key: cord-1056623-4nfrdriu
authors: Teramoto, Shinobu; Kaiho, Miki; Takano, Yasuo; Endo, Rika; Kikuta, Hideaki; Sawa, Hirofumi; Ariga, Tadashi; Ishiguro, Nobuhisa
title: Detection of KI polyomavirus and WU polyomavirus DNA by real‐time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues
date: 2011-06-28
journal: Microbiol Immunol
DOI: 10.1111/j.1348-0421.2011.00346.x
sha: 3bd36485a4c24fbe7ee0351e55a192ef44307e24
doc_id: 1056623
cord_uid: 4nfrdriu

Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real‐time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV‐positive and WUPyV‐positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.

mors in animal models, there is no evidence of their role as cancer-producing operants in humans. MCPyV is strongly suspected to be oncogenic because its DNA has been found to be integrated within the genome of Merkel cell carcinoma (23) . MCPyV has also been detected in patients with small cell lung cancer (24, 25) and in squamous cell carcinoma and basal cell carcinoma of the skin (26, 27) . However, little is known about the oncogenicity of KIPyV and WUPyV (28, 29) . In this study, we examined the prevalence of KIPyV and WUPyV in pediatric patients with RTIs to obtain epidemiological data for Japan. In addition, we investigated the prevalence of KIPyV and WUPyV in Japanese normal lung and lung adenocarcinoma tissues to evaluate the c 2011 The Societies and Blackwell Publishing Asia Pty Ltd (30) . The elution volume of the extractions was 90 μL. Thirty fresh sample pairs of lung adenocarcinoma and adjacent non-cancerous normal lung tissue were obtained from surgical material at the University of Toyama Hospital in 2002, with the informed consent of the patients. These patients included 20 men and 10 women aged from 45 to 77 years, with an average age of 61.7 years. None of the patients from whom samples were obtained for this study had immunodeficiency diseases. The tissue samples were frozen in liquid nitrogen and stored at −80 • C until used for DNA extrac- and 100 nmol/L of a probe. Amplification was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with the following instrument settings: 50 • C for 2 min, 95 • C for 10 min and then 50 cycles of 95 • C for 15 s and 60 • C for 1 min. The plasmids pKIPyV-real and pWUPyV-real containing the PCR products of the qualitative PCR in the vector pT7Blue (Novagene, Madison, WI, USA) were used as positive controls and for standard curves. All real-time PCR reactions were performed in duplicate and the results analyzed using ABI Prism 7000 SDS software. Viral loads were calculated from the CT values of individual samples with respect to the standard curve. The minimum concentrations of KIPyV and WUPyV genomes that would allow reproducible quantification were 10 copies per reaction. These correspond to 2 × 10 2 copies/μg (lung tissues and tumors) and 2 × 10 3 copies/mL (NPSs). As an internal control, β-actin was also amplified using TaqMan β actin detection reagents according to the manufacturer's instructions (Applied Biosystems). Nested PCR for KIPyV and PCR for WUPyV were also performed for lung tissue samples and NPSs as described previously (4, 5) . All specimens that were positive for KIPyV and WUPyV were also assayed for the presence of twelve other respiratory viruses: hRSV, hMPV, HRV, HBoV, PIV 1-3, influenza A and B viruses, HEV, HCoV, and adenoviruses. The PCR and RT-PCR protocols used for detecting these twelve viruses were the same as those previously described (33, 34) . The KIPyV genome was detected in 7 of the 232 NPSs (3.0%) ( Table 3) , consistent with previously reported detection rates (0.5 to 5%) (28) . The median viral load of all KIPyV-positive NPSs was 1.8 × 10 4 copies/mL, and the maximum value 4.1 × 10 5 copies/mL. The prevalence of KIPyV in NPSs determined by real-time PCR 

Each dot represents the copy number of WUPyV detected by real-time PCR. The first row shows the copy numbers of WUPyV that were negative by PCR and the second row shows the copy numbers of WUPyV that were positive by PCR. The black arrow shows the detection limit of realtime PCR (10 copies per reaction, which is equal to 2 × 10 3 copies per mL), and the white arrow shows the detection limit of PCR (100 copies per reaction, which is equal to 2 × 10 4 copies per mL).

was the same as that determined using nested PCR (4), and direct sequencing of PCR products of the seven samples showed that they were completely identical to the published sequence of KIPyV strain Stockholm 350 (Genbank accession number EF127907 bases 1536 to 1860) (4). The WUPyV genome was detected in 38 of the 232 NPSs (16.4%) by means of real-time PCR ( Table 3 ). The median viral load of all WUPyV-positive NPSs was 5.1 × 10 3 copies/mL, and the maximum value was 3.2 × 10 7 copies/mL. Three of the seven c 2011 The Societies and Blackwell Publishing Asia Pty Ltd KIPyV-positive samples were detected simultaneously with other viruses (two with hMPV and one with hMPV and HBoV). Among the 38 WUPyV-positive samples, 17 were detected simultaneously with other viruses (7 with hRSV, 4 with hMPV, 1 with HRV, 2 with HBoV, 1 with PIV1, 1 with hRSV and HBoV, and 1 with HRV and HBoV). One case was positive for KIPyV, WUPyV and hMPV (Table 4) The prevalence of WUPyV in NPSs in this study was obviously higher than previously reported prevalences (0.4 to 9%) (28) , though by switching the detection method from real-time PCR to PCR (5), the rate of WUPyV-positive NPSs fell from 16.4% (38 of 232) to 5.6% (13 of 232). The detection limit of PCR (5) (100 copies per reaction, which is equal to 2 × 10 4 copies per mL) and that of realtime PCR (10 copies per reaction, which corresponds to 2 × 10 3 copies per mL) explains the prevalence gap for WUPyV in NPSs in this study. The copy numbers of 13 NPSs that were real-time PCR-positive but PCR-negative were all below 2 × 10 4 copies per mL except for one (Fig. 1) . Direct sequencing of the PCR products of the 13 PCR-positive samples showed that 10 of the 13 sequences were completely identical to the published sequence of WUPyV strain B0 (Genbank accession number EF444549 bases 1331 to 1580), and 3 of the 13 sequences had a single-base-pair substitution (G1369C, A1396C and C1432A) in the VP3 gene (5) .

Clinical and laboratory features of the KIPyV-and WUPyV-positive patients are shown in Table 5 and Supplemental table. The ages of patients with KIPyV-positive samples ranged from 3 months to 2 years 11 months, and the ages of patients with WUPyV-positive samples ranged from 1 month to 4 years 11 months. All seven of the KIPyV-positive patients (100%) and 34 of the 38 WUPyVpositive patients (89.5%) were admitted to hospital for 3 to 11 days. The clinical diagnoses of the KIPyV-positive patients were wheezy bronchitis (three patients), bronchitis (three) and pneumonia (one). The clinical diagnoses of the WUPyV-positive patients were bronchitis (15 patients), wheezy bronchitis (14) , pneumonia (5), asthma (1), laryngotracheitis (1), acute pharyngolaryngitis (1), and acute pharyngitis (1). There was no obvious difference in clinical symptoms among KIPyV-and WUPyV-positive patients with or without coinfection.

Using real-time PCR, the KIPyV genome was detected in 1 of the 30 specimens of normal lung tissue (3.3%), whereas the WUPyV genome was not detected in any of the 30 specimens of normal lung tissues ( Table 3) . The viral load in KIPyV-positive normal lung tissue was 3.58 × 10 2 copies/μg. The sequence of KIPyV detected in normal lung tissue could not be determined because the KIPyV genome was not successfully amplified by nested PCR. On the other hand, neither the KIPyV genome nor the WUPyV genome was detected in 30 Japanese lung adenocarcinoma tissue samples ( Table 3 ). The mean β actin values of the normal and adenocarcinoma lung tissues were 3.6 × 10 5 DNA molecules (range, 7.6 × 10 4 to 6.9 × 10 5 ) and 2.8 × 10 5 DNA molecules (range, 9.8 × 10 4 to 6.6 × 10 5 ) per μg, respectively.

In a study in Italy, the KIPyV genome was detected in 1 of 20 specimens of normal lung tissue (5.0%) (29) , in agreement with our data. The oncogenic potential of KIPyV and WUPyV in human lung adenocarcinoma could not be determined in the present study. Although the KIPyV genome was detected in 9 of 20 specimens of lung cancer tissue (45.0%) in the study in Italy (29), because these authors did not provide the pathological classification of the lung cancer specimens, the relationship between KIPyV and lung adenocarcinoma remains unclear. Further studies on a larger number of specimens of malignant lung tissue of different types should be performed to evaluate the oncogenic properties of KIPyV and WUPyV.

To our knowledge, this is the first report of detection of KIPyV and WUPyV in Japanese children with RTIs. Results of nested PCR and PCR for KIPyV and WUPyV in NPSs from Japanese children with RTIs are consistent with the results of studies in other countries, and the sequence similarities to previous findings indicate worldwide distribution of the same virus lineage. The presence of KIPyV in normal lung tissue suggests that KIPyV may establish infection in lung tissue; alternatively it may be an innocent bystander. Our negative findings for KIPyV and WUPyV in lung adenocarcinoma tissue indicate that there may be no relationship between these viruses and lung adenocarcinoma transformation; alternatively the overall prevalence of the viruses might have been too low to be distinguished in our small number of samples. A larger sample size is needed to determine whether they are present in lung adenocarcinoma and their association with lung adenocarcinoma progression.

Clinical characteristics of KIPyV-poitive and WUPyV-positive patients.

Table S1 Seasonal distribution of KIPyV-and WUPyVpositive cases and numbers of samples collected.

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This research was supported in part by a Grant-in-Aid for Scientific Research (C), 2010 (22591174), from the Ministry of Education, Science, Sports and Culture of Japan. All of the necessary ethics approval for this study was obtained from the Institutional Review Board of Hokkaido University Hospital for Clinical Research. Nasopharyngeal swab samples were kindly provided by Yutaka Takahashi of KKR Sapporo Medical Center, Hiroyuki Sawada and Tsuguyo Nakayama of Hokkaido Social Insurance Hospital, Mutsuko Konno of Sapporo Kosei General Hospital, and Kunio Ozutusmi of Nemuro City Hospital. We thank Stewart Chisholm for proofreading the manuscript.