Novel human polyomaviruses, Merkel cell polyomavirus and human polyomavirus 9, in Japanese chronic lymphocytic leukemia cases
© Imajoh et al.; licensee BioMed Central Ltd. 2012
Received: 16 April 2012
Accepted: 1 June 2012
Published: 1 June 2012
Chronic lymphocytic leukemia (CLL) is the rarest adult leukemia in Japan, whereas it is the most common leukemia in the Western world. Recent studies from the United States and Germany suggest a possible etiological association between Merkel cell polyomavirus (MCPyV) and CLL, although no data have been reported from Eastern countries. To increase the volume of relevant data, this study investigated the prevalence and DNA loads of MCPyV and human polyomavirus 9 (HPyV9), another lymphotropic polyomavirus, in Japanese CLL cases.
We found that 9/27 CLL cases (33.3 %) were positive for MCPyV using quantitative real-time polymerase chain reaction analysis. The viral DNA loads ranged from 0.000017 to 0.0012 copies per cell. All cases were negative for HPyV9. One MCPyV-positive CLL case was evaluated by mutational analysis of the large T (LT) gene, which indicated the presence of wild-type MCPyV without a nucleotide deletion. DNA sequence analysis of the entire small T (ST) gene and the partial LT gene revealed that a Japanese MCPyV isolate, designated CLL-JK, had two nucleotide gaps when compared with the reference sequence of the North American isolate MCC350.
This study provides the first evidence that MCPyV is present in a subset of Japanese CLL cases with low viral DNA loads. MCPyV and HPyV9 are unlikely to contribute directly to the development of CLL in the majority of Japanese cases. MCPyV isolated from the Japanese CLL cases may constitute an Asian group and its pathogenicity needs to be clarified in future studies.
KeywordsChronic lymphocytic leukemia Merkel cell polyomavirus Human polyomavirus 9 Japanese study
Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western countries, where its frequency is ca. 3–8 new cases per 100,000 persons per year [1–3]. However, CLL is very rare in far Eastern countries. The incidence of CLL in Japan is 0.48 per 100,000 persons per year, although the proportion of T-CLL is relatively high . It has been suggested that genetic predisposition is a causal factor in CLL, but other largely unknown etiological factors may contribute to the geographical difference in the CLL frequency. Infectious agents may be correlated directly or indirectly with the pathogenicity of CLL, including viral infections.
Polyomaviruses are nonenveloped icosahedral viruses with a circular double-stranded DNA genome that encodes certain oncoproteins and they are putative oncogenic viruses . Currently, nine human polyomaviruses are known but their precise roles in carcinogenesis remain poorly understood . The fifth polyomavirus, Merkel cell polyomavirus (MCPyV), was reported in 2008 by Feng et al. . It was detected in approximately 80 % of Merkel cell carcinomas (MCCs), which is an aggressive neuroendocrine skin carcinoma. Human polyomavirus 9 (HPyV9) was identified recently in the serum of kidney transplant patients who were receiving immunosuppressive treatment . The presence of the HPyV9 genome in particular diseases has not been clarified. MCPyV and HPyV9, with a close phylogenetic relationship to the African green monkey-derived lymphotropic polyomavirus, have the potential to infect lymphoid cells [8, 9].
The search for hematologic neoplasias where MCPyV has a role in etiopathogenesis is currently an important issue . The possible association of MCPyV with various lymphoid malignancies was first demonstrated by US researchers, but they did not find a significant association between CLL and MCPyV [11, 12]. However, Canadian and German studies detected MCPyV-positive CLL cases and suggested the possible involvement of MCPyV in a subset of CLLs [13–15]. More recently, another US group reported MCPyV-positive CLLs with very low MCPyV copy numbers . Thus, all investigations of links between MCPyV and CLL have come from the Western world, whereas the prevalence of MCPyV in CLL cases in Eastern countries has not been investigated.
In this study, we aimed to determine the prevalence and DNA loads of MCPyV and HPyV9 in Japanese CLL cases, and we provide the first findings of a relationship between CLL and new human polyomaviruses in the Eastern world.
Patients and DNA preparation
We recruited 27 Japanese patients with CLL, including 25 B-CLLs and 2 T-CLLs (cases 3 and 17). The cohort comprised 18 males and nine females with a median age of 63 years at diagnosis (range 49–93). We also studied peripheral blood samples from 18 healthy Japanese donors with a median age of 48 years (range 27–55). A serum fraction containing abundant leukemia cells was separated using Ficoll–Conray density gradient and frozen at −80°C until use. Total DNA was extracted using the standard phenol–chloroform method. Approval was obtained from the ethics committee for this study.
Quantitative real-time polymerase chain reaction (PCR)
Primer sequences used in this study
Real-time PCR analysis
Forward (5′ → 3′)
Reverse (5′ → 3′)
Probe (5′ → 3′)
Human RNase P
Target gene (positions)
Forward (5′ → 3′)
Reverse (5′ → 3′)
MCPyV LT (1867–2221)
MCPyV ST-LT (183–828)
MCPyV ST-LT (571–1157)
PCR and DNA sequence analysis
MCPyV-positive samples identified by real-time PCR amplification were subjected to mutational analysis of the large T (LT) gene using standard PCR, as reported by Pantulu et al. . The primers shown in Table 1 yielded a 355-bp PCR product representing wild-type MCPyV, while the 120-bp product represented mutated MCPyV with a nucleotide deletion .
Nucleotide sequences at positions 183 to 1157 (based on the GenBank sequence EU375803), including the entire small T (ST) gene and the partial LT gene, were also investigated by standard PCR for 35 cycles using two overlapping primer sets (nucleotide 183 to 828 and nucleotide 571 to 1157) (Table 1). We expected amplicons of 646 bp and 587 bp with these PCR amplifications. The PCR products were gel purified and directly sequenced with a Big Dye Terminator Cycle Sequencing Kit (Life Technologies, Tokyo, Japan) on a 3130 Genetic Analyzer Instrument (Applied Biosystems, Tokyo, Japan). The nucleotide sequences that we obtained were analyzed using the BioEdit program and deposited in GenBank under accession number AB709861. The DNA sequence data were compared with reference sequences of MCPyV isolates from the National Center for Biotechnology Information Entrez Nucleotide database (MCC350, MCC339, MKL-1, TKS, and 16b) and from a previous report (B.C.) .
Results and discussion
DNA loads of MCPyV and HPyV9 detected by quantitative real-time PCR
Viral DNA load (copies per cell)
Viral DNA load (copies per cell)
We tested the nine MCPyV-positive cases to determine the presence of truncating mutations of the LT gene, which have been reported previously . However, LT sequences were not detectable in all samples by standard PCR, possibly because of the low viral DNA loads. The 355-bp amplicon was only detected in case 21 without the nucleotide deletion, indicating a wild-type MCPyV infection in this case. Pantulu et al.  detected the mutated LT gene in 6/19 MCPyV-positive German CLL patients, but it is uncertain whether such truncating LT mutations are prevalent in Japanese CLL cases.
We also aimed to determine whether the lymphotropic polyomavirus HPyV9 was associated with CLL. We found that no cases with CLL or healthy donors had amplifiable HPyV9 DNA according to the highly sensitive real-time PCR (Table 2). Our results appear to indicate that HPyV9 is not involved directly in the leukemogenesis of CLL.
In summary, this study provides the first evidence of MCPyV DNA in a subset of Japanese CLL cases. Mutational analysis of the MCPyV LT gene was not fully evaluated because of low viral DNA levels, but one MCPyV-positive CLL case had wild-type MCPyV. These findings suggest that MCPyV is unlikely to have a direct causal role in the majority of Japanese CLL cases. DNA sequence analysis revealed that the MCPyV isolates found in our CLL patients constituted an Asian group with two nucleotide gaps. We also showed that HPyV9 was not present in Japanese CLL cases. Further worldwide epidemiological and virological studies are required to determine the pathogenetic relevance of human polyomaviruses in CLL.
This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Science and Technology of Japan to MD.
- Sant M, Allemani C, Tereanu C, De Angelis R, Capocaccia R, Visser O, Marcos-Gragera R, Maynadié M, Simonetti A, Lutz JM, Berrino F: Incidence of hematologic malignancies in Europe by morphologic subtype: results of the HAEMACARE project. Blood. 2010, 116: 3724-3734. 10.1182/blood-2010-05-282632.View ArticlePubMedGoogle Scholar
- Dores GM, Anderson WF, Curtis RE, Landgren O, Ostroumova E, Bluhm EC, Rabkin CS, Devesa SS, Linet MS: Chronic lymphocytic leukaemia and small lymphocytic lymphoma: overview of the descriptive epidemiology. Br J Haematol. 2007, 139: 809-819. 10.1111/j.1365-2141.2007.06856.x.View ArticlePubMedGoogle Scholar
- Rozman C, Montserrat E: Chronic lymphocytic leukemia. N Engl J Med. 1995, 333: 1052-1057. 10.1056/NEJM199510193331606.View ArticlePubMedGoogle Scholar
- Tamura K, Sawada H, Izumi Y, Fukuda T, Utsunomiya A, Ikeda S, Uike N, Tsukada J, Kawano F, Shibuya T, Gondo H, Okamura S, Suzumiya J: Kyushu Hematology Organization for Treatment (K-HOT) Study Group: Chronic lymphocytic leukemia (CLL) is rare, but the proportion of T-CLL is high in Japan. Eur J Haematol. 2001, 67: 152-157. 10.1034/j.1600-0609.2001.5790514.x.View ArticlePubMedGoogle Scholar
- Gjoerup O, Chang Y: Update on human polyomaviruses and cancer. Adv Cancer Res. 2010, 106: 1-51.View ArticlePubMedGoogle Scholar
- Moens U, Ludvigsen M, Van Ghelue M: Human polyomaviruses in skin diseases. Patholog Res Int 2011. 2011, 123491: 12-Google Scholar
- Feng H, Shuda M, Chang Y, Moore PS: Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008, 319: 1096-1100. 10.1126/science.1152586.PubMed CentralView ArticlePubMedGoogle Scholar
- Scuda N, Hofmann J, Calvignac-Spencer S, Ruprecht K, Liman P, Kühn J, Hengel H, Ehlers B: A novel human polyomavirus closely related to the african green monkey-derived lymphotropic polyomavirus. J Virol. 2011, 85: 4586-4590. 10.1128/JVI.02602-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB: Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin. Cell Host Microbe. 2010, 7: 509-515. 10.1016/j.chom.2010.05.006.PubMed CentralView ArticlePubMedGoogle Scholar
- zur Hausen A: Opening new roads for MCPyV. Blood. 2011, 117: 6975-6976. 10.1182/blood-2011-05-348441.View ArticlePubMedGoogle Scholar
- Shuda M, Arora R, Kwun HJ, Feng H, Sarid R, Fernández-Figueras MT, Tolstov Y, Gjoerup O, Mansukhani MM, Swerdlow SH, Chaudhary PM, Kirkwood JM, Nalesnik MA, Kant JA, Weiss LM, Moore PS, Chang Y: Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors. Int J Cancer. 2009, 125: 1243-1249. 10.1002/ijc.24510.View ArticlePubMedGoogle Scholar
- Tolstov YL, Arora R, Scudiere SC, Busam K, Chaudhary PM, Chang Y, Moore PS: Lack of evidence for direct involvement of Merkel cell polyomavirus (MCV) in chronic lymphocytic leukemia (CLL). Blood. 2010, 115: 4973-4974. 10.1182/blood-2010-03-273177.PubMed CentralView ArticlePubMedGoogle Scholar
- Toracchio S, Foyle A, Sroller V, Reed JA, Wu J, Kozinetz CA, Butel JS: Lymphotropism of Merkel cell polyomavirus infection, Nova Scotia, Canada. Emerg Infect Dis. 2010, 16: 1702-1709.PubMed CentralView ArticlePubMedGoogle Scholar
- Pantulu ND, Pallasch CP, Kurz AK, Kassem A, Frenzel L, Sodenkamp S, Kvasnicka HM, Wendtner CM, Zur Hausen A: Detection of a novel truncating Merkel cell polyomavirus large T antigen deletion in chronic lymphocytic leukemia cells. Blood. 2010, 116: 5280-5284. 10.1182/blood-2010-02-269829.View ArticlePubMedGoogle Scholar
- Haugg AM, Speel EJ, Pantulu ND, Pallasch C, Kurz AK, Kvasnicka HM, Cathomas G, Wendtner CM, zur Hausen A: Fluorescence in situ hybridization confirms the presence of Merkel cell polyomavirus in chronic lymphocytic leukemia cells. Blood. 2011, 117: 5776-5777. 10.1182/blood-2011-03-339895.View ArticlePubMedGoogle Scholar
- Teman CJ, Tripp SR, Perkins SL, Duncavage EJ: Merkel cell polyomavirus (MCPyV) in chronic lymphocytic leukemia/small lymphocytic lymphoma. Leuk Res. 2011, 35: 689-692. 10.1016/j.leukres.2011.01.032.View ArticlePubMedGoogle Scholar
- Bhatia K, Goedert JJ, Modali R, Preiss L, Ayers LW: Merkel cell carcinoma subgroups by Merkel cell polyomavirus DNA relative abundance and oncogene expression. Int J Cancer. 2010, 126: 2240-2246.PubMed CentralPubMedGoogle Scholar
- Pancaldi C, Corazzari V, Maniero S, Mazzoni E, Comar M, Martini F, Tognon M: Merkel cell polyomavirus DNA sequences in the buffy coats of healthy blood donors. Blood. 2011, 117: 7099-7101. 10.1182/blood-2010-09-310557.View ArticlePubMedGoogle Scholar
- Katano H, Ito H, Suzuki Y, Nakamura T, Sato Y, Tsuji T, Matsuo K, Nakagawa H, Sata T: Detection of Merkel cell polyomavirus in Merkel cell carcinoma and Kaposi's sarcoma. J Med Virol. 2009, 81: 1951-1958. 10.1002/jmv.21608.View ArticlePubMedGoogle Scholar
- Touzé A, Gaitan J, Maruani A, Le Bidre E, Doussinaud A, Clavel C, Durlach A, Aubin F, Guyétant S, Lorette G, Coursaget P: Merkel cell polyomavirus strains in patients with merkel cell carcinoma. Emerg Infect Dis. 2009, 15: 960-962. 10.3201/eid1506.081463.PubMed CentralView ArticlePubMedGoogle Scholar
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