Generation of Merkel Cell Polyomavirus (MCV)-Like Particles and Their Application to Detection of MCV Antibodies

Abstract

The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited crossreactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.Polyomaviruses are small, nonenveloped DNA viruses, with a double-stranded circular DNA genome of ϳ5 kbp packaged within a capsid about 45 nm in diameter. The polyomavirus capsid is composed of three structural proteins: VP1, the major capsid protein, and VP2 and VP3, the minor capsid proteins. Twenty members of the polyomavirus family have been identified to date (24) and, with the exception of the murine pneumotropic polyomavirus and the avian polyomavirus, primary infection is generally asymptomatic. Five polyomaviruses infect humans, including the ubiquitous BK polyomavirus (BKV) and JC polyomavirus (JCV), which cause persistent and/or latent infections and the recently identified KI and WU polyomaviruses isolated from pulmonary secretions (1, 6). A new polyomavirus, the Merkel cell polyomavirus (MCV), was recently discovered in human Merkel cell carcinomas (MCC) (4). MCC is a relatively rare skin cancer in elderly or immunosuppressed patients and is one of the most lethal skin cancers (8). The annual incidence rate of this aggressive primary cutaneous neuroendocrine carcinoma in the United States was reported to be 0.44 per 100,000 inhabitants in 2001 and tripled between 1986 and 2001 (8), and this trend is continuing (7). An incidence of 0.13 cases per 100,000 was recently reported in France (15). Clonal integration of the MCV genome within the tumor genome (4) and the deletions and/or mutations observed within the T antigen gene (17) have suggested a direct oncogenic role for MCV. However, the prevalence and pathogenicity of this newly discovered MCV have yet to be fully investigated.The aim of the study was to produce MCV viruslike particles (VLPs) and to investigate the presence of MCV antibodies in the general population of Europe. MCV VLPs were obtained with only one of the three MCV VP1 strains investigated, and these VLPs were used to investigate cross-reactivity against other polyomaviruses and for the determination of the prevalence of MCV antibodies in the general European population. MATERIALS AND METHODSGeneration of VLPs for MCV, BKV, and LPV polyomaviruses. Expression of the VP1 protein was performed using the MCC350 VP1 prototype sequence and the VP1 sequences amplified from two French MCC patients (MKT-21 and MKT-26) (EMBL FM864207 and FM864209, respectively) (21). MCC350 VP1 codi...

Merkel Cell Carcinomas Arising in Autoimmune Disease Affected Patients Treated with Biologic Drugs, Including Anti-TNF

“…These data suggest that MCPyV infects a large proportion of the population, where it induces a physiological immune response (28,32,33).…”

“…27), were detected by ELISA using MCPyV virus-like particles (VLP) and the recombinant LT/ST. VLPs and LT/ST were generated and purified as previously described (27,28). Detection of VP1 and LT/ST antibodies by indirect ELISA was performed as previously described (27,28).…”

Human Merkel cell polyomavirus: virological background and clinical implications

“…Seroprevalence of 50-95% is reported in adults (7,(12)(13)(14)(25)(26)(27)(28)(29). The age-specific seroprevalence of MCPyV (Fig.…”

“…When expressed in mammalian or insect cells, VP1 proteins (or VP1 + VP2 proteins) self-assemble into 45-55 nm diameter virus-like particles (VLPs) (Fig. 2) that are used in serological assays (7,(12)(13)(14).…”

Seroprevalence of Human Malawi Polyomavirus

“…Purified MWPyV VLPs (100 ng/well in phosphate-buffered saline [PBS]) were used to sensitize microplates (Maxisorp; Nunc) overnight at 4°C. Briefly, sera were diluted 1:100, and peroxidase-conjugated anti-human IgG (Southern Biotech, Clinisciences, Nanterre, France) diluted 1:20,000 was used to detect human IgG binding (14). A histogram of the ELISA optical density (OD) values in 1-to 10-year-old children (data not shown) revealed a bimodal distribution of seroreactivity.…”