A random DNA sequencing, computer-based approach for the generation of a gene map of molluscum contagiosum virus.

The genome of Molluscum contagiosum virus (MCV) has a high G + C content, which largely differs from those of vaccinia virus (VAC) and other characterized poxviruses. This has precluded the use of DNA hybridization for the identification of MCV genes and the further establishment of the virus genetic map. To circumvent this problem, we have partially sequenced clones containing virus restriction endonuclease fragments, which were derived by either single or double-digestion of genomic DNA from the subtype I of MCV. The DNA sequences were translated and used to search protein data bases. This analysis resulted in the finding of high-scoring matches to data base entries, including forty-five VAC genes. In addition, MCV-specific sequences that encoded protein domains of known function (i.e. DNA J domain) were found. The locations of MCV clones were inferred from the presumed colinearity of both MCV and VAC genomes, and further confirmed by PCR technology. The data presented here led to the construction of a partial genetic map of MCVI, which revealed that the order and orientation of a large number of MCV genes were equivalent to those of their VAC homologues. The conserved gene arrangement was apparently disrupted in the terminal regions, where MCV sequences showing homologies with the VAC counterparts were not found.

Detection and typing of molluscum contagiosum virus in skin lesions by using a simple lysis method and polymerase chain reaction.

A polymerase chain reaction (PCR) assay for the rapid detection and typing of molluscum contagiosum virus (MCV) was developed. The target DNA was a 393 base pair (bp) segment, which is present in the coding region of the MCV p43K gene product. Release of MCV DNA from skin lesions was performed by using a simple procedure that provided suitable template DNA for amplification, and allowed detection of MCV directly in clinical material. The PCR yielded a unique 393 bp product when MCV DNA was used as template. This product was not shown with DNA from other viruses and bacterial pathogens causing skin diseases. The specific PCR product was obtained with individual lesions from all patients clinically diagnosed with MCV infection, whereas no products were detected with skin samples from healthy individuals. Sequencing of this PCR product allowed determination of the virus subtype on the basis of previously described nucleotide differences between subtypes MCVI and MCVII. To avoid the sequencing process, a second PCR assay was developed, in which the target DNA sequence included a MCVI-specific recognition site for the restriction endonuclease BamHI. This PCR assay yielded a unique 575 bp product with lesions from either MCVI- or MCVII-infected patients. However, only the MCVI-derived product was susceptible to BamHI digestion, which generated two fragments of 291 and 284 bp, respectively. Amplification of specific MCV DNA sequences from single, individual lesions provides a sensitive and reliable method for laboratory diagnosis and molecular epidemiology studies of molluscum contagiosum.

Sequence analysis of a Molluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization.

The nucleotide sequence of a near left-terminal region from the genome of Molluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacent BamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb). BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N'), which had been mapped 30-50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.