Merkel cell polyomavirus DNA sequences in the buffy coats of healthy blood donors

Abstract

Merkel cell polyomavirus (MCPyV), a DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukemia. MCPyV sequences have also been detected in various normal tissues in tumor-affected patients. Immunologic studies have detected MCPyV antibodies in as many as 80% of healthy blood donors. This high seroprevalence suggests that MCPyV infection is widespread in humans. In our study, buffy coats, which were examined for MCPyV DNA Tag sequences, showed a prevalence of 22%. Viral DNA load was revealed in blood samples from 10 to 100 molecules/100 000 cells. DNA sequencing confirmed that polymerase chain reaction amplicons belong to the IntroductionMerkel cell polyomavirus (MCPyV) is a small DNA tumor virus. 1 A recent investigation has reported MCPyV sequences in 27.1% of purified malignant cells from human chronic lymphocytic leukemia (CLL) samples. 2 CLL, the most common leukemia in the Western world, results from the expansion of a rare population of mature B-lymphocytes. 3 In a previous study, MCPyV DNA was identified in Merkel cell carcinoma (MCC). 4 This neoplasm is a rare but aggressive skin cancer of neuroendocrine origin. 5 MCC has been detected in immunosuppressed patients who have undergone organ transplantation or are affected by HIV infection, with Tlymphocyte immunosuppression resulting from CLL. 6 MCPyV DNA has been also identified in various tissue samples in patients affected by malignant or nonmalignant tumors. 4,[7][8][9] Moreover, the presence of MCPyV in peripheral blood at low copy number has also been reported. 4,10 Recent immunologic studies have detected MCPyV antibodies in 70% to 80% of adults. 11-13 Because human polyomavirus DNAs have been detected in tonsillar tissues, the high respiratory tract has been proposed as one of the entry portals for these viral agents. 14-16 However, the process by which polyomaviruses, and specifically MCPyV, gain access to and establish persistent infections in distal body compartments has not been well established. 15 Methods Samples and PCR techniquesBuffy coats (n ϭ 60) were obtained from healthy blood donors as recently described. 17 DNA was isolated as previously done, and its quality was confirmed by ␤-globin polymerase chain reaction (PCR). 17 The pUC57MC1 recombinant plasmid, with MCC 350 strain sequences, was used as a positive control in MCPyV PCR amplification experiments. 18 Two different MCPyV Tag regions, nucleotides (nt) 571 to 879 and nt 1709 to 1846, were investigated by single PCR analysis for 35 cycles using the primer sets, LT3F-LT3R and MCPyLT1709.FMCPyLT1864.R, respectively. Amplicons of 308 bp and 138 bp are expected from these sPCR amplifications. 4,18 However, no MCPyV Tag sequences were detected in our samples. Recently, similar data have been reported by other groups. 19,20 To verify whether these negative data were the result of a low viral DNA load in our buffy coats, the same MCPyV sequences were investigated by PCR reamplifications, using 5 L of the first PCR reaction, with the same ...

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

“…In a previous study, we detected MCPyV DNA sequences in 22% of PBMCs from healthy subjects (20), whereas other groups found MCPyV sequences in PBMCs from patients with MCC (29). Herein, MCPyV DNA was detected in all 3 MCC samples, whereas none of the 3 PBMCs analyzed showed MCPyV DNA sequences (Table 1).…”

“…Molecular analysis of MCPyV LT DNA sequences from the 3 cases showed high homology with previously published MCPyV sequences obtained from MCC specimens. Specifically, we identified the MKL-1 strain, suggesting that this strain is the main MCPyV isolate in Europe (20,22). Strong MCPyV LT antigen (LTA) expression in IHC, with diffuse nuclear localization, was evaluated in the 3 MCC tissues.…”

“…Total DNA was extracted from PBMCs belonging to patients (n ¼ 3) using QIAmp DNA Blood Mini Kit (Qiagen), as described (20). After DNA extraction, each sample was quantified by the spectrophotometric reading (NanoDrop 2000, Thermo Scientific).…”

“…To verify the presence of MCPyV DNA sequences, DNA from MCC and PBMC samples was analyzed by qualitative PCR with MCPyLT1709.F and MCPyLT1846.R primer set, which generate an amplicon of 138 bp (20). The forward and reverse primers are located at nucleotide positions 1,709 to 1,846, based on the GenBank sequence EU375803.…”

“…Recombinant plasmid pMCPyVLT.1, which contains 258 bp of the large T antigen (24) coding sequence (FJ472933), was used as positive control (25). Standard calibration curves were generated using 10-fold dilutions, from 10 1 to 10 7 copies, of pMCPyVLT.1 (20). Cellular RNase P gene was used to determine the human cell equivalents of each sample under analysis (26).…”