Detection of enteroviral genome in the myocardium from patients with viral myocarditis and dilated cardiomyopathy, and animals with Coxsackievirus myocarditis

为了研究扩张型心肌病（DCM）的病毒病因学，我们使用聚合酶链反应（PCR）和原位杂交（ISH）技术检测了DCM、心肌炎（MC）或其他心脏疾病患者的心肌活检中肠病毒RNA的存在，以及柯萨奇病毒（CV） B3型心肌炎动物模型的心肌组织。对154例患者进行心肌活检，采用RT-PCR检测NC区、NS区和POL区肠病毒基因组的存在。DCM、MC与其他心脏疾病的阳性率均为20%，差异无统计学意义。我们还通过PCR-SSCP分析检测了心肌活检中检测到的肠病毒的基因型。大多数基因型与CV B组的基因型不一致，DCM心脏中不存在特定类型的肠道病毒。在C3H/He小鼠的CVB3病毒心肌炎动物模型中，检测到接种后28天的病毒基因组。光镜下，CVB cDNA探针在心肌坏死和细胞浸润区显示出簇状阳性信号。电镜下，在心肌细胞质、肌原纤维之间、线粒体附近、管状或泡状结构中检测到CVB3-RNA。在坏死组织、巨噬细胞和成纤维细胞的细胞质中也检测到病毒RNA。这些结果表明，CVB3 RNA在心肌细胞质中复制，转移到管状或泡状结构中，释放到间质中，并被巨噬细胞吞噬。在与浸润淋巴细胞密切接触的心肌细胞胞质中也检测到一些阳性信号，提示淋巴细胞识别病毒感染的心肌细胞并引起细胞介导的免疫心肌损伤。

To study the viral etiology of dilated cardiomyopathy (DCM), we examined for the presence of enteroviral RNA in myocardial biopsics from patients with DCM,myocarditis (MC), or other cardiac discases, and myocardial fissues of animal models of coxasckievirus (CV) B3 myocarditis, using polymerase chain reaction (PCR) and in situ hybridization (ISH).Myocardial biopsies from 154 patients were examined for the presence of enteroviral genome in NC,NS and POL region by RT-PCR.The positive incidence was 20% and there was no significant difference among DCM,MC and other cardiac diseases. We also examined the genotypes of enteroviruses detected in myocardial biopsies by PCR-SSCP analysis. Most of the genotypes were not corresponded with those of CV group B and a particular type of enterovirus was not present in DCM heart. In animal models of CVB3 virus myocarditis, the viral genome was detected up to the 28th day of inoculation in C3H/He mice. Light microscopic ISH with a cDNA probe for CVB showed clusters of positive signals in the areas of myocardial necrosis and cell infiltration. With electron microscopic ISH,CVB3-RNA was detected in the cytoplasm of cardiocytes, between the myofibrils, near the mitochondria, and in tubular or vesicular structures. Viral RNA was also detected in necrotic debri, in the cytoplasm of macrophages and fibroblasts. These findings suggest that CVB3 RNA is replicated in the cytoplasm of cardiocytes, transferred into tubular or vesicular structures, released into the interstitium, and phagocytosed by macrophages. Some positive signals were also detected in the cytoplasm of cardiocytes showing close contact with infiltrating lymphocytes, suggesting that the lymphocytes recognized virus-infected cardiocytes and caused to cell-mediated immune cardiocyte damage.