Molluscum Contagiosum Virus Genome Research Articles

New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequences.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.

Immune evasion strategies of molluscum contagiosum virus.

Molluscum contagiosum virus (MCV) is the causative agent of molluscum contagiosum (MC), the third most common viral skin infection in children, and one of the five most prevalent skin diseases worldwide. No FDA-approved treatments, vaccines, or commercially available rapid diagnostics for MCV are available. This review discusses several aspects of this medically important virus including: physical properties of MCV, MCV pathogenesis, MCV replication, and immune responses to MCV infection. Sequencing of the MCV genome revealed novel immune evasion molecules which are highlighted here. Special attention is given to the MCV MC159 and MC160 proteins. These proteins are FLIPs with homologs in gamma herpesviruses and in the cell. They are of great interest because each protein regulates apoptosis, NF-κB, and IRF3. However, the mechanism that each protein uses to impart its effects is different. It is important to elucidate how MCV inhibits immune responses; this knowledge contributes to our understanding of viral pathogenesis and also provides new insights into how the immune system neutralizes virus infections.

Detection of Molluscum contagiosum Virus (MCV) Subtype I as a Single Dominant Virus Subtype in Molluscum Lesions from a Turkish Population

Molluscum contagiosum has a worldwide occurrence and its primary mode of transmission is via direct human contact including sexual means. The aim of the study was to implement a polymerase chain reaction-based assay for detection and subtyping of Molluscum contagiosum virus (MCV) in skin lesions diagnosed with molluscum contagiosum in a large regional teaching hospital in Turkey. For this purpose, a total of 61 patients were included in the study. Randomly selected single lesion from each patient was used to extract DNA material and a specific PCR reaction amplifying 393-bp- and 575-bp-long regions from MCV genome was used in the detection. Subtyping was carried out by digestion of the amplified 575-bp product with restriction endonuclease enzyme BamHI. Both amplified and restriction enzyme digested products visualized on agarose gel electrophoresis. All 61 molluscum cases (100%) included in the study contained MCV genetic material as demonstrated by the presence of 393- and 575-bp-long PCR amplified products. Restriction enzyme BamHI digestion of the 575-bp-long amplicon indicated that the infecting subtype in all the cases (100%) was MCV subtype I. Results of this study demonstrate that subtype I is the only infecting strain dominant in our region. Because the only consecutive molluscum patients admitted to our hospital were included in the study, our data do not rule out the possibility that other genotypes might be present in the Turkish population. However, it is not unreasonable to conclude that similar trends exist in the rest of the country. Results also show that a molecular-based diagnostic assay would be feasible in cases where diagnosis was deemed necessary.

Preparation and use of molluscum contagiosum virus from human tissue biopsy specimens.

Molluscum contagiosum virus (MCV) lesions are limited to the human epidermis and can persist for months, showing only weak signs of inflammation. Because a culture system has not yet been created to replicate MCV, this chapter describes how the virus can be isolated from patient specimens. From this material, we describe how MCV is purified and used for infection studies, electron microscopy, viral DNA extraction, and analyses of early mRNA synthesized by in vitro transcription of permeabilized virions. The complete MCV-1 genome has been sequenced, and a redundant MCV genome fragment library of MCV type 1 (available from American Type Culture Collection [ATCC]) is useful for the cloning and study of individual MCV genes.

Molluscum contagiosum: recent advances in pathogenic mechanisms, and new therapies.

Two poxviruses, Molluscum contagiosum virus (MCV) and Variola virus are specific to humans. MCV is present worldwide and is directly passed by direct skin to skin contact to produce cutaneous and, rarely, mucosal lesions. It occurs predominantly in preadolescent children, sexually active adults, participants in sports with skin to skin contact, and in individuals with impaired cellular immunity. MCV characteristically proliferates within the follicular epithelium, and with routine fixation produces an area of retraction artifact separating layers 1 to 3 of CD34+ stromal cells that immediately surround the follicle from the surrounding dermis. This feature may be obscured when the lesions are inflamed, usually after rupture into the surrounding dermis. MCV is a cytoplasmically replicating virus. MCV-infected cells grow in size, while internal organelles are dislocated and eventually obliterated by a large intracytoplasmic inclusion. Rupture and discharge of the virus-packed cells occurs in a process similar to membrane debris and MCV accumulate in the crater-like ostium; MCV infection is spread by contact with infectious debris. In HIV-1-positive patients the histologic features, as well as the clinical features, may be atypical in patients with MCV infections. Not only are the lesions often large, but they may be verrucous and markedly hyperkeratotic. Recent sequencing of the MCV genome has increased our understanding and investigations into its mechanisms for avoiding host defense mechanisms. These include regions which encode for homologues of cellular chemokines and chemokine-binding proteins, a homolog of MHC1 and a viral FLICE-like inhibitory protein. Treatment, until recently, has depended upon tissue destruction including curettage, cryotherapy, CO(2) laser, electrodesiccation, trichloracetic acid and cantharadin. Recently, topical immune modulators have been used with some success. Understanding of the MCV genome is providing the basis for the development of drugs for therapy and prevention of MCV infections.

Mapping of mRNA transcripts in the genome of molluscum contagiosum virus: transcriptional analysis of the viral slam gene family.

Molluscum contagiosum virus (MCV) is a member of the poxvirus family and causes benign skin tumors in children and immunocompromised individuals. The primary structure and coding capacity of MCV was previously determined by DNA nucleotide sequencing (Senkevich et al., Science 273, 813-816, 1996). Hypothetical genes were predicted based on (i) amino acid homologies with known genes, (ii) presence or absence of conserved transcription regulation signals, and (iii) algorithms based on learning sets of coding sequences. These methods provide a rational basis for the prediction of MCV coding sequences. However, the existence and exact size of MCV open reading frames and the precise position of transcription regulation signals can only be determined by MCV mRNA transcript mapping experiments. We developed methods for the characterization of the mRNA transcripts of MCV genes in infected skin tissue and abortively infected human fibroblast cell cultures. Using these methods the properties of the mRNA transcripts of the MCV SLAM (signaling lymphocytic activating molecule) gene family (mc002L, mc161R, and mc162R) were analyzed. The mRNA start site found for the mc161R transcript suggests that a second start codon is used leading to a mc161R open reading frame that is nine amino acid residues shorter than predicted.

Characterization of Early Gene Transcripts of Molluscum Contagiosum Virus

Molluscum contagiosum virus (MCV), a member of the family Poxviridae, replicates wellin vivobut cannot be propagated in cell culture. The coding capacity of the MCV genome was previously determined by DNA nucleotide sequence analysis. The objective of the present study was to establish experimental systems for the identification and characterization of early MCV gene transcripts. MCV mRNA was obtained in three ways: (1) MCV early mRNA was synthesizedin vitrousing permeabilized virions, (2) MCV mRNA was extracted from MCV-infected skin tissue, and (3) MCV mRNA was extracted from MCV-infected human embryonic fibroblasts. RNA/DNA hybridization experiments showed significant early transcriptional activity in two parts of the MCV genome. Transcripts of 11 early MCV genes located in these parts of the genome, including two subunits of the MCV DNA-dependent RNA polymerase (mc077R and mc079R), the MCV poly(A)+polymerase gene (mc076R), and the MCV MHC class I homolog (mc080R), were detected in reverse transcription-polymerase chain reaction experiments. Total RNA obtained from MCV-infected skin tissue was used to confirm these results. Three MCV early transcripts, mc002L, mc004.1L, and mc005L, produced distinct bands on rapid amplification of their 3′ ends (3′ RACE). The 5′ mapping of transcription start sites of MCV open reading frames (ORFs) mc002L, mc004.1L, mc005L, and mc148R revealed that the MCV RNA polymerase transcription start sites are consistently located between 11 and 13 nucleotides downstream of the early MCV consensus promoter signal. When cDNA from both 5′ and 3′ mapping experiments was analyzed, MCV ORFs mc004.1L and mc005L were found to be transcribed as a single bicistronic mRNA. The transcript from MCV ORF mc066L, encoding a glutathione peroxidase, was detected inin vitrosynthesized MCV mRNA as well as in total RNA from MCV-infected human embryonic fibroblasts and MCV-infected skin. This indicates that despite the lack of an early MCV consensus promoter signal immediately proximal to the start codon, this particular gene is transcribed early during MCV infection.

Molluscum Contagiosum

WITH THE eradication of smallpox, molluscum contagiosum (MC) virus (MCV) became the last remaining member of the Poxviridae family to specifically infect humans.1-3Unlike the related poxviruses variola and vaccinia, MCV received little attention, since infection resulted in benign cutaneous neoplasms that were usually self-limited or easily controlled. All that changed with the advent of the acquired immunodeficiency syndrome (AIDS), as extensive and recalcitrant MC emerged as an important cause of morbidity and disfigurement.4-8Physicians who treat MC in human immunodeficiency virus (HIV)— infected individuals have been frustrated by the lack of effective antiviral therapy and the inexorable progression of MC lesions as patients enter the late stages of HIV disease.5,6,8Fortunately, recent basic knowledge about poxviruses, through sequencing of the MCV genome,9as well as advances in the therapy of HIV-infected patients,10,11gives reasons for optimism. Poxviruses wereamong the first groups of infectious agents to be morphologically defined by their effect

The Genome of Molluscum Contagiosum Virus: Analysis and Comparison with Other Poxviruses

Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.

Recent advances in molluscum contagiosum virus research.

Molluscum contagiosum virus (MCV) and variola virus (VAR) are the only two poxviruses that are specific for man. MCV causes skin tumors in humans and primarily in children and immunocompromised individuals. MCV is unable to replicate in tissue culture cells or animals. Recently, the DNA sequence of the 190 kbp MCV genome was reported by Senkevich et al. MCV was predicted to encode 163 proteins of which 103 were clearly related to those of smallpox virus. In contrast, it was found that MCV lacks 83 genes of VAR, including those involved in the suppression of the host response to infection, nucleotide biosynthesis, and cell proliferation. However, MCV possesses 59 genes predicted to code for novel proteins including MHC-class I, chemokine and glutathione peroxidase homologs not found in other poxviruses. The MCV genomic data allow the investigation of novel host defense mechanisms and provide new possibilities for the development of therapeutics for treatment and prevention of the MCV infection.

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.

Genome Sequence of a Human Tumorigenic Poxvirus: Prediction of Specific Host Response-Evasion Genes

Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.

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.

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

Analysis of the genome of molluscum contagiosum virus by restriction endonuclease analysis and molecular cloning.

Virions of molluscum contagiosum virus (MCV), a member of the poxviridae, were isolated directly from lesions of individual patients and characterized by restriction enzyme analysis. The comparative analysis of the cleavage patterns and Southern blot hybridization of 14 independently isolated virus samples revealed that MCV isolates can be classified into two different types. The majority of MCV isolated from clinically typical skin lesions (13 of 14) showed similar DNA cleavage patterns and were termed MCV type 1, whereas one isolate derived from a vaginal lesion showed a completely different DNA cleavage pattern and therefore was termed MCV type 2. For detailed investigation of the viral genome, a defined gene library of MCV DNA sequences was established. The Bam HI DNA fragments of the viral genome of MCV type 1 prototype isolate 1/80 was inserted into the bacterial plasmid vector pAT153. With the exception of terminal fragments (fragments A and B) of the viral genome, all other DNA fragments were cloned. All cloned Bam HI DNA fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of plasmid DNA to viral DNA.

Structural characterization of the Molluscum contagiosum virus genome

The genome of Molluscum contagiosum virus (MCV) appears in an electron microscope as a linear, duplex DNA molecule having a mean contour length of 53.02 ± 1.87 μm. Vaccinia virus DNA, similarly processed and used for comparison, measures 53.03 ± 2.2 μm. The average molecular weight of MCV-DNA was calculated to be 118 × 10 6. Single-stranded circles measuring twice the length of linear molecules were observed with both poxvirus DNAs following high levels of denaturation. However, partial denaturation profiles of MCV and vaccinia virus DNA differed markedly when mounted for microscopy under similar conditions. MCV-DNA consistently required an additional 10° equivalents to achieve comparable levels of denaturation. In contrast to vaccinia virus DNA, the most easily denaturable regions of the MCV genome appeared to be the ends of the molecule. When MCV-DNA was cleaved by restriction endonuclease BAM · HI, 12 fragments were produced ranging in size from 4 × 10 5 to 31 × 10 6 daltons as determined by agarose-gel electrophoresis and contour length measurements. Vaccinia virus DNA, similarly treated, produced more than 23 fragments which did not appear to correspond in size to those of MCV. With the aim of comparing the genetic and structural heterogeneity of the MCV genome, 11 independently isolated virus samples were analyzed by restriction endonuclease digestion and coelectrophoresis. Only three cleavage profiles were detected, with some fragments present in all three profiles, comigrating in agarose gels. We conclude, therefore, that the genome of Molluscum contagiosum virus is a continuous polynucleotide chain which is base paired to form a linear, duplex DNA molecule. Three genetic types of MCV were isolated from clinically typical skin lesions and identified in this study.