RAPID COMMUNICATION |
Correspondence to: Hongyi Zhang, Molecular Pathology Section, Div. of Biomedical Sciences, SAF Building, ICSTM, South Kensington, London SW7 2AZ, UK. E-mail: h.zhang@ic.ac.uk
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Summary |
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The association of enterovirus infection and heart muscle diseases has been investigated extensively by detection of viral genomic RNA using nucleic acid hybridization and the reverse transcription-polymerase chain reaction. To further understand the role of enterovirus and its persistence in these diseases, an immunohistochemical technique was optimized to investigate the expression of viral capsid proteins in situ. A monoclonal antibody (5-D8/1) against an epitope in the N-terminus of capsid protein VP1, conserved in the enterovirus genus, was employed. To enhance sensitivity, the EnVison system was used to detect antigenantibody complex. VP1 was detected in formalin-fixed, paraffin-embedded endomyocardial biopsy or postmortem myocardial tissues and in liver, spleen, lung, kidney, and pancreas from patients with myocarditis or dilated cardiomyopathy, but not from controls. VP1 was localized in cytoplasm of myofibers, often adjacent to necrosis and infiltrate in myocarditis, and was clustered or scattered in dilated cardiomyopathy. This technique can be used for a definitive laboratory diagnosis of enterovirus-associated diseases and for studying the mechanisms of virus persistence in chronic myocardial disease.
(J Histochem Cytochem 48:579584, 2000)
Key Words: enterovirus, antigen, myocarditis, cardiomyopathy, immunohistochemistry, monoclonal antibody, EnVision
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Introduction |
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HUMAN ENTEROVIRUSES (Evs) of the picornavirus family include poliovirus, Group A and B Coxsackievirus, echovirus, and enterovirus 6871. The viral particles are 2530 nm in diameter with icosahedral symmetry, and consist of a single-stranded positive-sense RNA of 70007500 bases and a viral capsid. The capsid contains four proteins, VP1 through VP4. Serotype-specific antigenic sites recognized by neutralizing antibodies are located predominantly in VP1. Such epitopes have also been mapped to VP2 and VP3. Major neutralization epitopes are often conformational, involving interaction of discontinued residues, and are heat-sensitive (
Enteroviruses are one of the most common and important pathogens of humans. In the United States alone, the Evs are estimated to cause 10 million symptomatic infections annually (
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Materials and Methods |
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Human Tissue Samples
Autopsy or endomyocardial biopsy myocardial samples were obtained from 13 patients with histopathologically proven myocarditis (nine cases) or DCM (four cases). Cardiac control tissue was taken from 11 cases of accidental death or noncardiac diseases. Histologically, no abnormality was found in these control myocardial tissues. Tissue samples of other organs, i.e., liver, spleen, lung, kidney, and pancreas from some autopsy cases, were also collected. Myocardial tissues were not available from three fatal myocarditis cases, but their pancreas tissue samples were made available for etiological investigation and were included in this study. All specimens were fixed in 10% formalin and paraffin-embedded for routine histopathology. Some of them were fixed for over 1 year before embedding. Most of the blocks had been stored for 110 years before the present examination. These samples were collected from Shanghai Medical University (P. R. China) and consent was obtained from patients or their relatives. Clinically, these patients had a history of suspected enteroviral infection or died during outbreaks of enteroviral heart muscle disease. The histopathologic diagnosis of myocarditis or DCM is based on Dallas criteria (
Immunohistochemical Procedures
Enteroviral group-specific MAb 5-D8/1, peroxidase-conjugated rabbit anti-mouse or swine anti-rabbit Ig, normal mouse IgG2a, pronase, pepsin, serum-free protein, blocking reagents, antibody diluent, EnVision Plus detection, and labeled streptavidinbiotin (LSAB) systems were purchased from DAKO (Cambridge, UK). Anti-Coxsackievirus B3 MAb (IgG2a) and polyclonal antibodies were from Chemicon International (Harrow, UK) and Central Public Health Laboratory (Colindale, UK), respectively. Trypsin with buffer salt tablets was from Sigma Chemicals (Dorset, UK). Immunohistochemical procedures included exposure of antigens, blocking, incubation with primary antibody, incubation with conjugated secondary antibody or with detection systems (EnVison or LSAB), and appropriate wash between steps with Tris-buffered saline (TBS). All incubations were performed at room temperature (RT).
Briefly, tissue sections (5 µm) were dewaxed with xylene and rehydrated with graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 15 min. Antigen exposure was achieved by heat-mediated antigen retrieval. Sections were immersed in 10 mM citrate buffer (pH 6.0) within a plastic slide container, heated three times for 5 min in a microwave oven at full power, and left at RT for cooling (
Expression of Viral Capsid Proteins in Eukaryotic or Insect Cells
The capsid protein PV1 of Coxsackievirus B3 expressed in either eukaryotic or insect cells was used to confirm the specificity of MAb 5-D8/1 by immunofluorescence and western blot. Appropriate viral coding sequences were amplified by polymerase chain reaction (PCR) with high-fidelity pfu polymerase (Stratagene; Amsterdam, The Netherlands) from a cDNA clone of CVB3 (
Western Blotting
A cell lysate was prepared for SDS-PAGE from pCMV/ VP1-transfected Vero cells, recombinant baculovirus-infected insect cells, and CVB3-infected Vero cells. VP1 proteins were seperated in a 12% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, and subjected to immunostaining with MAb 5-D8/1 according to methods described previously (
Immunofluorescence
Transfected or infected cells expressing CVB3 proteins were fixed in cold acetone and subjected to immunofluorescence for PV1 detection using MAb 5-D8/1 according to standard protocols (DAKO).
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Results |
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Confirmation of Specificity of MAb 5-D8/1 Using In Vitro-expressed VP1 Protein
The specificity of MAb 5-D8/1 to a broad range of enteroviral serotypes, but not to other viruses and cellular antigens, has been documented (
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Detection and Localization of Enteroviral VP1 in Myocardium from Patients with Heart Muscle Diseases
Using MAb 5-D8/1 and the improved techniques involving combination of the microwave-based epitope unmasking and EnVision system, enteroviral capsid protein VP1 was detected in duplicate myocardial tissue sections from six of nine myocarditis and three of four DCM cases. The results were reproducible when further sections were tested 24 weeks later. Substitution of primary antibody with antibody diluent only or with isotype- and concentration-matched normal mouse IgG2a produced negative results on consecutive sections. Pre-absorption of the MAb 5-D8/1 (0.22 µg/ml) with CVB3 in twofold dilution from 1:2 to 1:32 (106 3 x 104 plaque forming units) for 30 min abolished the immunoreactivity. All control tissues from accidental death or noncardiac diseases were negative. Immunostaining signals were localized in cytoplasm of cardiac myocytes/myofibers. Where there were necrotic and infiltrating lesions in myocardial sections from myocarditis, VP1 was often localized near or around, but not in the center of, necrotic tissue (Fig 2). In general, there were two staining patterns of VP1: strong signals were distributed focally or confluently (in fulminent cases) involving a large area of sections in myocarditis, whereas VP1 signals were found in scattered myocytes in DCM cases (Fig 2).
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Detection of VP1 in Other Tissues
Tissue samples of spleen, lung, kidney, pancreas, and liver were collected from a limited number of cases and investigated similarly. VP1 immunostaining was positive in one of two spleen, two of three lung, two of two kidney, three of five pancreas tissue samples from myocarditis. VP1 was also detected in one liver sample from a DCM case. A lung tissue sample from another DCM case was negative. The heart tissue samples from the above cases were all positive for VP1 except for three myocarditis cases, whose pancreas samples were positive but heart samples were not available for the present investigation. VP1 signals were localized in the cytoplasm of various cell types in these different tissues: lymphoid cells in spleen, endothelia in the lung, acinar cells in the pancreas, hepatocytes in the liver, and tubule cells in the kidney. VP1 detection was negative in the spleen, lung, pancreas, liver, and kidney from a control.
Method Comparison
Permeabilization of sections by conventional protease treatment was compared with that by heat treatment in a microwave oven. Consecutive sections were treated with 1 mg/ml trypsin, 0.5 mg/ml pronase, or 4 mg/ml pepsin at 37C for 1520 min or with microwaving. VP1 signals were seen in sections treated with microwaving as described above, but not in sections treated with any of these proteolytic enzymes. A different CVB3-specific MAb (IgG2a; Chemicon International, Temecula, CA) and a CVB3-specific rabbit neutralizing antiserum (Public Health Laboratory) were used to compare with the MAb 5-D8/1, and both failed to detect viral antigens in sections pretreated with proteolytic enzymes or microwaving. When different detection systems were compared, signals of VP1 immunostaining in the myocardium generated by the three-stage immunoperoxidase method or LSAB were comparable to that produced by the EnVision system, but a higher concentration of primary antibody was needed and more nonspecific background staining was generated by the former. The EnVision system is simpler, faster, more sensitive, and results in clearer background.
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Discussion |
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Enteroviral capsid protein VP1 was detected reproducibly in heart and other tissue samples from patients with myocarditis or DCM. The high incidence of VP1 detection in this study does not represent a true incidence of enteroviral infection in the heart because the patient groups were highly selected: all had a history of enteroviral infection. Similar results have been reported in selected pediatric patients with fatal neonatal myocarditis (
Pathological changes have been found in organs other than the heart in experimental animal models of viral myocarditis. Pancreatitis is common in mice after inoculation with Coxsackievirus (
In summary, the improved immunohistochemical method, involving MAb 5-D8/1 and EnVision detection system, pioneered in our laboratory is an enteroviral group-specific, rapid, easy, and sensitive assay. It can be used to detect VP1 in archival fixed tissues as old as 10 years. It will become an indispensable measure in routine laboratory diagnosis as well as in mechanistic study of diseases associated with enteroviral infection.
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Acknowledgments |
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Supported by the Wellcome Trust (052954Z97), by the University of London Central Fund, and in part by the British Heart Foundation.
Received for publication January 19, 2000; accepted January 19, 2000.
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