxsackievirus B3 (CVB3) myocarditis is characterized by persistence of viral RNA and chronic inflammation primarily mediated by macrophages and T cells. Activated macrophages produce anti-viral effector molecules comprising reactive nitrogen intermediates; however, reactive nitrogen intermediates also contribute to host tissue damage. Controlled activation of macrophages depends on interferon (IFN)-gamma and interleukin (IL)-10. To evaluate mechanisms involved in CVB3-induced pathogenesis of myocarditis, we determined the relationship of inducible nitric-oxide synthase (iNOS) mRNA expression with IFN-gamma and IL-10 secretion during CVB3 infection in different mouse strains. We found in susceptible A.BY/SnJ mice that develop ongoing myocarditis, a low and delayed IFN-gamma secretion and highly diminished IL-10 production compared with resistant C57BL/6 mice. Consequently, iNOS mRNA synthesis was delayed but clearly prolonged in susceptible mice. IL-10 gene-deficient mice confirmed the regulatory role of IL-10 in the outcome of CVB3 myocarditis. These mice did not establish a persistent cardiac infection and revealed IFN-gamma secretion kinetics similar to resistant mice but showed a slightly elongated cardiac iNOS mRNA expression resulting in extended myocarditis. We conclude that coordinated secretion of IFN-gamma and IL-10 is crucial for the effective resolution of CVB3 myocarditis. Moreover, lack of regulatory IL-10 leads to uncontrolled iNOS mRNA production, thus contributing to ongoing myocardial injury.     

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.     

Persistence of virus and viral genome in myocardium after coxsackievirus B3-induced murine myocarditis.

Following infection with Coxsackievirus B3 (CVB3), A-strain mice develop ongoing myocarditis that persists after the virus ceases to be cultivatable from heart tissue. We studied the natural history of this virus-induced but apparently autoimmune inflammation by means of in situ hybridization (ISH) and by polymerase chain reaction (PCR). Both ISH and culture allowed detection of virus up to 2 weeks post-infection in virtually all heart tissues. In contrast, PCR revealed the presence of viral genome for a substantially longer period of time, i.e. at least 34 days after CVB3 infection. Similarly, the majority of mice showed myocardial inflammation at this time point. However, the persistence of virus did not correlate with ongoing myocarditis, and vice versa. Most mice with ongoing myocarditis produced heart myosin autoantibodies, most probably as a result of tissue damage. The lack of correlation between presence of ongoing inflammation and persistence of virus supports our previous view that the late phase of CVB3-induced myocarditis is mediated by autoimmunological mechanisms.     

Amelioration of coxsackievirus B3-mediated myocarditis by inhibition of tissue inhibitors of matrix metalloproteinase-1

Coxsackievirus B3 (CVB3) is a major cause of acute myocarditis, a serious condition that is refractory to treatment. Myocardial damage results in tissue remodeling that, if too extensive, may contribute to disease. Remodeling is achieved by extracellular proteolysis mediated by the matrix metalloproteinases (MMPs), and MMP activity is counterbalanced by tissue inhibitors of MMPs (TIMPs). We show herein that TIMP-1 expression is induced in the myocardium by CVB3 infection. Surprisingly, TIMP-1 knockout mice exhibited a profound attenuation of myocarditis, with increased survival. The amelioration of disease in TIMP-1 knockout mice was not attributable to either an altered T-cell response to the virus or to reduced viral replication. These data led us to propose a novel function for TIMP-1: its highly localized up-regulation might arrest the MMP-dependent migration of inflammatory cells at sites of infection, thereby anatomically focusing the adaptive immune response. The benefits of TIMP-1 blockade in treating viral myocarditis were confirmed by administering, to wild-type animals, TIMP-1-specific siRNA or polyclonal antisera, both of which diminished CVB3-induced myocarditis. These unexpected findings indicate that increased TIMP-1 expression exacerbates, rather than ameliorates, CVB3-induced myocarditis and, thus, that TIMP-1 may represent a target for the treatment of virus-induced heart disease.     

beta2-microglobulin-associated regulation of interferon-gamma and virus-specific immunoglobulin G confer resistance against the development of chronic coxsackievirus myocarditis

To gain insight into the strategies of the immune system to confer resistance against the development of chronic coxsackievirus B3 (CVB3) myocarditis we compared the course of the disease in C57BL/6 mice, beta2-microglobulin knockout (beta2m(-/-)) mice, and perforin-deficient (perforin(-/-)) mice. We found that perforin(-/-) mice as well as immunocompetent C57BL/6 mice reveal a resistant phenotype with complete elimination of the virus from the heart in the course of acute myocarditis. In contrast, myocardial CVB3 infection of beta2m(-/-) mice was characterized by a significantly higher virus load associated with a fulminant acute inflammatory response and, as a consequence of virus persistence, by the development of chronic myocarditis. Interferon-gamma secretion of stimulated spleen cells was found to be significantly delayed in beta2m(-/-) mice compared to perforin(-/-) mice and C57BL/6 control mice during acute myocarditis. In addition, generation of virus-specific IgG and neutralizing antibodies were found to be significantly decreased in beta2m(-/-) mice during acute infection. From these results we conclude that protection against the development of chronic myocarditis strongly depends on the expression of beta2m, influencing the catabolism of IgG as well as the production of protective cytokines, such as interferon-gamma. Moreover, CVB3-induced cardiac injury and prevention of chronic myocarditis was found to be unrelated to perforin-mediated cytotoxicity in our model system.     

Outbred mice infected by an encephalomyocarditis virus variant: A model for studying chronic viral heart disease

Male 8 to 20-week-old NMRI mice (an outbred strain) infected with the encephalomyocarditis virus (EMCV) plaque variant (PV) 7 consistently develop a distinct myocarditis with a relatively low mortality (21%). Myocarditis occurs in essence independent of the virus dose applied, and other internal organs are not affected. Nevertheless, 3.5-week-old NMRI mice perished within 5 days of virus inoculation and exhibited disseminated myofibrillar degeneration (MFD); this obviously virus-induced myocardial damage was accompanied by scanty inflammatory infiltrates. EMCV PV7 infection of adult male C57B1/6 and DBA/2 mice causes myocarditis comparable to that seen in NMRI mice. In DBA/2 mice, however, the virus-induced myocardial necrosis is complicated by subtotal calcification. This strain has a genetically determined spontaneous calcification of the myocardium, as shown by the study of uninfected controls. EMCV PV7-infected NMRI mice appear a promising model for study of long-term effects of viral myocarditis, possibly including cardiomyopathy. Furthermore, this outbred mouse strain offers the possibility of examining the pathogenesis of direct viral cytolysis and its relation to MFD as well as immunologically mediated cell damage.     

Coxsackievirus B3-induced myocarditis in MHC class II-deficient mice

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in immunocompetent C57BL/6 and MHC class II knockout mice with identical genetic backgrounds. STUDY DESIGN/METHODS: We analyzed the histology and immunohistology of myocardial injury, the replicating virus titer, and antibody response in the early and late phase of disease. RESULTS: CVB3-infected C57BL/6 mice showed acute myocarditis, with spontaneous healing, virus elimination, anti-CVB3 IgM/IgG production, and neutralizing antibody response. In contrast, MHC class II knockout mice developed less severe acute myocarditis, persistence of infiltrations and strong fibrosis, virus persistence, and weak IgG response, with absence of virus neutralizing antibodies. CONCLUSIONS: Immunodeficient organisms are more susceptible to long-term heart muscle injuries after infection with CVB3. The presence of CD4+ T cells are necessary to prevent the development of chronic disease.     

汉黄芩苷对柯萨奇B3病毒诱导的病毒性心肌炎小鼠炎症反应的影响

目的:探讨汉黄芩苷对柯萨奇B3病毒(CVB3)诱导的病毒性心肌炎小鼠炎症反应的影响及其可能的调控机制。方法:用CVB3感染BALB/c小鼠构建病毒性心肌炎动物模型。取40只BALB/c小鼠将其随机分为4组:正常对照组、CVB3感染的病毒性心肌炎组、CVB3感染后给予汉黄芩苷处理的治疗组以及CVB3感染后同时给予汉黄芩苷和AKT激动剂处理的激动剂组,每组10只。于药物治疗7 d后处死各组小鼠。用HE染色检测前3组小鼠心肌组织内炎症细胞浸润情况。用ELISA检测前3组小鼠血清中白细胞介素1β(IL-1β)和IL-6含量。用Western blot实验检测前3组小鼠心脏组织中炎症因子蛋白表达水平以及AKT/NF-κB通路的活化情况。最后,通过Western blot实验检测全部4组小鼠心肺组织中AKT/NF-κB通路的活化情况。结果:与正常组相比,病毒性心肌炎小鼠心脏组织内存在大量炎症细胞浸润,而汉黄芩苷治疗显著降低CVB3病毒诱导的心脏组织炎症细胞浸润(P<0.05)。CVB3病毒感染后小鼠血清中IL-1β和IL-6含量较正常组显著升高(P<0.05),而给予汉黄芩苷治疗后小鼠...     

RIP3/CaMKⅡ信号通路对重症病毒性心肌炎小鼠心脏功能及生存曲线的影响

目的:观察RIP3/CaMKⅡ信号通路对柯萨奇病毒B3(CVB3)诱导的重症病毒性心肌炎小鼠心脏功能及生存曲线的影响,并探讨CaMKⅡ特异性抑制剂KN-93对心肌损伤的作用。方法:将60只雄性BALB/c小鼠分为正常对照组、CVB3组、CVB3+KN-93组和CVB3+KN-93+Ti(活性氧簇特异性抑制剂)组,以上小鼠腹腔及尾静脉注射药物连续7 d。采用双抗体夹心免疫法检测小鼠心肌细胞坏死标志物,心脏超声技术检测小鼠心脏结构和功能,绘制Kaplan-Meier生存曲线,观察KN-93对小鼠心肌的保护作用。结果:CVB3+KN-93组小鼠心肌细胞坏死较CVB3组显著减轻,心脏功能和生存曲线改善(P<0.01);CVB3+KN-93组小鼠心肌组织H2O2含量较CVB3组显著下降(P<0.01);加入Ti后,CVB3+KN-93+Ti组H2O2含量较CVB3+KN-93组轻度下降(P<0.05),但无论是心脏功能还是生存曲线均无显著差异。结论:RIP3/CaMKⅡ信号通路在CVB3诱导的急性重症病毒性心肌炎小鼠心肌细胞损伤中起到主导作用;KN-93能阻断这种作用并可能产生新的治疗方式,其效果可能是通过对CaMKⅡ的直接抑制和对活性氧簇的间接抑制而实现的。     

Gene therapy with CCL2 (MCP-1) mutant protects CVB3-induced myocarditis by compromising Th1 polarization

Viral myocarditis, which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, affects about 5-20% of the world population and lacks efficient treatments. We previously reported that monocyte chemotactic protein-1 (MCP-1, CCL2) was significantly induced during CVB3 infection and greatly contributed to the myocardic inflammation and injury. Herein a CCL2 mutant with removed chemotactic activity was administrated and its therapeutic effect on CVB3-induced myocarditis was explored. A dominant negative CCL2 mutant, lacking the N-terminal amino acids 2-8 (CCL2^Δ2-8), was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection, severity of myocarditis was evaluated by weight loss, survival rate, serological indices and pathological observation. Systemic and local Th1/Th2 cytokine profiles were also assessed. Mice receiving pCCL2^Δ2-8 exhibited a profound attenuation of myocarditis compared to pcDNA3.1 or non-treated mice, as evidenced by invariant body weight, decreased serum CK-MB level, reduced myocardial inflammatory infiltration and increased survival. This effect was not attributable to the efficient viral clearance, but associated with weakened Th1 immune responses, as evidenced by significantly reduced CD4⁺IFN-γ⁺ T cell frequency and Th1 cytokine level systemically and locally. Strategy of blocking in vivo CCL2 activity could effectively alleviate the severity of CVB3-induced myocarditis and may present an alternative therapeutic approach against viral myocarditis.     

Recombinant coxsackievirus vectors for prevention and therapy of virus-induced heart disease

Cardiovascular diseases are the major cause of human death and have been linked to many different risk factors. Among them, coxsackievirus B3 (CVB3), as a member of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Despite the fact that the molecular structure of this pathogen has been characterized very precisely, there is no virus-specific preventive or therapeutic procedure against CVB3-induced heart disease in clinical use today. A promising approach to prevent CVB3-caused myocarditis represents the mutation of the viral genome in a way that coding sequences of cytokines are integrated into the viral RNA. Recombinant cytokine-expressing CVB3 variants were established to increase the local cytokine concentration and to modulate TH1-/TH2-specific immune responses. Especially protective against CVB3-induced murine myocarditis is the application of an interferon-γ (IFN-γ)-expressing recombinant coxsackievirus variant. The local and simultaneous expression of an immuno-relevant cytokine by the virus itself induces a strong and long-lasting immune response which protects laboratory animals against lethal infections.     

Evaluation of the effects of low molecular weight heparin on inflammation and collagen deposition in chronic coxsackievirus B3-induced myocarditis in A/J mice.

Coxsackievirus, Group B, type 3 (CVB3) infection of A/J male mice induces chronic myocarditis with increased interstitial fibrosis and collagen deposition. Heparin, a naturally occurring sulfated glycosaminoglycan, has both anti-inflammatory and antifibrotic activities besides its well-known anticoagulant activity. This study determined whether heparin treatment could decrease either cardiac inflammation or fibrosis in chronic CVB3-induced myocarditis. Control mice were either untreated or treated with heparin (4 micrograms/g body weight, subcutaneously 5 times weekly) beginning 2 days before infection of other groups. Additional groups received either virus only (1 x 10(4) plaque-forming units [PFU]), virus followed by heparin beginning 14 days after CVB3 inoculation, or virus and heparin beginning 2 days before CVB3 inoculation. Animals were sacrificed 14, 28, and 58 days after infection. Heparin treatment begun either before or after virus inoculation reduced animal mortality by approximately 20%. Heparin did not alter virus infection or replication in the heart. Histologically, only animals treated with heparin before virus inoculation showed reduced myocardial inflammation, and only at day 58. However, heparin treatment begun either before or after virus infection significantly decreased collagen deposition in the heart (fibrosis).     

The Role of B Lymphocytes in Coxsackievirus B3 Infection

Coxsackieviruses are important human pathogens, frequently causing myocarditis, pancreatitis, and a variety of less severe diseases. B lymphocytes appear central to the interaction between these viruses and their mammalian hosts, because agammaglobulinemic humans, genetically incapable of antibody production, are susceptible to chronic infections by coxsackieviruses and related enteroviruses, such as poliovirus and echovirus. However, recent studies show that Type B coxsackievirus (CVB) infects B lymphocytes soon after infection, suggesting the possibility that these cells may play some role in virus dissemination and/or that the virus may be able to modulate the host immune response. We analyzed the role of B lymphocytes in CVB infection and confirmed that CVB infects B lymphocytes, and extended these findings to show that this is a productive infection involving approximately 1 to 10% of the cells; however, infectious center assays show that other splenocytes are infected at approximately the same frequency. Virus is readily detectable by in situ hybridization in the spleen of immunocompetent mice but is difficult to detect in mice deficient in B cells (BcKO mice), consistent with much of the splenic signal being the result of B cell infection. Surprisingly, given the extent of their infection, B cells express barely detectable levels of the murine coxsackievirus-adenovirus receptor (mCAR), suggesting that another means of cell entry may be used. We found no evidence of B cell depletion following CVB infection, indicating that this is not the explanation for the transient immunosuppression previously reported. Virus replication and dissemination are slightly delayed in BcKO mice, consistent with B cells' playing a role as an important early target of infection and/or a means to distribute the virus to many tissues. In addition, we show that BcKO mice recapitulate a central feature of human agammaglobulinemia: CVB establishes chronic infection in a variety of organs (heart, liver, brain, kidney, lung, pancreas, spleen). In most of these tissues the viral titers remain high (10(5)-10(8) plaque forming units (pfu) per gram of tissue) for the life of the mouse, and in several there is severe pathology, particularly severe myocardial fibrosis with ventricular dilation, reminiscent of the dilated cardiomyopathy seen in humans with chronic enteroviral myocarditis. Transfer of B and/or T cells from non-immune mice had no discernible effect, whereas equivalent transfers from immune mice often resulted in transient or permanent disappearance of detectable CVB.