In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease.

Abstract

We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.In North America and Europe, acute myocarditis is most commonly associated with infections by coxsackie B viruses (types 1 to 5). Other members of the human enteroviruses comprising at present over 70 serotypes (e.g., various coxsackie A viruses and echoviruses) are also considered to be relatively frequent causes of human viral heart disease (1-4). These agents appear to be capable of producing dilated cardiomyopathy of acute onset or lead to a variety of cardiac arrhythmias. Some of the acute cases may also evolve into a chronic form of dilated cardiomyopathy.The difficulty of establishing a specific diagnosis of viral heart disease is a major problem in clinical cardiology. Confirmation ofthe clinical suspicion of viral heart disease demands demonstration of replicating virus inside myocardial cells, which is exceedingly difficult by conventional methods (5). In addition, the increasing availability of potential antiviral agents has accentuated the need for methods by which endomyocardial biopsy specimens of patients with suspected viral heart disease can be diagnosed conclusively (6, 7).In order to introduce in situ nucleic acid hybridization (8-10) as a diagnostic tool in suspected enteroviral heart disease, we have recently cloned the single-stranded RNA genome of coxsackievirus B3 (CVB3) (11) that had been propagated in cultured human heart cells (12). Full-length reverse-transcribed cloned viral cDNA generated infectious CVB3 upon transfection into mammalian cells, demonstrating the molecular cloning of a faithful transcript ofthe original viral RNA. We now report the use of radioactively labeled cloned CVB3 cDNA as a diagnostic probe for the in situ detection of viral RNA in infected cultured cells and in myocardial tissue sections of CVB3-infected T-cell-deficient mice. We show that the molecular hybridization approach permits the detection of infected myocardial cells at the single-cell level, thus providing a unique possibility for an unequivocal diagnosis.A further advantage of the nucleic acid hybridization approach is provided by the high degree of genetic homology shared among the different serotypes of the human enterovirus group (11, 13-17), making possible their detection in a single hybridization assay. Detectio...

Expression of Ksp-cadherin during kidney development and in renal cell carcinoma

“…Until further use, sections could be stored at À801C in 100% ethanol. After rehydration in 100, 70 and 40% ethanol and finally in distilled water, in situ hybridisation was performed as described previously (Kandolf et al, 1987). In brief, slides were pretreated with 0.2 M HCl, heated and digested with proteinase K. Then, the slides were dehydrated in graded ethanol series, dried over night and hybridised at 421C for 18 h with 500 ng ml À1 35 S-labelled RNA.…”

Hepatitis C Virus from the Hearts of Patients with Myocarditis and Cardiomyopathy

“…Persistent enterovirus infection of the myocardium has been reported (Bowles et al, 1986;Kandolf et al, 1987), and the existence of a causative role of enteroviruses in acute myocarditis, particularly in children, finds support in a metaanalysis. On the other hand, some of the studies examining the association of enteroviruses with dilated cardiomyopathy have been negative (Baboonian and Treasure, 1997).…”

Acute heart failure after allogeneic blood stem cell transplantation due to massive myocardial infiltration by cytotoxic T cells of donor origin

“…[4][5][6] Additionally, viral infection of the heart by EV and PVB19 was excluded by means of radioactive in situ hybridization. 4,7 Immunohistochemical studies For immunohistochemical characterization of inflammatory cells in the myocardium, 4-m dewaxed paraffin-embedded tissue sections were tested by an avidin-biotin immunoperoxidase method according to the manufacturer's protocol (Vectastain Elite ABC Kit, Vector, Burlingame, CA, USA) applying the following monoclonal antibodies, CD3 (T cells, Novocastra Laboratories, Newcastle upon Tyne, UK), CD8 (Dako, Glostrup, Denmark), CD68 (Dako). Controls with normal mouse serum were performed to exclude nonspecific staining.…”