Journal of Medical Virology 83:2220–2224 (2011) Merkel Cell Polyomavirus DNA in Immunocompetent and Immunocompromised Patients With Respiratory Disease Bahman Abedi Kiasari,1,3* Pamela J. Vallely,1 and Paul E. Klapper1,2 1Department of Virology, Genomic Epidemiology Research Group, School of Translational Medicine, University of Manchester, Manchester, United Kingdom 2Clinical Virology, Manchester Medical Microbiology Partnership, Manchester Royal Infirmary, Oxford Road, Manchester, United Kingdom 3Human Viral Vaccine Department, Razi Vaccine & Serum Research Institute, Hesarak, Karaj, Iran Merkel cell polyomavirus (MCPyV) was identi- INTRODUCTION fied originally in association with a rare but aggressive skin cancer, Merkel cell carcinoma. In the past few years, a number of new human poly- The virus has since been found in the respirato- omaviruses, KI, WU, human polyomavirus 6 (HPyV6), ry tract of some patients with respiratory human polyomavirus 7 (HPyV7), trichodysplasia spi- disease. However, the role of MCPyV in the nulosa virus (TSV), human polyomavirus 9 (HPyV9), causation of respiratory disease has not been and Merkel cell polyomavirus (MCPyV) have been established. To determine the prevalence of discovered [Allander et al., 2007; Gaynor et al., 2007; MCPyV in 305 respiratory samples from Feng et al., 2008; Schowalter et al., 2010; van der immunocompetent and immunocompromised Meijden et al., 2010; Scuda et al., 2011]. MCPyV was patients and evaluate their contribution to re- discovered by digital transcriptome subtraction from a spiratory diseases, specimens were screened human skin cancer, Merkel cell carcinoma [Feng for MCPyV using single, multiplex, or real-time et al., 2008]. The finding of MCPyV in human Merkel PCR; co-infection with other viruses was exam- cell carcinoma suggests a role for this virus in the ined. Of the 305 samples tested, 10 (3.27%) causation of this cancer. were positive for MCPyV. The virus was found In confirmation of this, researchers were able to de- in two groups of patients: in 6 (2%) nasopha- tect MCPyV DNA in 77% [Germany; Kassem et al., ryngeal aspirate samples from children aged 2008], 84.9% [Europe; Becker et al., 2009], 43% [North 26 days to 7 months who were immunocompe- America; Garneski et al., 2009], and 88% [France; tent; and in 4 (1.3%) of nasopharyngeal aspirate Foulongne et al., 2008] of Merkel cell carcinoma speci- samples taken from patients aged 41 to 69 years mens. More recently, studies have reported detection who were severely immunosuppressed from of MCPyV in lymphoid tissues [Sharp et al., 2009] leukemia or transplant therapy. Both groups and in respiratory specimens [Bialasiewicz et al., had upper or lower respiratory tract infection. 2009; Goh et al., 2009; Kantola et al., 2009; Babakir- Co-infections with other viruses were found in Mina et al., 2010]. 30% of the MCPyV positive samples. The data The aim of this study was to determine the preva- present a pattern of infection similar to that lence of MCPyV in respiratory specimens collected seen with the polyomaviruses JC and BK in from immunocompetent and immunocompromised which the virus is acquired during childhood, patients and evaluate the possible of contribution of probably by the respiratory route. The viruses the virus in respiratory disease alone, or in combina- then establish latency and become reactivated tion with other respiratory viruses. in the event of immunosuppression. J. Med. Virol. 83:2220–2224, 2011. ß 2011 Wiley Periodicals, Inc. *Correspondence to: Bahman Abedi Kiasari, 3rd Floor Clini- cal Sciences Building, Manchester Royal Infirmary Oxford Road, KEY WORDS: Merkel cell polyomavirus; Manchester M13 9WL, United Kingdom. @ @ respiratory infection; PCR; E-mail: babedik yahoo.com, b.abedikiasari rvsri.ir co-infection; immunocom- Accepted 26 July 2011 promised DOI 10.1002/jmv.22222 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2011 WILEY PERIODICALS, INC. Merkel Cell Polyomavirus in Respiratory Tract 2221 METHODS provided by Dr. Vincent Foulongne, University of Montpellier, Montpellier, France) was also used as a Specimen Collection and Processing positive control. Amplified products were analyzed A total of 305 specimens from patients with respira- by electrophoresis on 2% agarose gel, visualized with tory tract disease were randomly selected from among ultraviolet (UV) light and compared for size with 1,335 nasopharyngeal aspirates submitted to the Clin- E-Gel Low Range Quantitative DNA Ladder (Invitro- ical Virology Laboratory, Manchester Royal Infirmary, gen, Life Technologies, Paisley, UK). A strongly posi- between October 2006 and February 2007. The speci- tive MCPyV sample (5 Â 107 molecules/ml DNA mens were stored at À708C until use. Specimens were copies) was used as a positive control in subsequent re-used for this study in accordance with current experiments (VP1 PCR assay). To confirm the results, Royal College of Pathologists Guideline (G035) (www. all positive specimens were subjected to a second rcpath.org/index.asp?PageID¼38) on re-use of diag- PCR analysis using primers targeting a different nostic specimens. Specimens and associated clinical genomic region of MCPyV [VP1 PCR assay; Feng data were collected; the specimens were anonymised et al., 2008]. Using tenfold serial dilutions of posi- by renumbering and removal of all patient identifiers tive controls, the sensitivity of the LT PCR and VP1 from the data before use in this study. PCR were determined as 1 copy/ml and 50 copies/ml, respectively. Positivity was defined as being definitely Other Virus Screening established when the results of both PCR assays 1 agreed. Specimens were extracted using the QIAamp 1 MinElute Virus Spin kit (Qiagen, Crawley, West Sequencing and Phylogenetic Analysis Sussex, UK) following the manufacturers instructions. To allow detection of RNA viruses, viral RNA was Sequencing was carried out using the Big Dye reverse transcribed to complementary DNA (cDNA) Terminator Cycle Sequencing kit (Applied Bio- utilizing Appplied Biosystems Taq Man1 Reverse systems) and the ABI 3100 Genetic Analyzer (Applied Transcription Kit (Applied Biosystems, Warrington, Biosystems). Sequences were assembled, analyzed, UK) and random hexamer priming prior to real-time and edited using Sequencher software version 4.6 PCR amplification using in-house assays for Influenza (Gene Codes Corporation, Ann Arbor, MI). Nucleotide A, B, and C viruses; parainfluenza viruses types 1–3; sequences were aligned with Clustal W (University PCRs for respiratory syncytial virus (RSV) types A College Dublin, Eire). Phylogenetic analysis was per- and B, human bocavirus (hBoV), and human meta- formed on sequences derived from the VP1 region. All pneumovirus (HMPV) [Al-Hammadi, 2008]. For detec- phylogenetic trees were visualized using Molecular tion of the polyomaviruses KI, WU, BK, JC, SV40, Evolutionary Genetics Analysis (Mega) version 4.0 and LPV in-house assays DNA PCRs were utilized as [Tamura et al., 2007]. A bootstrap test with 1,000 rep- previously described [Abedi Kiasari et al., 2008; Abedi licates was used to estimate the confidence of the Kiasari, 2009]. branching pattern of the trees. RESULTS Merkel Cell Polyomavirus PCR Assay Patient Characteristics Nucleic acid was extracted from nasopharyngeal aspirates using the QIAampDNA Blood BioRobot The median age of the 305 patients was 7 months MDx Kit (Qiagen) according to the manufacturer’s (mean 7 years; range 7 days–79 years), and the male instructions and stored at À208C until use. To avoid to female ratio was 1.31:1 (173:132). Specimens tested cross-contamination, all pre-PCR processing was un- in this study were from two groups of patients; Group der taken in a separate location from PCR and post- I consisted of 250 nasopharyngeal aspirate samples PCR analysis. For all PCR assays, standard precau- obtained from paediatric patients who were im- tions to avoid amplicon and nucleic acid contamina- munocompetent (age range 7 days–14 years; mean tion were taken. All samples were tested individually 7 months; median 4 months). Group II included 55 adding 5 ml nucleic acid to 45 ml master mix. Each nasopharyngeal aspirate samples obtained from adult reaction mix contained 200 pmol of each primer, patients who were either solid organ or bone-marrow 200 mM of dNTPs, 1Â PCR buffer, and 2.5 U of Ampli- transplant patients or patients being treated for leu- Taq Gold DNA polymerase (Applied Biosystems). PCR kemia (age range 15–79 years; mean 45 years; median assays were performed on a GenAmp PCR System 46 years). Both groups had upper or lower respiratory 9700 thermal cycler (Applied Biosystems). Samples infection. were subjected to 1 cycle of 948C for 10 min followed Prevalence of Merkel Cell Polyomavirus and by 40 cycles of 958C for 1 min, 568C for 1 min, 728C Clinical Findings for 1 min, and a final extension of 5 min at 728C. Negative controls were included in each experiment. Sixteen (5.2%) samples produced positive results Samples were initially analyzed using the LT PCR as- by the LT PCR assay. Of these, 10 (3.27%) were say described by Feng et al. [2008]. Plasmid pGEMT, confirmed by VP1 PCR assay. Positive samples were containing Large T antigen of MCPyV (kindly investigated further by bidirectional sequencing of J. Med. Virol. DOI 10.1002/jmv 2222 Abedi Kiasari et al. TABLE I. Age Distribution of MCPyV Infected Patients Age group No. of sample MCPyV (years) tested positive no. (%) <1 188 6 (3.19) 1–5 50 0 (0) 6–14 12 0(0) 15–29 9 0 (0) 30–44 11 0 (0) 45–60 20 3 (15) >60 15 1 (6.66) Total 305 10 (3.27) their LT3 and VP1 assay amplification products. The age distribution of patients with MCPyV infection is shown in Table I. MCPyV was identified in 6 (1.96%) Fig. 1. Phylogenetic analysis of nucleotide sequences of MCV VP1 samples from children aged 26 days to 7 months partial genes from positive nasopharyngeal aspirate samples. The (mean: 5 months) who were immunocompetent. tree was built with the MEGA 4.0.