Enterovirus related metabolic myopathy: a postviral fatigue syndrome.

OBJECTIVE: To detect and characterise enterovirus RNA in skeletal muscle from patients with chronic fatigue syndrome (CFS) and to compare efficiency of muscle energy metabolism in enterovirus positive and negative CFS patients. METHODS: Quadriceps muscle biopsy samples from 48 patients with CFS were processed to detect enterovirus RNA by two stage, reverse transcription, nested polymerase chain reaction (RT-NPCR), using enterovirus group specific primer sets. Direct nucleotide sequencing of PCR products was used to characterise the enterovirus. Controls were 29 subjects with normal muscles. On the day of biopsy, each CFS patient undertook a subanaerobic threshold exercise test (SATET). Venous plasma lactate was measured immediately before and after exercise, and 30 minutes after testing. An abnormal lactate response to exercise (SATET+) was defined as an exercise test in which plasma lactate exceeded the upper 99% confidence limits for normal sedentary controls at two or more time points. RESULTS: Muscle biopsy samples from 20.8% of the CFS patients were positive for enterovirus sequences by RT-NPCR, while all the 29 control samples were negative; 58.3% of the CFS patients had a SATET+ response. Nine of the 10 enterovirus positive cases were among the 28 SATET+ patients (32.1%), compared with only one (5%) of the 20 SATET- patients. PCR products were most closely related to coxsackie B virus. CONCLUSIONS: There is an association between abnormal lactate response to exercise, reflecting impaired muscle energy metabolism, and the presence of enterovirus sequences in muscle in a proportion of CFS patients.

Enterovirus replication in valvular tissue from patients with chronic rheumatic heart disease.

AIMS: To investigate the involvement of enterovirus infection in chronic, rheumatic heart disease. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded, surgical samples of valve tissue were examined for the presence of enteroviral RNA and virus capsid protein VP1 by in situ hybridization and immunostaining. Of 53 cases, 33 were patients with chronic rheumatic heart disease and 20 had Marfan's syndrome or degenerative valve disease. Enterovirus RNA was detected in 8 (24.2%) of 33 patients with chronic rheumatic heart disease by in situ hybridization using strand-specific oligonucleotide probes, complementary to conserved sequences in enterovirus genomic (positive strand) RNA. The replication template (negative strand) RNA also was found in seven of these eight cases. The viral capsid protein VP1 was detected in 16 (48.5%) of 33 patients with chronic rheumatic heart disease by immunohistochemistry and correlated with viral RNA detection. Virus was localized generally to valvular tissue. Neither viral RNA nor capsid protein VP1 were found in valvular tissue from any of the 20 comparison cases. CONCLUSIONS: This is the first demonstration of detection and localization of both enterovirus RNA and capsid protein in chronic rheumatic heart disease. The presence of negative strand RNA and VP1 indicates enteroviral RNA replication and protein synthesis and suggests an aetiological role of enterovirus in the pathogenesis of chronic rheumatic heart disease.

A vector with transcriptional terminators increases efficiency of cloning of an RNA virus by reverse transcription long polymerase chain reaction.

Full-length cDNA clones of RNA viruses are advantageous for maintaining the genomic sequence without the generation of diversity by accumulation of sequence mutations during productive virus replication. They permit in vitro manipulation of the genomic clone to test the effect of sequence changes on the phenotype of reactivated virus. Infectious cDNA clones have been produced by ligation of subgenomic clones but are sometimes difficult to generate in a single cloning operation. We used reverse-transcription to synthesize full-length cDNA from genomic RNA of Coxsackievirus B3 of the Picornavirus family and enzymatically amplified this by long PCR. Five different cloning vectors were used to clone the long PCR product, including the vector Lorist6 which contains transcriptional terminators on either side of the cloning site to prevent transcription of inserts in E. coli. No recombinant colonies were obtained from any of the vectors lacking transcriptional terminators but three full-length clones were obtained using Lorist6. The results suggest that transcriptional terminators increase the recovery of cDNA clones of the 7.4 kb Coxsackie virus genome in this cosmid vector, without resort to phage packaging, representing an advance over previous methods and advantages in the molecular manipulation of these viruses.

Nitric oxide and Coxsackievirus B3 myocarditis: differential expression of inducible nitric oxide synthase in mouse heart after infection with virulent or attenuated virus.

Increased expression of inducible nitric oxide synthase (iNOS) has been found in inflammatory myocardial disease and increased production of nitric oxide (NO) has both an inhibitory effect on virus replication and a cytotoxic effect on host cells. To investigate the relationship between severity of enteroviral myocarditis and iNOS expression, a characterised murine model was infected with either cardiovirulent or an attenuated Coxsackievirus B3 and myocardial samples were collected on Day 7. The ability of these viruses to induce NOS expression was compared by measurement of iNOS enzyme activity and localisation of iNOS protein or peroxynitrite, a product of excessive NO production. In accordance with previous reports, high expression of iNOS was detected in mice infected with the cardiovirulent virus. The iNOS protein was located mainly in infiltrating macrophages in and around foci of necrotic myofibres where viral genomic RNA was detected. In contrast, the level of iNOS expression was significantly lower in mice infected with the attenuated virus. This correlates with fewer and smaller myocarditic lesions and less infiltrating cells in the heart. iNOS was not detected in mock-infected mice by the above assays. These findings suggest that one mechanism of attenuation may be associated with the reduced ability of the variant to induce NOS expression in the heart. This also confirms a cytotoxic role for NO in the pathogenesis of Coxsackievirus B3-induced myocarditis.

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.

Characterization of enterovirus isolates from patients with heart muscle disease in a selenium-deficient area of China.

An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural areas of southwestern China has been established. Enteroviruses have been isolated from patients with KD or during outbreaks of myocarditis in last two decades. Six of these isolates grew readily in cell lines (Vero or HEp-2) and were investigated by a novel molecular typing method apart from serotyping and pathogenicity. A neutralization assay identified two isolates from KD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isolates, also from myocarditis. Direct nucleotide sequencing of reverse transcription-PCR products amplified from the 5' nontranslated region (5'NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisting of coxsackie B-like viruses, including some echovirus serotypes. Sequence analysis of the coding region for viral capsid protein VP1 showed that two isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknown serotypes, are most closely related to the sequence of CVB6. Sequences among these isolates varied from 82.3 to 99% in the 5'NTR and from 69 to 99% in VP1, indicating no cross contamination. The pathogenicity of these viruses in adult and suckling mice was assessed. None caused pathologic changes in the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominant in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogenicity.

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

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.

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.

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.

Induction of myocardial nitric oxide synthase by Coxsackie B3 virus in mice.

BACKGROUND: Inducible nitric oxide synthase (iNOS) expression is regulated by cytokines. This study investigated whether Coxsackie group B virus (CVB) myocarditis resulted in an environment suitable for induction of NOS in the murine heart. MATERIALS AND METHODS: Myocardium was removed from mice infected with CVB3 and from controls. Histology, reverse transcriptase polymerase reaction (RT-PCR) for murine iNOS, NOS enzyme activity and immunohistochemistry were assessed. RESULTS: Histology revealed severe myocarditis 7 days after infection with CVB3 but not in controls. RT-PCR using primers for murine iNOS detected iNOS mRNA in infected mice but not in controls. Calcium-independent NOS activity increased by day 5 after infection with a peak at day 7. Calcium-dependent NOS activity was present throughout, with a trend to lower levels during peak calcium-independent activity. Immunohistochemistry revealed iNOS to be localized to inflammatory cells rather than to myocytes. CONCLUSION: This study demonstrates the development of calcium-independent NOS activity and de novo gene transcription for iNOS in the murine myocardium in response to CVB3 infection. The nitric oxide produced at such high output may act at times as part of the immune defence as an antiviral agent and may be toxic to host tissue.

Characterization of anti-heart antibodies in mice after infection with coxsackie B3 virus.

Coxsackie virus B3 (CVB3) infection results in a marked inflammatory response and the production of autoantibodies to cardiac antigens, with cardiac myosin heavy chain documented to be the most immunogenic antigen. The present study investigated the temporal appearance of anti-heart antibodies in mice after mock infection or infection with an attenuated variant of CVB3 or wildtype CVB3 by SDS-PAGE and Western blotting. Further characterization of the autoantigens was carried out using 2D electrophoresis followed by Western blotting. Mice infected with wildtype CVB3 demonstrated high levels of IgG anti-heart antibodies, reacting predominantly with myosin heavy chain but also with numerous other myocardial proteins. Significant increases in anti-myosin heavy chain, anti-actin, and anti-tropomyosin antibodies were seen in wildtype-infected mice as early as day 7 postinfection compared to those mice that were mock infected or infected with attenuated virus. Characterization of other antigens revealed novel reactivities against myosin subfragments, heat shock proteins, and desmin and its subfragments.

Cardiac protein abnormalities in dilated cardiomyopathy detected by two-dimensional polyacrylamide gel electrophoresis.

The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two-dimensional (2-D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p<0.01). The most prominent changes occurred in the contractile protein myosin light chain 2 and in a group of proteins identified as desmin. These changes do not appear to be artefactual degradation events occurring during sample processing. These proteins are not apparent in electrophoretic separations of vascular tissue or cultured endothelial cells, mesothelial cells or cardiac fibroblasts, which are clearly distinguishable from the 2-D protein patterns of whole heart and of isolated cardiac myocytes and do not appear to reflect variations in the cellular composition of biopsy samples. The different protein patterns observed in cardiomyopathy showed no obvious relationship with New York Heart Association (NYHA) functional class or haemodynamic parameters. The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function. These changes might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.

Nucleotide sequence of an attenuated mutant of coxsackievirus B3 compared with the cardiovirulent wildtype: assessment of candidate mutations by analysis of a revertant to cardiovirulence.

BACKGROUND: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed. OBJECTIVES: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype. STUDY DESIGN: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5'-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant. RESULTS: There are five candidates for attenuation: (1) A-G at base 580 in the 5'-NTR; (2) A-T at base 690 in the 5'-NTR; (3) CG-GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA-GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A-G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5'-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation.

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.

Muscle fibre characteristics and lactate responses to exercise in chronic fatigue syndrome.

OBJECTIVES: To examine the proportions of type 1 and type 2 muscle fibres and the degree of muscle fibre atrophy and hypertrophy in patients with chronic fatigue syndrome in relation to lactate responses to exercise, and to determine to what extent any abnormalities found might be due to inactivity. METHODS: Quadriceps needle muscle biopsies were obtained from 105 patients with chronic fatigue syndrome and the proportions of type 1 and 2 fibres and fibre atrophy and hypertrophy factors were determined from histochemical preparations, using a semiautomated image analysis system. Forty one randomly selected biopsies were also examined by electron microscopy. Lactate responses to exercise were measured in the subanaerobic threshold exercise test (SATET). RESULTS: Inactivity would be expected to result in a shift to type 2 fibre predominance and fibre atrophy, but type 1 predominance (23%) was more common than type 2 predominance (3%), and fibre atrophy was found in only 10.4% of cases. Patients with increased lactate responses to exercise did have significantly fewer type 1 muscle fibres (p<0.043 males, p<0.0003 females), but there was no evidence that this group was less active than the patients with normal lactate responses. No significant ultrastructural abnormalities were found. CONCLUSION: Muscle histometry in patients with chronic fatigue syndrome generally did not show the changes expected as a result of inactivity. However, patients with abnormal lactate responses to exercise had a significantly lower proportion of mitochondria rich type 1 muscle fibres.

Coxsackievirus B3-induced myocarditis. Characterization of stable attenuated variants that protect against infection with the cardiovirulent wild-type strain.

Coxsackievirus B3 (CVB3) is the enterovirus most frequently involved in human myocarditis or dilated cardiomyopathy. Attenuated variants were derived from a cardiovirulent CVB3 reactivated from a sequenced, full-length cDNA clone. The prophylactic potential of these variants was assessed in SWR/Ola (H-2q) mice. Animals immunized with attenuated variants of CVB3 were protected from myocarditis when challenged subsequently with the cardiovirulent wild-type virus. In contrast to nonimmunized controls, the wild-type virus was not isolated from myocardium of protected mice, nor was viral RNA detected in myocardium by reverse transcription nested polymerase chain reaction. Specific antibody to CVB3 was demonstrated by virus neutralization assay and by indirect immunofluorescence. The attenuated phenotype of one variant, p14V-1, remained stable throughout 20 consecutive passages in SWR mice and induced a markedly lower level of autoantibody against mouse cardiac myosin heavy chain than the cardiovirulent wild type. These data demonstrate that attenuated strains protect against CVB3-induced myocarditis in mice, that the attenuated phenotype is stable, and that they do not persist in myocardium nor induce a significant level of anti-heart anti-body against myosin heavy chain. These attenuants may be the basis of a live vaccine against CVB3 in the prevention of enteroviral heart muscle disease.

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.

Similarity in genome organization between Molluscum contagiosum virus (MCV) and vaccinia virus (VV): identification of MCV homologues of the VV genes for protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3beta-hydroxysteroid dehydrogenase.

Molluscum contagiosum virus (MCV) and vaccinia virus (VV) are serologically unrelated poxviruses with a disparate genome composition (MCV, 66% G+C; VV, 33% G+C). Molecular studies of MCV have been hindered by the inability to propagate the virus in cells cultured in vitro. We sequenced 7765 bp of MCV DNA cloned from four widely spaced regions throughout the MCV genome and identified a total of 11 potential open reading frames (ORF), designated CX1-11. These include MCV homologues of the VV genes encoding protein kinase 2, structural protein VP8, RNA polymerase 35 kDa subunit and 3beta-hydroxysteroid dehydrogenase. The position and orientation of the MCV ORFs was collinear to the VV genome, with the exception of the region around ORF CX11 which is inverted in the MCV genome.