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. 2000 Jun;83(6):696–701. doi: 10.1136/heart.83.6.696

High prevalence of enteroviral genomic sequences in myocardium from cases of endemic cardiomyopathy (Keshan disease) in China

Y Li 1, T Peng 1, Y Yang 1, C Niu 1, L Archard 1, H Zhang 1
PMCID: PMC1760852  PMID: 10814633

Abstract

OBJECTIVE—To verify the aetiological involvement of enterovirus and identify the viral genomic sequences in Keshan disease.
DESIGN—Formalin fixed, paraffin embedded myocardial necropsy tissue samples were collected in Keshan disease endemic regions. Fourteen cases with a histologically confirmed diagnosis of subacute or chronic Keshan disease were studied. Control tissue included 10 samples of myocardium from cases of cerebral trauma and one from accidental acid intoxication. One sample from a case of enteroviral myocarditis was used as a positive control. The presence of viral genomic RNA was investigated using an established reverse transcription nested polymerase chain reaction (PCR) coupled with direct nucleotide sequencing. Further investigations of PCR positive samples included in situ antigen detection or hybridisation to confirm positive results.
RESULTS—Nine of 14 myocardial samples from Keshan disease cases and the positive control were positive for the enteroviral RNA. All the controls were negative. Six of the PCR positive samples were investigated further by in situ enteroviral antigen or RNA detection and all were positive. DNA sequencing of six representative PCR products confirmed that they were homologous to the 5' non-translated region of enteroviral genomic RNA. Five had highest homology to coxsackievirus B genotypes and one was identical to poliovirus type 3.
CONCLUSIONS—These results support an aetiological role for enteroviral infection in Keshan disease. Nucleotide sequence data suggest that coxsackievirus B or coxsackie B like viruses are often involved in Keshan disease.


Keywords: enterovirus; coxsackievirus; cardiomyopathy; Keshan disease

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Figure 1  .

Figure 1  

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Agarose gel electrophoresis of representative reverse transcription nested polymerase chain reaction (RT-NPCR) products. (A) Enteroviral RT-NPCR products amplified with primers F24/R253 (215 bp) or F3/R8 (511 bp). (B) Representative RT-NPCR products of house keeping gene, β actin, from Keshan disease or control heart tissue samples. The first round PCR was with the primer pair HBAF1/R1 and the nested PCR was with the primer pair HBAF2/R2. DNA bands in the 1.5% agarose gel were stained with ethidium bromide. M, 100 bp markers; W, water and reagent control.

Figure 2  .

Figure 2  

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Agarose gel electrophoresis of enterovirus reverse transcription nested polymerase chain reaction (RT-NPCR) products from patients 3, 8, and 9. The RNA extracts from these three patients were subjected to DNase (D) or RNase (R) treatment before RT-NPCR. PCR was also performed on these samples directly without reverse transcription (RT−).

Figure 3  .

Figure 3  

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Photomicrographs of heart sections of Keshan disease. (A) Trichrome staining of a section from patient 3 with subacute Keshan disease (× 100, cytoplasm and muscle fibres stain red, nuclei stain black, and collagen stains blue). (B) Immunohistochemistry staining of a section from the same patient. The brown staining indicates the presence of enteroviral capsid protein VP1 reactive with Mab 5-D8/1 (haematoxylin counterstaining × 200). (C) Immunohistochemistry staining of a section from patient 8 with chronic Keshan disease. The brown staining indicates the presence of enteroviral capsid protein VP1 reactive with Mab 5-D8/1 (haematoxylin counterstaining × 200). (D) In situ hybridisation on a section from patient 10 with chronic Keshan disease. The dark purple signals indicate the presence of enteroviral genomic RNA probed by the digoxigenin labelled primer mixture (no counterstaining × 100).

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