Lack of evidence for a direct involvement of muscle infection by parvovirus B19 in the pathogenesis of inflammatory myopathies: a follow-up study

G. Chevrel, J. P. Borsotti1 and P. Miossec

Department of Immunology and Rheumatology, Hôpital E. Herriot and
1 Department of Pathology, Centre Hospitalier Universitaire, Dijon, France


    Abstract
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 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 
Objective. To clarify the association between parvovirus B19 and myositis.

Methods. Biopsy samples of muscle from eight patients with inflammatory myopathies were studied for the presence of B19 DNA by polymerase chain reaction. Expression of VP1 and VP2 capsid proteins was evaluated by immunohistochemistry. Interleukin 6 (IL-6) production was measured in the supernatant of myoblasts following incubation with parvovirus B19.

Results. In seven samples, detection of B19 DNA was negative. The expression of VP1 and VP2 capsid proteins was not observed by immunohistochemistry. In one patient, detection was transiently positive but became negative despite a flare-up of muscle disease. In vitro, parvovirus B19 was not able to induce IL-6 production by myoblasts.

Conclusion. Our results do not support the direct implication of parvovirus B19 in the pathogenesis of myositis.

KEY WORDS: Myositis, Autoimmune disease, Parvovirus B19, Myoblast, Muscle biopsy.


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 
Parvovirus B19 is the causative agent of transient aplastic crisis, erythema infectiosum, non-immune hydrops fetalis and chronic pure red cell aplasia. It has also been associated with rheumatoid arthritis (RA) and various autoimmune connective tissue diseases [1]. However, the direct contribution to disease induction or perpetuation remains to be clarified.

We have previously reported the first case of dermatomyositis (DM), a muscle autoimmune disease, where B19 DNA was present in two sequential muscle biopsies [2]. Accordingly, the presence of parvovirus B19 in the muscle of our patient suggested the potential association between this viral infection and myositis.

Here we provide a follow-up of the same case where disease relapse was not associated with the presence of parvovirus B19 in a new muscle biopsy. Samples from additional patients were also negative.


    Case report
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 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 
This case has been reported previously in detail [2]. Briefly, a 48-yr-old woman presented with muscle weakness associated with a purple rash of the upper eyelids in December 1996. An electromyographic study showed a myogenic pattern. A first biopsy showed perivascular inflammatory infiltration and muscular necrosis. The erythrocyte sedimentation rate (ESR) was 100 mm. Serum creatinine phosphokinase (CK) was 724 U/l (N: 30–125). In February 1997, treatment with prednisone (20 mg/day) and intramuscular methotrexate (12.5 mg/week) was started. Three months later, she still complained of proximal lower limb muscle weakness. A second muscle biopsy was performed in June 1997. In this biopsy, the initial lesions were less severe. In June 1997, serum CK was normal. The doses of prednisone and methotrexate could be gradually decreased from June to September 1997. Stable titres of specific IgG antibodies were present against a synthetic peptide derived from VP1+VP2 antigen and recombinant VP2 antigen; no specific IgM antibodies were detected. None of the serum samples was positive for parvovirus B19 DNA. The two muscle biopsies contained parvovirus B19 VP1 sequences and the second one was also positive for NS1. After being treated with prednisone and methotrexate for 2 yr, she stopped the treatment by herself in June 1999. She remained inactive until April 2000 when she developed asthenia and cramps in the legs and arms, signs compatible with a relapse of myositis 11 months after stopping methotrexate. The ESR was 56 mm and serum CK was 479 U/l. An electromyographic study did not show evidence of an abnormal muscle pattern. Stable titres of specific IgG antibodies were still present. To confirm DM relapse, a deltoid muscle biopsy was performed, which showed a typical inflammatory infiltrate. However, this third biopsy was negative for B19 DNA in contrast to the first two biopsies performed in 1997 [2].


    Patients and methods
 Top
 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 
Patients
To clarify the possible association between parvovirus B19 and myositis, a short pilot study of muscle biopsy samples was performed. Seven frozen and paraffin-embedded muscle biopsy samples were obtained from patients with myositis, four female and three male, mean age 50 yr, range 7–73 (Table 1Go). All five DM patients and two patients with polymyositis (PM) fulfilled the criteria of Bohan and Peter [3]. All biopsies showed abnormalities compatible with the respective diagnosis. In addition, muscle biopsies from three osteoarthritis patients undergoing hip replacement were used as controls. Frozen biopsies were used for PCR and paraffin-embedded biopsies for immunohistochemistry.


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TABLE 1. Details of patients included in the study

 
A paraffin section of a placenta from a baby with B19-associated hydrops fetalis was used as a positive control for immunohistochemistry. B19-positive sera from patients with transient aplastic crises associated with sickle-cell anaemia were used for the biological assay and as a positive control for the PCR. The same B19-positive sera have already been used as positive controls in our previous report [2].

Polymerase chain reaction (PCR)
The same procedure was used as in our previous study [2]. Viral DNA was extracted by the tissue protocol of the QIAamp® DNA Mini Kit (Qiagen), using ATL buffer and proteinase K solution. The first set of oligonucleotide primers consisted of a sense nucleotide 2445 to 2470 (5'-TGA GTA AAA AAA GTG GCA AAT GGT GG-3') and of an antisense nucleotide 2634 to 2608 (5'-GCA ACT AAG TCA AAC AGA GAG GAT GGG-3'). It amplifies a 189 bp segment, corresponding to the beginning of VP1 [4]. The reaction mixture for PCR amplification contained deoxynucleoside triphosphate with uracil, and heat-labile uracil-N-glycosidase (UNG Roche). Amplification was obtained with a Hybaid Omnigene system (35 cycles at an annealing temperature of 57°C). Amplified products were detected by ethidium bromide staining after agarose gel electrophoresis. Specific identification was performed with a 5'-biotin-labelled probe (5'-AAT ATT AAA AGA TCA TTA TAA TAT TTC TTT AGA TAA TCC CC-3', nucleotide 2560 to 2600) according to hybridization procedures of GEN-ETI-KTM (DEIA DiaSorin, Saluggia, Italy).

The second set of primers was selected to amplify a 208 bp fragment corresponding to NS1 coding sequence: sense nucleotide 1539 to 1560 (5'-GGT GGT CTG GGA TGA AGG TAT T-3'), antisense nucleotide 1747 to 1724 (5'-GCT CTT TTA AGG CTT TAG CAT GTA-3') [5]. Amplification used 40 cycles at an annealing temperature of 55°C. The specificity of the PCR positive products was confirmed with a 5'-biotin-labelled probe (5'-CGG GAA CAC TAC AAC AAC TG-3', nucleotides 1704 to 1723).

Immunohistochemistry
Paraffin-embedded sections of control and inflammatory muscle or placenta were treated in xylene and dipped in a gradient of ethanol (once in 99% ethanol, once in 95% ethanol, and once in water). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The sections were then incubated with a monoclonal antibody specific for human parvovirus B19 (VP1 and VP2 capsid proteins) obtained from Novocastra (Newcastle upon Tyne, UK) with 1% normal human serum. Antibody dilution was 1:20 as suggested by the manufacturer. After overnight incubation at 4°C and washing, the sections were incubated with biotinylated goat anti-mouse IgG antibody for 30 min at room temperature, followed by streptavidin–peroxidase complex (Dako, Glostrup, Denmark) for 15 min and 3,3'-diaminobenzidine tetrahydrochloride (Dako) for 20 min. The sections were then counterstained with Mayer's haematoxylin.

Induction of IL-6 production assay
The possible effect of the parvovirus B19 infection on interleukin 6 (IL-6) production by myoblasts was studied in a biological assay. Normal skeletal myoblasts were obtained from muscle biopsy and used between passage 3 and 5. Myoblasts (104 cell/well) were incubated in 8-well LabTekTM chamberslides (Nunc, Rochester, NY) with 40 µl of a B19 PCR positive serum or a serum from a healthy donor with or without 10 pg/ml of human rIL-1ß (Sigma, St. Louis, MO) in a final volume of 200 µl of complete medium. After 72 h, supernatants were removed and IL-6 production analysed by enzyme-linked immunosorbent assay (ELISA) as previously described [6]. Myoblasts were then fixed and stained with the anti-B19 antibody.


    Results and discussion
 Top
 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 
The contribution of parvovirus B19, a single-stranded DNA virus, to various autoimmune diseases is difficult to interpret [1]. In particular, in RA a large proportion of synovial biopsies was found to contain B19 DNA, but the significance of this finding remains controversial [7, 8].

We have reported the first case of DM associated with B19 DNA in muscle biopsies [2]. However, the presence of B19 DNA was not found in a third biopsy performed during a relapse associated with concordant muscle pathology findings. This dissociation did not support a direct link between myositis and B19 infection. Interestingly, the presence of B19 DNA has sometimes been suspected but rarely demonstrated in skeletal muscle (Table 1Go) [914].

To extend this single case report suggesting the absence of a link between parvovirus B19 and myositis, we studied seven other cases of myositis (Table 1Go). First, we analysed seven other muscle biopsies by PCR and immunohistochemistry. None of these was positive by PCR or by immunohistochemistry. In particular, there was no staining of muscle cells or inflammatory cells in any of these samples. Patients were not selected on the basis of positive antibodies since these are commonly observed in controls and patients in the absence of any muscle symptoms. Thus, a viral detection directly at the site of disease by PCR or Southern hybridization was necessary. Although the size of our population is small, our results suggest the absence of direct implication of the virus in the pathogenesis of myositis. However, parvovirus B19 could still initiate the myositis process but disappear in the chronic phase. Obviously the association between viral detection and disease could also be fortuitous. Details in Table 1Go show the available clinical data from patients with myositis. No particular subset of patients with or without the presence of B19 DNA in muscle could be identified.

Parvovirus B19 infection has been found in several cell types including macrophages, follicular dendritic cells, T and B lymphocytes and endothelial cells, all potentially implicated in inflammatory muscle diseases. Surprisingly, infection of synoviocytes from RA patients following contact with parvovirus B19-infected cells induced or enhanced IL-6 production [8]. The mode of infection of these synoviocytes has not been elucidated [15], but in a recent study, parvovirus B19 induced invasive properties in normal human synoviocytes [16]. In addition, IL-6 production can be induced in human haemopoietic cell lines stably transfected with DNA encoding the NS1 protein [17].

These findings suggest the possible association between B19 infection and IL-6 production by mesenchymal cells. We tested the possible increase of IL-6 production by myoblasts stimulated with a B19-positive serum. IL-6 production was not increased in the presence of a B19-positive serum. Addition of IL-1, a pro-inflammatory monocyte-derived cytokine used as a positive control for IL-6 production, had no costimulatory effect. In the same conditions, the expression of VP1 and VP2 capsid proteins by normal myoblasts was not induced as determined by immunohistochemistry.

Our previous case of DM indicated a temporal association between parvovirus B19 infection and active myositis. Our present study and the published cases do not demonstrate the implication of parvovirus B19 in a particular phase of the disease or a subset of patients. If parvovirus B19 does contribute to the pathogenic process leading to myositis, it is probably through indirect mechanisms in patients with a particular genetic or risk factor.


    Acknowledgments
 
We are grateful to J. F. Sauzon from the Department of Virology, Domaine Rockefeller, Lyon, for his technical assistance. Dr Chevrel's work was supported by a grant from the Association Française contre les Myopathies.


    Notes
 
Correspondence to: P. Miossec, Department of Immunology and Rheumatology, Hôpital E. Herriot, 5 place d'Arsonval, 69437 Lyon Cedex 03, France. E-mail: miossec{at}laennec.univ-lyon1.fr Back


    References
 Top
 Abstract
 Introduction
 Case report
 Patients and methods
 Results and discussion
 References
 

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Submitted 14 December 2001; Accepted 2 August 2002





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