Annals of the Rheumatic Diseases 1993; 52: 575-578 575 Polymyositis and dermatomyositis: no persistence Ann Rheum Dis: first published as 10.1136/ard.52.8.575 on 1 August 1993. Downloaded from of enterovirus or encephalomyocarditis virus RNA in muscle Peter J H Jongen, Gerrit J Zoll, Marielle Beaumont, Willem J G Melchers, Levinus B A van de Putte, Jochem M D Galama Abstract autoimmune origin is supported by the Objectives-A persistent infection of presence of various autoantibodies, the enteroviruses and cardioviruses has been evidence of myotoxicity mediated by cytotoxic implicated in polymyositis and dermato- T cells or complement mediated microangio- myositis, but conventional hybridisation pathy, their response to immunotherapies, and studies ofthe presence ofenterovirus RNA their association with diseases of which the and encaphalomyocarditis (EMC) virus autoimmune character has been established RNA in affected muscle have yielded more firmly.'-3 The agents initiating self conflicting results. To investigate further sensitisation and the specific antigens against the possibility of viral persistence, the which the immune reactions are directed are presence of viral RNA in muscle from unknown, however.' patients with adult onset polymyositis and Viruses can induce autoimmunity in mice"6 dermatomyositis was investigated using a and, probably, in humans.4 7 Electron polymerase chain reaction (PCR) microscopic8 and serological9 10 studies have technique. implicated the enterovirus and cardiovirus Methods-Muscle tissue was obtained subgroup ofpicomaviruses in polymyositis and from 10 patients with polymyositis and dermatomyositis, but reports of the isolation of five patients with dermatomyositis, all the viruses from muscle tissue are rare.' Dot with adult onset active disease. A PCR was spot hybridisation studies of the presence of performed using primers with high enterovirus specific RNA in affected muscle specificity for enterovirus and EMC virus have yielded conflicting results. Bowles et al RNA, followed by Southern blot hybrid- found coxsackievirus B specific RNA in muscle isation with an oligonucleotide probe tissues from patients with polymyositis or directed against the internal portion ofthe dermatomyositis," whereas Bunn and Walport http://ard.bmj.com/ amplified product. A PCR directed could not detect this virus in patients with against the Abelson tyrosine kinase mRNA polymyositis.12 Unconfirmed in situ hybrid- served as an internal control for the isation studies in patients with polymyositis presence and quality of RNA. and dermatomyositis have found enterovirus Results-A specific amplification for specific'3 or encephalomyocarditis (EMC) virus enterovirus or for EMC virus could not be specific RNA.14 on September 28, 2021 by guest. Protected copyright. Department of seen in any of the muscle biopsy samples, Persistent infection of enterovirus and EMC Neurology, despite a sensitivity of about 30 plaque virus as a pathogenic factor in polymyositis and University Hospital forming units for enterovirus and of 100 dermatomyositis is therefore a subject of Nijmegen, Nijmegen, plaque forming units for EMC virus. controversy. To investigate further the The Netherlands Southern blot hybridisation confirmed possibility of viral RNA persistence in poly- P J H Jongen these results in that positive controls myositis and dermatomyositis, we extracted M Beaumont hybridised with the oligonucleotide probe, RNA from muscle biopsy samples from Department ofMedical but no signal was obtained with the muscle patients with active disease, and used a highly Microbiology, University Hospital specimens. specific and sensitive polymerase chain Nijmegen, Conclusion-A sensitive and specific PCR reaction (PCR) technique for the detection of Nijmegen, technique showed no evidence of the enterovirus and EMC virus RNA. The Netherlands G J Zoll presence of enterovirus or EMC virus W J G Melchers RNA in muscle samples from patients J M D Galama with polymyositis or dermatomyositis. Patients and methods Department of These data do not support the proposal PATIENTS Rheumatology, that viral RNA persistence plays a We studied 10 patients with adult onset University Hospital Nijmegen, part in these idiopathic inflammatory polymyositis and five patients with adult onset Nijmegen, myopathies. dermatomyositis attending the centre for The Netherlands neuromuscular diseases of the University of L B A van de Putte (Ann Rheum Dis 1993; 52: 575-578) Nijmegen. All patients fulfilled the diagnostic Correspondence to: Dr Jochem M D Galama, criteria for definite polymyositis or definite PO Box 9101, dermatomyositis, as formulated by Dalakas.' 6500 HB Nijmegen, Polymyositis and dermatomyositis are specific Of the patients with polymyositis five were The Netherlands. were their Accepted for publication diagnoses in the group of inflammatory men and five women; ages ranged 10 May 1993 myopathies.1 2 In these two disorders an from 34 to 73 years. The patients with 57656ongen, Zoll, Beaumont, Melchers, van de Putte, Galama Diagnosis, age, sex, duration ofsymptoms, and associated diseases ofthe patients with polymyositis (PM) and dermatomyositis (DM) conserved 5'NCR of enterovirus and have been shown to detect 60 of the 66 different Patient Diagnosis Age Sex Disease Associated disease enterovirus tested. 16 Ann Rheum Dis: first published as 10.1136/ard.52.8.575 on 1 August 1993. Downloaded from No (years) * duration types * For amplification of EMC RNA we used an 1 PM 66 F Four years antisense primer (5'CACGTGGCTT-l-l- 2 PM 70 F One month Primary biliary cirrhosis 3 PM 59 M Six weeks GGCCGCAGAGG3') and a sense primer 4 PM 73 M 16 years Dilated cardiomyopathy (5'CGAAGCCGCTTGGAATA3') specific for 5 PM 63 M Three months - 6 PM 51 M One year Adenocarcinoma (site unknown) cardioviruses and Theiler's murine encephalo- 7 PM 73 F 2.5 months - myocarditis virus (TMEV) (Zoll G 8 PM 59 F 13 years J, 9 PM 34 M 34years - Galama J M D, Melcher W J G, unpublished 10 PM 58 F Nine years - data). For Southern blot analysis we used an 11 DM 63 F One month Interstitial lung fibrosis 12 DM 24 F Three months EMC/TMEV specific oligonucleotide probe 13 DM 34 F Eight years (5'CGTGTTACCAGGTGGGG3'). A 14 DM 42 F Five years frag- 15 DM 22 F Seven years ment of 282 base pairs is obtained with positive specimens. *At time ofmuscle biopsy. As an internal control for the presence and quality of RNA, we performed a PCR directed against the Abelson tyrosine kinase mRNA, dermatomyositis were all women; their ages which is expressed in muscles.17 A positive ranged from 22 to 63 years. The table gives the PCR results in a fragment of 218 base pairs. diagnosis, age, sex, duration of symptoms at time of examination, and associated diseases of the patients. Biopsy samples were obtained Results from a quadriceps muscle for diagnostic When a titrated stock of virus was serially reasons. At the time of examination all patients diluted and added to negative muscle samples had progressive muscle weakness and before RNA extraction, we obtained a increased serum creatine kinase concen- sensitivity of approximately 30 plaque forming trations. For RNA extraction and viral RNA units for enterovirus (fig 1A) and of 100 plaque detection, tissue samples of approximately forming units for EMC (fig 1B). A specific 25-30mm2 were immediately frozen in liquid amplification for enterovirus (fig 2A) or for nitrogen and stored at -80°C until use. Fifteen EMC virus (fig 2B) was not observed in any patients with non-inflammatory non-immune of the muscle biopsy samples. A specific mediated neuromuscular disorders were amplification product of 155 base pairs was studied as controls. seen from enterovirus RNA and from enterovirus cDNA, and of 282 base pairs from EMC virus RNA, which were used as positive SAMPLE PROCESSING controls. In all samples the reverse RNA from muscle samples was purified using transcriptase PCR on the non-relevant mRNA http://ard.bmj.com/ a single extraction with guanidinium- of the Abelson tyrosine kinase gene resulted in thiocyanate-phenol-chloroform according to a specific amplification product of 218 base the method of Chomczynski and Sacchi. 15 pairs, confirming the presence of adequate RNA was dissolved in 50 ,ul distilled water RNA in the tissues. Southern blot hybrid- treated with diethylpyrocarbonate. To all isation with an oligonucleotide probe directed samples, human placenta RNAse inhibitor against the internal portion of the amplified (RNasin; Promega) was added to a confirmed the results as product observed on September 28, 2021 by guest. Protected copyright. concentration of 0.4 U/,ul. The samples were directly on gel electrophoresis: the positive stored at -80°C. The reverse transcriptase controls hybridised with the oligonucleotide PCR and the determination of the amplified probe, but no signal was obtained with the products were performed as described muscle specimens. previously."6 In brief, for enteroviral RNA amplification, the RNA was transcribed in cDNA using reverse transcriptase and an Discussion antisense primer (3'ATTGTCACCATAA- The aetiology of polymyositis and dermato- GCAGCCA5'). After completing the reverse myositis is unknown, but evidence is transcriptase reaction, the buffer conditions accumulating that viruses belonging to the were adapted for the PCR, and the sense enterovirus and cardiovirus subgroup of primer (5'TCCTCCGGCCCCTGAATG- picornaviruses play a part. 811 13 14 Immuno- CG3') was added. RNA/cDNA hybrids were pathogenic studies in experimental murine denaturated