Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy.

BACKGROUND: There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS: Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS: Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Altered expression of Bag-1 in Coxsackievirus B3 infected mouse heart.

OBJECTIVE: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. METHODS: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. RESULTS: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. CONCLUSION: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease.

Specific transcription of orthopox virus DNA by HeLa cell RNA polymerase II.

A HeLa cell extract was used to transcribe DNA isolated from cowpox virus. Truncated templates generate accurately initiated run-off transcripts of discrete sizes and whose sensitivity to inhibition by alpha-amanitin indicates synthesis by cell RNA polymerase II. A mapped restriction fragment of wild-type cowpox DNA contains specific sites of initiation which are not detected in the geographically equivalent fragment from a cowpox mutant having a defined sequence rearrangement in this region.

The role of Coxsackie B viruses in the pathogenesis of myocarditis, dilated cardiomyopathy and inflammatory muscle disease.

Coxsackie B viruses are members of the family Picornaviridae which have been associated by retrospective serology with a range of muscle diseases, particularly myocarditis, dilated cardiomyopathy and epidemic pleurodynia (epidemic myalgia or Bornholm disease). It has been proposed that virus-induced myocarditis disposes to the development of idiopathic dilated cardiomyopathy. However, despite many attempts, isolation of infectious virus or immunofluorescent detection of virus-specific antigens in the affected tissue is rare, although virus may be found in faeces early in infection. This discrepancy awaited the development of nucleic acid probes to resolve the problem of whether virus was present consistently in myocardium or other muscle tissues. We report here the synthesis of Coxsackie B virus-specific complementary DNA (cDNA) probes and their use in molecular hybridizations to quantitative slot-blots of RNA prepared from either endomyocardial or skeletal muscle biopsy specimens. Of 50 patients with histologically proven myocarditis or dilated cardiomyopathy, 28 (56%) had an endomyocardial biopsy specimen positive for the presence of Coxsackie B virus-specific RNA. Twenty-two patients with other cardiac diseases of known aetiology, unrelated to virus infection, were all negative. Multiple biopsies were obtained from 20 patients with myocarditis or dilated cardiomyopathy and 15 of these (75%) had at least one biopsy specimen positive, indicating the focal nature of the disease. In analogous investigations, Coxsackie B virus-specific RNA was detected in four out of seven single skeletal muscle biopsy specimens from patients suffering from juvenile dermatomyositis, and one out of two patients with adult polymyositis. Ten muscle controls, either normal or Duchenne muscular dystrophy, were negative for virus RNA.

Localization of enteroviral antigen in myocardium and other tissues from patients with heart muscle disease by an improved immunohistochemical technique.

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 antigen-antibody 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.

Characterization of Coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products.

This study was performed to detect and characterize the enterovirus present in myocardium of some patients with heart muscle disease by nucleotide sequencing of polymerase chain reaction (PCR) products after amplification with enterovirus group-specific primers. Enterovirus sequences have been detected previously in myocardium of patients with myocarditis or dilated cardiomyopathy and seem causal, although the particular virus serotypes involved have not been identified. In a prospective study of endomyocardial biopsy specimens from 35 consecutive patients with suspected heart muscle disease, enterovirus sequences from the 5' nontranslated region were amplified by reverse transcription-nested PCR using group-specific primers. This region contains both conserved and variable sequence motifs, characteristic of particular enterovirus serotypes. The nucleotide sequences of individual PCR products were determined by cycle sequencing and compared with all known sequences (GenBank/EMBOL), using the GCG software package. Endomyocardial biopsy specimens from 9 of 21 (42.9%) patients with a histologically confirmed diagnosis of dilated cardiomyopathy were positive for enterovirus by PCR, compared with only 1 of 14 (7.1%) patients with other myocardial pathological conditions (Fisher's exact probability=0.0275: odds ratio=9.75; 95% confidence interval=1.31-72.78). The nucleotide sequence of the PCR products differed, indicating no cross-contamination. However, computerized comparison showed that each had greatest homology with the 5' nontranslated region of Coxsackie B virus but contained up to 11% sequence variations compared with the prototype Coxsackie B3 strain Nancy. Parallel investigation of tissue from our mouse model of Coxsackievirus B3-induced myocarditis showed that nucleotide sequence changes are not introduced by reverse transcription or PCR. These data support the link between enteroviral infection and dilated heart muscle disease and suggest that Coxsackie B serotypes are the enteroviruses most frequently involved.

Characterisation of genomic RNA of Coxsackievirus B3 in murine myocarditis: reliability of direct sequencing of reverse transcription-nested polymerase chain reaction products.

SWR mice develop viral myocarditis histologically similar to the human disease following inoculation with a cardiovirulent Coxsackievirus B3 (CVB3), reactivated from a sequenced cDNA clone of Nancy strain. A sequence of 215 nucleotides, or 628 nucleotides in representative cases, of the 5'non-translated region (5'NTR) of CVB3 genome was amplified from myocardial samples of the infected mice by reverse transcription-nested polymerase chain reaction (RT-NPCR). In order to verify the viral nucleotide sequence and detect the mutation frequency of the viral RNA, the nucleotide sequence of NPCR products were determined by direct sequencing in both orientations. The amplified products from mouse heart on day 1-13 post-inoculation were sequenced and, in each case, the consensus sequence was identical to the published sequence of CVB3 (Nancy strain). To evaluate further the reproducibility of these techniques, three tissue samples from the same infected mouse heart were processed independently. Sequences of their RT-NPCR products were identical to each other as well as to the published sequence. When two attenuated CVB3 mutants were amplified and sequenced, single mutations were detected. To evaluate the overall fidelity of these two combined techniques, genomic RNA of a different CVB3 Nancy strain stock, Coxsackievirus A9 or poliovirus sabin 1 was amplified and the NPCR products sequenced. Each product showed 100% homology with its published sequence. These results demonstrate that the coupled technique of the enterovirus RT-NPCR with direct sequencing of NPCR products generates accurate consensus sequence data and this technique proved to be useful in verification of enteroviral amplicons and in detection of nucleotide mutations. In addition, a low mutation frequency was found in the 5'NTR of CVB3 detected in myocardial samples of immunocompetent mice up to 13 days.

Conserved gene sequences for species identification: PCR analysis of the 3' UTR of the SON gene distinguishes human and other mammalian DNAs.

In this paper we show that the polymerase chain reaction (PCR) can be used on regions of highly conserved genes, such as the 3' untranslated region (3' UTR) of the SON gene, to identify the mammalian origin of a sample. Using this test, we have been able to distinguish human, monkey, cat, dog, mouse and hamster DNAs. We have also determined the DNA sequence of these different PCR products, which can be used to reinforce species identification. The advantages of this test are that: (i) no prior information is required on the possible species origin of a sample, (ii) the reaction produces a single PCR product which varies in size according to the species of origin, making the test simple to interpret, and (iii) the target region of DNA amplified in these experiments is small and can easily be sequenced and sized using automated techniques. This small size has enabled us to successfully amplify this product from DNA extracted from compromised material (human bone samples) and so demonstrate that the test is valuable for the characterisation of remains in which DNA is degraded.

Postviral fatigue syndrome: persistence of enterovirus RNA in muscle and elevated creatine kinase.

Enterovirus-specific probes have been prepared by reverse transcription of conserved sequences in purified Coxsackie B2 virus genomic RNA and molecular cloning techniques. These probes were used in quantitative slot blot hybridizations to test for the presence of enterovirus-specific RNA in skeletal muscle biopsy specimens from 96 patients who had suffered from the postviral fatigue syndrome myalgic encephalomyelitis for up to 20 years. Biopsy specimens from 20 patients were positive for the presence of virus-specific RNA with hybridization signals more than three standard deviations greater than the mean of the normal muscle controls. Biopsies from the remaining 76 patients were indistinguishable from the controls. These data show that enterovirus RNA is present in skeletal muscle of some patients with postviral fatigue syndrome up to 20 years after onset of disease and suggest that a persistent virus infection has an aetiological role.

De novo synthesis of two classes of DNA induced by vaccinia virus infection of HeLa cells.

Equilibrium density gradient centrifugation in CsCl confirms that DNA synthesized after vaccinia virus infection of HeLa cells is homogeneous in buoyant density and thus in base composition and is similar in this respect to bulk HeLa cell DNA. In contrast, rate sedimentation in alkaline sucrose gradients distinguishes two main classes of virus-induced DNA, neither of which can be equated with cell DNA synthesized in the same cultures prior to infection. The slower sedimenting class of virus-induced DNA co-sediments with DNA from purified virus particles: the second class sediments faster than pre-labelled cell DNA. Heterogeneity of virus-induced DNA does not result from fragmentation of radioactively labelled DNA, virus-mediated breakdown of cell DNA or association with either proteins or polyamines. Both slow and fast sedimenting classes of virus-induced DNA contain sequences complementary to all restriction endonuclease Hind III-specific fragments of the virus genome. The multiple species of DNA synthesized after infection are distinguished further by the effect of ethidium bromide. At a concentration which prevents the formation of infectious progeny virus, this compound inhibits selectively the de novo synthesis of that class of virus-induced DNA which sediments faster in alkaline sucrose gradients.

Synthesis of full-length, virus genomic DNA by nuclei of vaccinia-infected HeLa cells.

It is well established that vaccinia virus infection induces the synthesis of virus-specific DNA in cytoplasmic 'factories', which are sites of virus-specific transcription. The present study demonstrates that vaccinia virus-specific DNA is synthesized also in the nuclei of infected cells with a similar time course. Direct observation and radiolabelling confirm the integrity of isolated nuclei. Reconstitution experiments and inhibitor studies demonstrate that virus-induced DNA is synthesized de novo within nuclei and does not result from cytoplasmic contamination. Cell-specific DNA synthesis is inhibited completely after infection and nuclei of infected cells then synthesize DNA which co-sediments with virus genomic DNA in denaturing gradients. Restriction endonuclease cleavage and hybridization with a virus-specific probe indicate that this is full-length, virus genomic DNA. The biological implications of this are discussed.

Conservation and variation in Orthopoxvirus genome structure.

Orthopoxvirus DNA from representative strains of rabbitpox, vaccinia, monkeypox, variola, cowpox and ectromelia viruses was analysed by cleavage with restriction endonucleases HindIII, XhoI or SmaI. Genome mol. wt. vary from about 120 x 10(6) for rabbitpox to about 145 x 10(6) for cowpox. Physical maps of cleavage sites are similar and characteristic for strains of the same Orthopoxvirus type. The distribution of HindIII sites suggests that an internal region of mol. wt. about 30 x 10(6) is highly conserved between Orthopoxvirus genomes although some type-specific differences occur within this region, especially with strains of ectromelia virus. Conservation of internal sequences is less marked following analysis with XhoI although cleavages within this central region of particular genomes appear to represent a subset of preferred sites. Endonuclease SmaI cleaves exceptionally infrequently and distinguishes variola, monkeypox, vaccinia, cowpox or ectromelia viruses. Type specific differences result largely from extensive, near terminal variations in length and sequence. Representative Orthopoxvirus genomes have rapidly renaturing terminal restriction fragments confirming the presence of near terminal, covalent cross-links. Terminal restriction fragments from the same or different genomes generally cross hybridize indicating the presence of near terminal repetitions of mol. wt. up to 6 x 10(6) and which share at least a subset of common sequences. Variola strains however, appear to lack such sequences from one specific terminus which maps shorter than that of related viruses.

Comparison of white pock (h) mutants of monkeypox virus with parental monkeypox and with variola-like viruses isolated from animals.

Monkeypox mutants arising spontaneously or after serial, high multiplicity passage were characterized phenotypically and by restriction endonuclease mapping. Some resemble "whitepox" and variola viruses in several of the markers tested but all are distinguishable phenotypically from these. None resembles "whitepox" viruses in genome structure although near-terminal deletions or symmetrical, terminal rearrangements, relative to parental monkeypox, occurred. "Whitepox" viruses isolated from animals closely resemble variola in both phenotype and genome structure.

Restriction endonuclease analysis of red cowpox virus and its white pock variant.

The DNA of red cowpox virus strain Brighton or its white pock variant was analysed by cleavage with restriction endonucleases HindIII, XhoI, PstI or KpnI. Physical maps were constructed and the genomes compared with that of vaccinia virus strain DIE. The size of the red cowpox genome is 23 to 29 megadaltons greater than that of vaccinia and results from the presence of additional, near terminal sequences. An internal region of about 75 megadaltons appears to be highly conserved between the two viruses. The red cowpox genome contains near terminal, repetitive sequences which have some homology with those of vaccinia virus DNA. Rapid renaturation of red cowpox terminal restriction fragments indicates that these are covalently cross-linked. Viable white pock variants arise continually and map as deletion mutants lacking similar sequences from one specific terminus only of the parental genome. The deletion represents 11 to 12% of the red cowpox DNA and includes the terminal repetition which therefore is not required for replication. The deleted terminus of the white pock variant genome does not appear to be cross-linked.

Characterisation by restriction mapping of three subtypes of molluscum contagiosum virus.

DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease digestion, identifying three virus subtypes. The structural features of MCV DNA are typical of poxviral DNA. Physical maps of cleavage sites for BamHI, CIaI, and HindIII were constructed for single isolates of each subtype. These differ extensively, indicating the independence of the three subtypes. However, they are closely related, as determined by molecular hybridisation and nucleotide sequence analysis, and their genomes are essentially colinear. There is marked geographical variation in the relative incidence of MCV I and II, whilst MCV III is uniformly rare.

Characterization and physical mapping of Molluscum contagiosum virus DNA and location of a sequence capable of encoding a conserved domain of epidermal growth factor.

DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease cleavage, revealing two virus subtypes. Physical maps of cleavage sites for BamHI, ClaI and HindIII were constructed, and found to differ extensively between the two subtypes. MCV DNA was similar to Orthopoxvirus DNA with respect to size, terminal cross-linking and the presence of inverted terminal repetitions, but did not hybridize with vaccinia virus DNA. The genomes of the two MCV subtypes cross-hybridized and were colinear except for two small regions. There was sequence homology between DNA from corresponding map regions of the MCV subtypes but, in contrast to Orthopoxvirus DNA, no conservation of restriction sites. A synthetic oligonucleotide probe representing a conserved domain of epidermal growth factor, alpha-transforming growth factor and the vaccinia growth factor identified equivalent regions of both MCV genomes as having the potential to encode this domain. This locus is similar to the position of the vaccinia growth factor gene in vaccinia virus DNA. Thus MCV may induce epidermal cell proliferation and tumourigenesis by expression of an epidermal growth factor-like polypeptide.