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

为探讨扩张型心肌病(DCM)的病毒病原学，采用聚合酶链式反应(PCR)和原位杂交(ISH)技术检测了扩张型心肌病(DCM)、心肌炎(MC)或其他心脏病患者的心肌活检标本和柯萨奇病毒(CV)B3心肌炎动物模型心肌组织中肠道病毒RNA的存在。我们还通过PCR-SSCP分析检测了心肌活检组织中检测到的肠道病毒的基因类型。大多数基因分型与B组不符，DCM心脏中不存在一种特殊的肠道病毒。在CVB3型病毒性心肌炎动物模型中，直到接种C3H/He小鼠第28天仍可检测到病毒基因组。光镜下以CVB为探针的ISH显示心肌坏死区和细胞浸润区有成串的阳性信号。电镜ISH显示，CVB3-RNA存在于心肌细胞的胞浆、肌原纤维之间、线粒体附近以及管状或囊泡结构中。在坏死性胚泡、巨噬细胞和成纤维细胞的细胞质中也检测到病毒RNA。这些发现表明，CVB3RNA在心肌细胞的细胞质中复制，转移到管状或囊泡结构，释放到间质，并被巨噬细胞吞噬。在心肌细胞胞浆内也检测到与浸润性淋巴细胞密切接触的阳性信号，提示淋巴细胞识别病毒感染的心肌细胞，导致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.