Secondary Heterotypic Versus Homotypic Infection by Coxsackie B Group Viruses: Impact on Early and Late Histopathological Lesions and Virus Genome Prominence

The impact of prior exposure to a different or identical strain of Coxsackievirus B (CVB) on murine CVB myocarditis was studied using a susceptible murine host (A/J[H-2 ^a]) and myocarditic CVB3 or avirulent CVB2 as primary or secondary infectants. The effects of secondary heterotypic infection (CVB2 followed by CVB3) and homotypic infection (CVB3 followed by CVB3) 28 days after primary inoculation, versus CVB2 or CVB3 alone, on injury and viral genomic replication, both early (day 7) and late (days 28 and 56), were evaluated. After the primary infection by CVB2, trivial viral RNA was present in the heart and other organs, and a substantial positivity was observed with CVB3 infection. Seven days after secondary heterotypic (CVB2-CVB3) infection, the quantity of CVB genome in heart, pancreas, liver, and spleen was increased compared with the virus genome in the CVB3-CVB3 group and in the group with primary CVB3 infection alone. This phenomenon was seen in the heart and spleen up to day 28 postsecondary infection. Tissue inflammation and necrosis in heart and pancreas were prominent 7 days postsecondary infection with CVB2-CVB3 and correlated well with an increased quantity of CVB genome. Virus genome was present in heart and spleen 28 days after CVB3 infection alone. Serum CVB3 neutralization titer was increased to 1:128 in CVB2-CVB3 group at days 7 and 28 postsecondary infection, and serum completely neutralized cytopathological effects of CVB3 in the CVB3-CVB3 group at day 7 and 28 postsecondary infection. Our results indicate that secondary heterotypic infection by CVB causes increased injury, inflammation, and CVB replication in target organs such as the heart and pancreas, as well as in immune compartments like the spleen. Compared with CVB3 alone, the intense inflammatory infiltrate in the CVB2-CVB3 group is as not due solely to postviral sensitization of the immune system, but rather to the inability of the host to eradicate the virus.     

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.     

Coxsackievirus infection of mice. I. Viral kinetics and histopathological changes in mice experimentally infected with coxsackieviruses B3 and B4 by oral route

We followed the viral kinetics and histopathological changes in different organs of immunocompetent mice infected orally with coxsackieviruses B3 (CVB3) Nancy strain and B4 (CVB4) JVB strain separately. The viruses used were not adapted to mouse organs. In the acute phase of infection, the viral kinetics indicated virus replication in the heart, spleen, thymus, pancreas, and small and large intestines. This was accompanied by histopathological changes, mild infiltration of mononuclear cells and fibrosis in the heart. The necrotic changes with mononuclear infiltration and fibrosis in the myocard was observed on days 56 and 71 p.i. in the CVB4-infected animals only. In the mice infected with CVB3 and CVB4 a prolonged presence of infectious virus was shown in the spleen and small intestine; in the latter viral antigen was localized in smooth muscles of the muscular wall immunohistochemically. This is the first report on prolonged replication of coxsackieviruses (CV) in the spleen and small intestine in orally infected mice.     

Differential expression of coxsackievirus and adenovirus receptor on alveolar epithelial cells between fetal and adult mice determines their different susceptibility to coxsackievirus B infection

Coxsackievirus B (CVB) can cause aseptic meningitis, myocarditis and respiratory disease, especially in newborn infants. To compare the susceptibility to CVB infection of fetal and adult mice, we prepared primary alveolar epithelial cells (AECs) from lungs of BALB/c mice. In contrast to fetal mouse AECs, those of adults were less susceptible to CVB3 infection, as indicated by decreased cytopathic effects, and reduced levels of viral particles bound at the cell surface. In adult mouse AECs, amplification of the viral genome and virus capsid protein VP1 synthesis were concomitantly reduced. In addition, the cell-surface expression of coxsackievirus and adenovirus receptor (CAR), which plays a key role in the initiation of CVB and pulmonary infection, was downregulated in adult mouse AECs. These findings demonstrate that adult mouse AECs are less susceptible to CVB3 due to decreased CAR levels. Thus, these findings strongly indicate that the level of virus receptors on AECs is one of the crucial determinants for the age-dependence of CVB virulence in the mouse lung.     

Coxsackievirus infection of the pancreas: evaluation of receptor expression, pathogenesis, and immunopathology

Coxsackievirus type B (CVB) infection of the pancreas induces a massive cellular infiltrate composed of natural killer cells, T cells, and macrophages and leads to the destruction of exocrine tissue. The physiological manifestations of pancreatic CVB infection are correlated with viral tropism; the virus infects acinar cells but spares the islets of Langerhans. Here we evaluate the mechanisms underlying pancreatic inflammation and destruction and identify the determinants of viral tropism. T-cell-mediated immunopathology has been invoked, along with direct virus-mediated cytopathicity, to explain certain aspects of CVB-induced pancreatic disease. However, we show here that in the pancreas, the extent of inflammation and tissue destruction appears unaltered in the absence of the cytolytic protein perforin; these findings exclude any requirement for perforin-mediated lysis by natural killer cells or cytotoxic T cells in CVB3-induced pancreatic damage. Furthermore, perforin-mediated cytotoxic T-cell activity does not contribute to the control of CVB infection in this organ. In addition, we demonstrate that the recently identified coxsackie-adenovirus receptor is expressed at high levels in acinar cells but is barely detectable in islets, which is consistent with its being a major determinant of virus tropism and, therefore, of disease. However, further studies using various cell lines of pancreatic origin reveal secondary determinants of virus tropism.     

中药心康口服液抗柯萨奇B3病毒RNA复制作用的研究

目的研究探讨中药心康口服液对感染心肌中柯萨奇B3病毒RNA复制的影响。方法小鼠感染柯萨奇病毒B3(CVB3)诱发急性病毒性心肌炎,于急性期治疗后提取鼠心肌总RNA,用逆转录-聚合酶链反应技术检测心肌CVB3RNA含量,经琼脂糖凝胶电泳后,在凝胶成像系统下观察。结果中药心康口服液能明显降低心肌CVB3-RNA的含量,与病毒对照组比较差异有统计学意义,P<0.01;感染期不同阶段心肌病毒RNA含量的检测显示,心康口服液2个治疗组的病毒含量的检出,并没有随着用药时间的延长而显著降低。结论中药心康口服液能有效地影响心肌CVB3RNA的复制,其对病毒的作用是抑制复制而非杀灭。     

Melanoma differentiation-associated protein-5 (MDA-5) limits early viral replication but is not essential for the induction of type 1 interferons after Coxsackievirus infection

Coxsackievirus infections are associated with severe diseases such as myocarditis, meningitis and pancreatitis. To study the contribution of the intracellular viral sensor melanoma differentiation-associated protein-5 (MDA-5) in the host immune response to Coxsackievirus B3 (CVB3) we infected C57BL/6 and 129/SvJ mice lacking mda-5. Mice deficient in MDA-5 showed a dramatically increased susceptibility to CVB3 infection. The loss of MDA-5 allowed the virus to replicate faster, resulting in increased liver and pancreas damage and heightened mortality. MDA-5 was not absolutely required for the induction of type 1 interferons (IFNs), but essential for the production of maximal levels of systemic IFN-α early after infection. Taken together, our findings indicate that MDA-5 plays an important role in the host immune response to CVB3 by preventing early virus replication and limiting tissue pathology.     

Coxsackie virus B4 infection of the mouse pancreas: I. Detection of virus-specific RNA in the pancreas by in situ hybridisation.

The pathology of Coxsackie virus B4 (CVB4) infection in a murine model was investigated by in situ hybridisation using a biotinylated cDNA probe derived from CVB4. During the acute phase of infection virus RNA sequences were detected in the exocrine pancreas of 60% of mice infected with a pancreotropic variant of CVB4. A positive hybridisation signal was observed in other organs in some animals including the heart and liver of 1 mouse 28 days after infection. The cellular distribution of virus RNA sequences corresponded well with the histological findings in most tissues. Possible causes for failure of hybridisation in some infected pancreases are discussed in conjunction with potential application of the technique in human pancreas biopsy samples.     

The viral genetic background determines the outcome of coxsackievirus B3 infection in outbred NMRI mice

The reasons for the different outcome of coxsackievirus B3 (CVB3)-induced heart disease in humans are not well understood. Since there are no experimental data on the course of disease after infection with genetically different CVB3 in a natural variable population until now, we studied the outcome of virus infection in outbred NMRI mice after inoculation of genetically different CVB3 variants. Adult male mice were inoculated with seven closely related CVB3 variants. The histopathological changes of heart and pancreas tissue, antibody induction, virus titers, and persistence of viral positive- as well as negative-strand RNA in spleen and heart tissue were compared at day 7 or day 28 after infection to detect prerequisites and predictive factors for chronic myocarditis. Six CVB3 variants infected NMRI mice. CVB3 infection (i) did not induce detectable myocardial injury, (ii) caused signs of healing up acute myocarditis or (iii) ongoing chronic myocarditis. Neither IgG antibody responses nor the extent of destruction of exocrine pancreatic tissue or viral RNA load in spleen did correlate with myocardial histopathology. In contrast, a high persistent viral RNA load in heart tissue specimens was characteristic for mice developing chronic myocarditis. The results of the present study corroborate high viral load in the acute stage of myocarditis and high amounts of persisting CVB3 RNA in heart tissue as predictive marker of chronic myocarditis. The outcome of CVB3-induced heart disease in outbred NMRI mice depends strongly on the viral genetic background. In particular an important role of viral capsid proteins is suggested.     

Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells

Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica.     

Coxsackievirus B3 infection and type 1 diabetes development in NOD mice: insulitis determines susceptibility of pancreatic islets to virus infection

Group B coxsackieviruses (CVB) are believed to trigger some cases of human type 1 diabetes (T1D), although the mechanism by which this may occur has not been shown. We demonstrated previously that inoculation of young nonobese diabetic (NOD) mice with any of several different CVB strains reduced T1D incidence. We also observed no evidence of CVB replication within islets of young NOD mice, suggesting no role for CVB in T1D induction in the NOD mouse model. The failure to observe CVB replication within islets of young NOD mice has been proposed to be due to interferon expression by insulin-producing beta cells or lack of expression of the CVB receptor CAR. We found that CAR protein is detectable within islets of young and older NOD mice and that a CVB3 strain, which expresses murine IL-4, can replicate in islets. Mice inoculated with the IL-4 expressing CVB3 chimeric strain were better protected from T1D onset than were mock-infected control mice despite intraislet viral replication. Having demonstrated that CVB can replicate in healthy islets of young NOD mice when the intraislet environment is suitably altered, we asked whether islets in old prediabetic mice were resistant to CVB infection. Unlike young mice in which insulitis is not yet apparent, older NOD mice demonstrate severe insulitis in all islets. Inoculating older prediabetic mice with different pathogenic CVB strains caused accelerated T1D onset relative to control mice, a phenomenon that was preceded by detection of virus within islets. Together, the results suggest a model for resolving conflicting data regarding the role of CVB in human T1D etiology.     

Virus-receptor interactions of coxsackie B viruses and their putative influence on cardiotropism

Specific virus-receptor interactions are important determinants in the pathogenesis of viral infections, influencing the location and initiation of primary infection as well as the viral spread to other target organs in the postviremic phase. Coxsackieviruses of group B (CVB) specifically interact with at least two receptor proteins, the coxsackievirus-adenovirus receptor (CAR) and the decay-accelerating factor (DAF), and cause a broad spectrum of diseases, including acute and chronic myocarditis. In the human heart, CAR is predominantly expressed in intercalated discs, regions of utmost importance for the functional integrity of the heart. Since DAF is abundantly expressed in epithelial and endothelial cells, interaction of cardiotropic CVB with the DAF coreceptor protein, in addition to CAR, could therefore be advantageous to the virus by enhancing viral entry into the heart.     

Direct interferon-gamma-mediated protection caused by a recombinant coxsackievirus B3

Coxsackievirus B3 (CVB3) is one of the most important causes of viral myocarditis. Cytokines are involved in the control of CVB3 replication and pathogenesis. Local expression of specific cytokines by recombinant CVB3 confers prevention of virus-caused myocarditis. Expression of IFN-gamma by CVB3(IFN-gamma) protected BALB/c and C57BL/6 mice when the lethal infection with the highly pathogenic CVB3H3 variant was given directly after or prior to CVB3(IFN-gamma) inoculation by decreasing the viral load and spread as well as tissue destruction. This direct effect was not restricted to the homologous virus. In vitro, cocultivation of CVB3(IFN-gamma)-infected cells induced a reduction of CVB3H3 replication and virus-induced cytopathogenicity.     

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.     

Suppression of proinflammatory cytokines and induction of IL-10 in human monocytes after coxsackievirus B3 infection

Coxsackievirus B3 (CVB3) causes acute and chronic myocarditis, which is accompanied by an intense mononuclear leukocyte infiltration. Because myocardial tissue damage may either result from viral infections or from a dysregulated immune response, the susceptibility of human monocytes and macrophages to CVB3 was examined in this study with regard to virus replication, virus persistence, and release of cytokines. Monocytes were infected by CVB3 as shown by the intracellular appearance of plus- and minus-strand viral RNA, which was also capable of persisting for more than 10 days. Fresh monocytes were not permissive for full virus replication whereas monocyte-derived macrophages yielded a low amount of new viruses, which led to cell death. Although CVB3 infection induced the mRNA for the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6, only little cytokine production occurred. When infected monocytes were stimulated in addition by lipopolysaccharides (LPS), cytokine production was partially suppressed. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 on the mRNA and the protein level. These data show a dysregulated cytokine response in CVB3-exposed human monocytes and macrophages, which is characterized by a suppression of proinflammatory cytokines and a dominance of IL-10. This viral strategy may aid CVB3, causing chronic myocardiopathy.     

Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice

Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.