Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackievirus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5′ noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothyocyanate extraction from tissue stored frozen at -80 °C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrilamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 10⁵ to 10⁶ cells starting from 1 μg total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1. The VP1 gene primer set produced positive amplification only with RNA of Coxsackievirus B3-infected cells. Coxsackievirus B3-specific amplification products were distinguished from those of related enteroviruses by hybridization with specific oligoprobes. However, Coxsackievirus B2, B4 and poliovirus 1-specific PCR products showed positive hybridization if probed with Coxsackievirus B3 genomic probes. All total RNAs from blood and myocardial samples examined by our PCR assay failed to reveal any amplification product that could be related to Coxsackievirus B3 or to enteroviruses in general, after gel electrophoresis and low stringency Southern blot hybridization with the Coxsackievirus B3 specific oligoprobes or the Coxsackievirus B3 genomic probes. The negative results obtained in our series question the hypothesized widespread persistence of enteroviral RNA in hearts with idiopathic dilated cardiomyopathy.