Leonard C. Archard

Molluscum contagiosum virus types in genital and non-genital lesions.

The endonuclease digest patterns of viral DNA from 48 genital and 45 non‐genital molluscum contagiosum virus (MCV) lesions were examined. The overall ratio of MCVI to MCVII was 3.23:1. There was no predominance of either MCV type in genital lesions. No obvious morphological differences were seen between MCVI and MCVII lesions. MCVII was not found in any patient under 15 years old.

The Effect of Arginine Deprivation on the Replication of Vaccinia Virus

Summary Arginine has been shown to be essential for the replication of vaccinia virus in HeLa cell cultures. The yield of infective virus was dependent upon the concentration of arginine in the medium, maximum yield being obtained with 0.09 mm-arginine. There were marked reductions in the synthesis of DNA, RNA and protein in infected and control cultures deprived of arginine. The yield of infective virus from cell cultures infected in the presence of sub-optimal concentrations of arginine was increased following further addition of arginine at a time after the completion of virus DNA synthesis. Arginine has been shown to be incorporated into the virus particle. It is concluded that there are requirements for arginine demonstrable at both early and late stages in the replication of vaccinia virus in this system. While the early requirement was satisfied by 0.015 mm-arginine, later events showed a greater requirement up to a maximum of 0.09 mm-arginine.

Segmented Arrangement of Borrelia duttonii DNA and Location of Variant Surface Antigen Genes

The DNA of an isolate of Borrelia duttonii, an agent of relapsing fever is present as seven major species ranging in size from 10 kb to greater than 150 kb. Additionally, this isolate contains low copy number species, both smaller and larger than these seven major elements. No one of these individual DNA species obviously corresponds to the bacterial chromosome, unlike the situation in Borrelia hermsii, another relapsing fever Borrelia. Thus it appears that B. duttonii has a unique segmented arrangement of its genetic material. Cloned DNA fragments containing coding sequences specific for variant surface antigens of B. duttonii hybridize to a closely migrating, high copy number subset of these genetic elements.

Detection of enterovirus capsid protein VP1 in myocardium from cases of myocarditis or dilated cardiomyopathy by immunohistochemistry: further evidence of enterovirus persistence in myocytes.

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected χ2=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.

Herpes Simplex Virus Type 1 Infection Associated with Atrial Myxoma

Some findings suggest an infectious factor in cardiac myxoma and certain histopathological features indicate herpes simplex virus type 1 (HSV-1) infection. We hypothesized that HSV-1 may be involved in the pathogenesis of cardiac myxoma. Paraffin-embedded tissue samples from 17 patients with atrial myxoma were investigated for HSV-1 antigen by immunohistochemistry and viral genomic DNA by nested polymerase chain reaction. The histogenesis and oncogenesis of atrial myxoma were assessed by the expression of calretinin, Ki67, and p53 protein, respectively. Autopsy myocardial samples, including endocardium from 12 patients who died by accident or other conditions, were used for comparison. HSV-1 antigen was detected in atrial myxoma from 12 of 17 patients: 8 of these 12 samples were positive also for HSV-1 DNA. No HSV-1 antigen or DNA was found in tissue from the comparison group. Antigens of HSV-2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus were not found in atrial myxoma. Calretinin was found in myxoma cells of all 17 cases but Ki67 was present only in smooth muscle cells or infiltrating cells in some cases. p53 was not detectable in any myxoma. Most infiltrating cells were cytotoxic T lymphocytes. These data suggest that HSV-1 infection is associated with some cases of sporadic atrial myxoma and that these may result from a chronic inflammatory lesion of endocardium.

A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model.

SummaryWe previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5′ nontranslated region (5′NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5′NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.

Molecular and cell biology of sexually transmitted diseases

Contributors. Preface. Introduction A.D.B. Malcolm. Molecular mechanisms of antigenic variation in Neisseria gonorrhoea H.S. Seifert. Molecular biology of chlamydiae M.A. Monnickendam. Sexually transmitted mycoplasmas in humans A. Blanchard, L.D. Olson, M.F. Barile. Molecular biology of Treponema pallidum L.M. Schouls. Molecular biology of Candida pathogenesis D.R. Soll. Molecular analysis of Trichomonas vaginalis surface protein repertoires J.F. Alderete, R. Arroyo, D.C. Dailey, J. Engbring, M.A. Khoshnan, M.W. Lehker, J. McKay. Hepatitis B virus and Hepatitis delta virus T.J. Harrison, G.M. Dusheiko. Molluscum contagiosum virus C.D. Porter, N.W. Blake, J.J. Cream, L.C. Archard. Molecular biology of herpes simplex virus D.E. Barker, B. Roizman, M.B. Kovler. Anti-idiotypic therapeutic strategies in HIV infection D. Wilks, A. Dalgleish. Index.

Molluscum contagiosum virus

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign skin tumours in man, consisting of a localized mass of hypertrophied and hyperplastic epidermis due to enhanced basal cell division. The disease was first described in 1814 (Bateman, 1814). The intracytoplasmic inclusion bodies (molluscum or Henderson-Paterson bodies) were described in 1841 (Henderson, 1841; Paterson, 1841) and the viral nature of the disease was eventually established in 1905 (Juliusberg, 1905). MCV is one of two poxviruses regarded as having specificity for the human host, the other being variola, the agent of smallpox. There have been occasional reports of molluscum contagiosum in other species (Brown et al., 1981) although these have not been substantiated by molecular analysis. MCV is also one of a small number of poxviruses which induce tumour formation in their natural hosts.

Characterization of an rRNA gene-specific cDNA probe: applications in bacterial identification.

Discontinuous DNA complementary to Escherichia coli 16S + 23S ribosomal RNA was synthesized by random oligonucleotide priming using reverse transcriptase. cDNA generated from native or denatured rRNA template was labelled by incorporation of either [alpha-32P]dCTP or digoxigenin-labelled dUTP during synthesis, followed by template hydrolysis. The specific activity of the radiolabelled cDNA was 10(7)-10(8) c.p.m. (micrograms rRNA template)-1 with 60-92% incorporation after 5 h. The length of the reverse transcript was between 20 and 1140 nucleotides and was unaffected by exclusion of primer. The cDNA probe could detect 3 pg rRNA by quantitative slot blot. In the non-radiolabelling digoxigenin system 3 micrograms template gave 0.5-2.0 micrograms cDNA after 24 h with a length of between 100 and 1225 bases. This probe could detect 50 pg rRNA. Probes were evaluated in the comparison of Pasteurella haemolytica biotypes by hybridization to Southern blots of restriction-endonuclease-digested total DNA. The digoxigenin-labelled probe was used to identify clinical isolates of Campylobacter jejuni to demonstrate its potential use in laboratories requiring high-sensitivity detection without the use of radioisotopes.

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.