- Short Report
- Open Access
No evidence for WU polyomavirus infection in chronic obstructive pulmonary disease
https://doi.org/10.1186/1750-9378-4-12
© Ringshausen et al; licensee BioMed Central Ltd. 2009
- Received: 13 November 2008
- Accepted: 28 August 2009
- Published: 28 August 2009
Abstract
Human polyomaviruses are known to cause persistent or latent infections, which are reactivated under immunosuppression. Polyomaviruses have been found to immortalize cell lines and to possess oncogenic properties. Moreover, the recently discovered Merkel cell polyomavirus shows a strong association with human Merkel cell carcinomas. Another novel human polyomavirus, WU polyomavirus (WUPyV), has been identified in respiratory specimens from patients with acute respiratory tract infections (ARTI). WUPyV has been proposed to be a pathogen in ARTI in early life and immunocompromised individuals, but so far its role as a causative agent of respiratory disease remains controversial.
The objective of our study was to determine the prevalence of WUPyV infections in adult hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) and to establish its potential clinical relevance by comparison to patients with stable COPD hospitalized for other reasons than acute exacerbation of COPD (AE-COPD).
A total of 378 respiratory specimens, each 189 induced sputum and nasal lavage samples from 189 patients, who had been recruited in a prospective 2:1 ratio case-control set-up between 1999 and 2003, were evaluated for the presence of WUPyV DNA by real-time PCR.
In the present study we could not detect WUPyV DNA in 378 respiratory specimens from 189 adult hospitalized patients with AE-COPD and stable COPD in four consecutive years.
Persistence of viral replication or reactivation of latent WUPyV infection did not occur. WUPyV may not play a major role in adult immunocompetent patients with AE-COPD and stable COPD.
Keywords
- Chronic Obstructive Pulmonary Disease
- Chronic Obstructive Pulmonary Disease Patient
- Acute Respiratory Tract Infection
- Merkel Cell Carcinoma
- Stable Chronic Obstructive Pulmonary Disease
Findings
Polyomaviruses are small, non-enveloped viruses with a circular double-stranded DNA genome of approximately 5,000 base pairs. They are known to be capable of immortalizing animal and human cell lines. Their oncogenic potential has been demonstrated in vitro and in various animal cancer models and is accomplished by the integration of viral DNA into the host cell genome. Expression of the viral T-antigen is mandatory for cell transformation [1]. In the last years there has been a re-emergence of interest in human polyomaviruses as possible carcinogens as three novel polyomaviruses have been described in humans. While KI and WU polyomavirus have initially been detected in respiratory specimens [2, 3], the Merkel cell polyomavirus (MCPyV) was observed to be clonally integrated into Merkel cell carcinomas (MCC), a rare but aggressive human skin cancer of neuroendocrine origin [4]. Meanwhile, MCPyV has also been described in respiratory specimens [5, 6], small cell lung cancer tissue [7], and there is increasing evidence for a strong association between MCPyV and MCC [8–12]. Recently a first study reported the detection of KIPyV DNA in lung cancer specimens [13], and many more studies targeting different human polyomaviruses, cancer entities and populations may be anticipated in the near future. However, the detection in the respiratory tract is not a unique feature of KI-, MC- and WUPyV and transmission by the respiratory route has already been suggested for the first two human polyomaviruses, BK and JC virus [14–16]. In 2007, WU polyomavirus (WUPyV) was identified in respiratory specimens from patients with acute respiratory tract infections (ARTI) [2]. It has been proposed to be a relevant pathogen in ARTI in early life and immunocompromised individuals, but so far its role as a causative agent of respiratory disease remains controversial as it was also found in healthy asymptomatic individuals [17, 18]. WUPyV infections appear endemic worldwide [2], detection frequencies vary from 0.4% [19] to 7% [20] and coinfections with other respiratory viruses are common [21].
The aim of the present study was to determine the prevalence of WUPyV infections in adult hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) and to establish its potential clinical relevance by comparison to patients with stable COPD hospitalized for other reasons than acute exacerbation of COPD (AE-COPD).
A total of 378 respiratory specimens, each 189 induced sputum and nasal lavage samples from 189 patients were retrospectively evaluated for the presence of WUPyV DNA. Subjects with AE-COPD and stable COPD had been recruited in a prospective case-control study in a 2:1 ratio between October 1999 and April 2003. Underlying criteria, definitions and procedures have been the same as described previously [22]. Notably, patients with thoracic malignancies were excluded from the study. The induced sputum and nasal lavage samples were neatly stored in aliquots at -70°C until further processing. DNA was extracted from the samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and stored at -20°C for further testing. Although the amplifiability of the assayed sample DNA was recently demonstrated by the detection of another novel respiratory agent, the human bocavirus [23], the human β-globin gene was amplified as a cellular gene on a LightCycler PCR platform (LightCycler® Control Kit DNA, Roche, Mannheim, Germany) in order to demonstrate the integrity and sufficient quality of DNA in the assayed specimens. Primers and probe for the WUPyV real-time PCR assay were selected from the highly conserved C-terminal region of the large T-antigen that has been used for a qualitative WUPyV PCR previously [24]. Blasting of primer and probe sequences against GenBank excluded significant homologies with other organisms. The real-time PCR was performed in a final volume of 25 μl consisting of 5 μl of extracted DNA, the primers WU2958s (CCTGTTAGTGATTTTCACCCATGTA) and WU2865a (TGTCAGCAAATTCAGTAAGGCCTATATAT) at a final concentration of 400 nM, the probe WU2925s-TM (6FAM-AAAGTTGTGTATTGGAAAGAACTGTTAGACA-TAMRA) at a final concentration of 100 nM, and 1× Quantitect probe master mix (Qiagen, Hilden, Germany) as described previously [25]. The cycling conditions were 50 cycles with 30 s at 95°C and 60 s at 60°C after a preheating step of 15 min at 95°C. A plasmid containing the PCR product obtained with the primers AG0048 and AG0049 [2] cloned into the vector pCR2.1-TOPO (Invitrogen, Karlsruhe, Germany) was used as positive control and for the standard curve. Strict laboratory procedures were implemented to prevent PCR contamination. One negative control was amplified for every five samples. Plasmid spiking experiments were conducted in order to exclude PCR inhibition by induced sputum and nasal lavage samples. Data analysis was performed using SPSS, version 11.5 (SPSS Inc., Chicago, Illinois). Categorical data were compared by Pearson's chi-squared or Fisher's exact test, where appropriate. Normal distribution in continuous variables was determined with the Kolmogorov-Smirnov test and differences were subsequently determined either with the student's t-test or the Mann-Whitney-U test. All p values were calculated two-sided with statistical significance set to p < 0.05. The study was approved by the ethics committee of the Ruhr University, Bochum, Germany. All study participants gave their written informed consent prior to study inclusion.
Demographic and clinical characteristics of the study population
Variables | All | AE-COPD | Stable COPD | P valuea | |||
---|---|---|---|---|---|---|---|
n | % | n | % d | n | % d | ||
Patients | 189 | 100 | 123 | 65.1 | 66 | 34.9 | |
n | % e | n | % e | n | % e | ||
Sex | 0.35 | ||||||
Female | 39 | 20.6 | 28 | 22.8 | 11 | 16.7 | |
Male | 150 | 79.4 | 95 | 77.2 | 55 | 83.3 | |
Smoking behavior | 0.34 | ||||||
Active smokers | 53 | 28.0 | 32 | 26.0 | 21 | 31.8 | |
Non-smoker | 26 | 13.8 | 20 | 16.3 | 6 | 9.1 | |
Ex-smoker | 110 | 58.2 | 71 | 57.7 | 39 | 59.1 | |
Oral steroid medication | 0.52 | ||||||
Yes | 127 | 67.2 | 85 | 69.1 | 42 | 63.6 | |
No | 62 | 32.8 | 38 | 30.9 | 24 | 36.4 | |
Inhaled corticosteroids | 0.63 | ||||||
Yes | 126 | 66.7 | 80 | 65.0 | 46 | 69.7 | |
No | 63 | 33.3 | 43 | 35.0 | 20 | 30.3 | |
Mean | SD | Mean | SD | Mean | SD | ||
Age (years) | 67 | 10 | 68 | 9 | 65 | 11 | 0.17 |
Body mass index (kg/m2) | 26.9 | 5.1 | 26.8 | 5.0 | 27.2 | 5.2 | 0.60 |
Median | Range | Median | Range | Median | Range | ||
Pack yearsb | 30 | 2-120 | 30 | 2-120 | 30 | 2-120 | 0.71 |
FEV1ad (L) | 1.0 | 0.4-2.6 | 1.0 | 0.4-2.2 | 1.2 | 0.5 - 2.6 | < 0.0001 |
FEV1ad (% predicted) | 38.0 | 16.7-79.0 | 36.7 | 16.7-79.0 | 42.9 | 19.4-77.3 | 0.003 |
FEV1dis (L) | 1.2 | 0.5-2.9 | 1.2 | 0.6-2.9 | 1.2 | 0.5-2.6 | 0.53 |
FEV1dis (% predicted) | 43.6 | 18.5-78.9 | 44.3 | 18.5-78.9 | 42.9 | 19.4-77.3 | 0.90 |
CRP (mg/dL) | 0.8 | 0.0-39.8 | 1.0 | 0.0-39.8 | 0.6 | 0.0-12.9 | 0.0002 |
Leukocytes/nL | 10.5 | 0.7-27.2 | 10.9 | 0.7-27.2 | 10.1 | 5.1-24.0 | 0.016 |
Oral steroid dose (mg)c | 7.5 | 0-150 | 10 | 0-150 | 5 | 0-150 | 0.098 |
Our findings are in agreement with two recent studies from China [26] and the UK [27], which failed to detect WUPyV DNA in immunocompetent adults. The initial investigation by Gaynor et al. found four adults with altered immune status or multiple comorbidities to be positive for WUPyV [2]. None of the mentioned studies explicitly included patients with AE-COPD or stable COPD. The present population consisted of predominantly elderly COPD patients with severely impaired lung function and concomitant low dose oral steroid medication. In a previous study performed on a comparable population we demonstrated that AE-COPD was significantly associated with the detection of common respiratory viruses, foremost human rhinovirus, influenza virus A and respiratory syncytial virus, and that induced sputum had a higher viral yield than upper respiratory tract specimens in patients with AE-COPD [22]. Though our plasmid spiking experiments showed that PCR reactions were not inhibited by the assayed specimens, the use of diluted plasmid DNA instead of virus titers as a positive control and for the generation of the standard curve is an inevitable limitation of the present study. Infectious WU polyomavirus has yet to be isolated and cell lines susceptible to infection still need to be identified [28]. However, in the present study we could not detect WUPyV DNA in 378 respiratory specimens from 189 adult hospitalized patients with AE-COPD and stable COPD in four consecutive years between 1999 and 2003, whereas recent reports found WUPyV circulating in German, predominantly pediatric populations within and beyond our study period [24, 25, 29].
Our findings support the hypothesis that primary WUPyV infection is acquired in early life rather than adulthood and suggest that persistence of viral replication or reactivation of latent WUPyV infection is not a common phenomenon in the adult COPD population. Hence, WUPyV may not play a major role in adult immunocompetent patients with AE-COPD and stable COPD. A clear linkage between WUPyV and human disease still remains to be determined.
Declarations
Acknowledgements
This work was supported by an unrestricted research grant (2007-pneumo-574) provided to FCR by the scientific committee of the University Hospital Bergmannsheil, Bochum, Germany. The authors are grateful to B. Schaerling, A. Wagner and M. Ulbrich for their excellent technical assistance in our lab.
Authors’ Affiliations
References
- zur Hausen H: Novel human polyomaviruses - re-emergence of a well known virus family as possible human carcinogens. Int J Cancer. 2008, 123: 247-250. 10.1002/ijc.23620.PubMedView ArticleGoogle Scholar
- Gaynor AM, Nissen MD, Whiley DM, Mackay IM, Lambert SB, Wu G, Brennan DC, Storch GA, Sloots TP, Wang D: Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS Pathog. 2007, 3: e64-10.1371/journal.ppat.0030064.PubMedPubMed CentralView ArticleGoogle Scholar
- Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G, Persson MA, Dalianis T, Ramqvist T, Andersson B: Identification of a third human polyomavirus. J Virol. 2007, 81: 4130-4136. 10.1128/JVI.00028-07.PubMedPubMed CentralView ArticleGoogle 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.PubMedPubMed CentralView ArticleGoogle Scholar
- Goh S, Lindau C, Tiveljung-Lindell A, Allander T: Merkel cell polyomavirus in respiratory tract secretions. Emerg Infect Dis. 2009, 15: 489-491. 10.3201/eid1503.081206.PubMedPubMed CentralView ArticleGoogle Scholar
- Kantola K, Sadeghi M, Lahtinen A, Koskenvuo M, Aaltonen LM, Mottonen M, Rahiala J, Saarinen-Pihkala U, Riikonen P, Jartti T, Ruuskanen O, Söderlund-Venermo M, Hedman K: Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples: Implications for respiratory transmission and latency. J Clin Virol. 2009, 45 (4): 292-5. 10.1016/j.jcv.2009.04.008. Epub 2009 May 22.PubMedView ArticleGoogle Scholar
- Helmbold P, Lahtz C, Herpel E, Schnabel PA, Dammann RH: Frequent hypermethylation of RASSF1A tumour suppressor gene promoter and presence of Merkel cell polyomavirus in small cell lung cancer. Eur J Cancer. 2009, 45 (12): 2207-11. 10.1016/j.ejca.2009.04.038. Epub 2009 May 25.PubMedView ArticleGoogle Scholar
- Kassem A, Schopflin A, Diaz C, Weyers W, Stickeler E, Werner M, Zur Hausen A: Frequent detection of Merkel cell polyomavirus in human Merkel cell carcinomas and identification of a unique deletion in the VP1 gene. Cancer Res. 2008, 68: 5009-5013. 10.1158/0008-5472.CAN-08-0949.PubMedView ArticleGoogle Scholar
- Shuda M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, Moore PS, Chang Y: T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus. Proc Natl Acad Sci USA. 2008, 105: 16272-16277. 10.1073/pnas.0806526105.PubMedPubMed CentralView ArticleGoogle Scholar
- Duncavage EJ, Zehnbauer BA, Pfeifer JD: Prevalence of Merkel cell polyomavirus in Merkel cell carcinoma. Mod Pathol. 2009, 22: 516-521. 10.1038/modpathol.2009.3.PubMedView ArticleGoogle Scholar
- Sastre-Garau X, Peter M, Avril MF, Laude H, Couturier J, Rozenberg F, Almeida A, Boitier F, Carlotti A, Couturaud B, Dupin N: Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis. J Pathol. 2009, 218: 48-56. 10.1002/path.2532.PubMedView ArticleGoogle Scholar
- Wetzels CT, Hoefnagel JG, Bakkers JM, Dijkman HB, Blokx WA, Melchers WJ: Ultrastructural proof of polyomavirus in Merkel cell carcinoma tumour cells and its absence in small cell carcinoma of the lung. PLoS One. 2009, 4: e4958-10.1371/journal.pone.0004958.PubMedPubMed CentralView ArticleGoogle Scholar
- Babakir-Mina M, Ciccozzi M, Campitelli L, Aquaro S, Lo Coco A, Perno CF, Ciotti M: Identification of the novel KI Polyomavirus in paranasal and lung tissues. J Med Virol. 2009, 81: 558-561. 10.1002/jmv.21417.PubMedView ArticleGoogle Scholar
- Goudsmit J, Wertheim-van Dillen P, van Strien A, Noordaa van der J: The role of BK virus in acute respiratory tract disease and the presence of BKV DNA in tonsils. J Med Virol. 1982, 10: 91-99. 10.1002/jmv.1890100203.PubMedView ArticleGoogle Scholar
- Sundsfjord A, Spein AR, Lucht E, Flaegstad T, Seternes OM, Traavik T: Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients. J Clin Microbiol. 1994, 32: 1390-1394.PubMedPubMed CentralGoogle Scholar
- Monaco MC, Jensen PN, Hou J, Durham LC, Major EO: Detection of JC virus DNA in human tonsil tissue: evidence for site of initial viral infection. J Virol. 1998, 72: 9918-9923.PubMedPubMed CentralGoogle Scholar
- Abed Y, Wang D, Boivin G: WU polyomavirus in children, Canada. Emerg Infect Dis. 2007, 13: 1939-1941.PubMedPubMed CentralView ArticleGoogle Scholar
- Norja P, Ubillos I, Templeton K, Simmonds P: No evidence for an association between infections with WU and KI polyomaviruses and respiratory disease. J Clin Virol. 2007, 40: 307-311. 10.1016/j.jcv.2007.09.008.PubMedView ArticleGoogle Scholar
- Lin F, Zheng M, Li H, Zheng C, Li X, Rao G, Wu F, Zeng A: WU polyomavirus in children with acute lower respiratory tract infections, China. J Clin Virol. 2008, 42: 94-102. 10.1016/j.jcv.2007.12.009.PubMedView ArticleGoogle Scholar
- Han TH, Chung JY, Koo JW, Kim SW, Hwang ES: WU polyomavirus in children with acute lower respiratory tract infections, South Korea. Emerg Infect Dis. 2007, 13: 1766-1768.PubMedPubMed CentralView ArticleGoogle Scholar
- Le BM, Demertzis LM, Wu G, Tibbets RJ, Buller R, Arens MQ, Gaynor AM, Storch GA, Wang D: Clinical and epidemiologic characterization of WU polyomavirus infection, St. Louis, Missouri. Emerg Infect Dis. 2007, 13: 1936-1938.PubMedPubMed CentralView ArticleGoogle Scholar
- Rohde G, Wiethege A, Borg I, Kauth M, Bauer TT, Gillissen A, Bufe A, Schultze-Werninghaus G: Respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study. Thorax. 2003, 58: 37-42. 10.1136/thorax.58.1.37.PubMedPubMed CentralView ArticleGoogle Scholar
- Ringshausen FC, Tan AY, Allander T, Borg I, Arinir U, Kronsbein J, Hauptmeier BM, Schultze-Werninghaus G, Rohde G: Frequency and clinical relevance of human bocavirus infection in acute exacerbations of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2009, 4: 111-117.PubMedPubMed CentralView ArticleGoogle Scholar
- Neske F, Blessing K, Ullrich F, Prottel A, Wolfgang Kreth H, Weissbrich B: WU polyomavirus infection in children, Germany. Emerg Infect Dis. 2008, 14: 680-681. 10.3201/eid1404.071325.PubMedPubMed CentralView ArticleGoogle Scholar
- Neske F, Blessing K, Prottel A, Ullrich F, Kreth HW, Weissbrich B: Detection of WU polyomavirus DNA by real-time PCR in nasopharyngeal aspirates, serum, and stool samples. J Clin Virol. 2009, 44: 115-118. 10.1016/j.jcv.2008.12.004.PubMedView ArticleGoogle Scholar
- Ren L, Gonzalez R, Xie Z, Zhang J, Liu C, Li J, Li Y, Wang Z, Kong X, Yao Y, Hu Y, Qian S, Geng R, Yang Y, Vernet G, Paranhos-Baccalà G, Jin Q, Shen K, Wang J: WU and KI polyomavirus present in the respiratory tract of children, but not in immunocompetent adults. J Clin Virol. 2008, 43 (3): 330-3. 10.1016/j.jcv.2008.08.003. Epub 2008 Sep 14.PubMedView ArticleGoogle Scholar
- Abedi Kiasari B, Vallely PJ, Corless CE, Al-Hammadi M, Klapper PE: Age-related pattern of KI and WU polyomavirus infection. J Clin Virol. 2008, 43: 123-125. 10.1016/j.jcv.2008.05.003.PubMedView ArticleGoogle Scholar
- Dalianis T, Ramqvist T, Andreasson K, Kean JM, Garcea RL: KI, WU and Merkel cell polyomaviruses: a new era for human polyomavirus research. Semin Cancer Biol. 2009, 19: 270-275. 10.1016/j.semcancer.2009.04.001.PubMedView ArticleGoogle Scholar
- Kleines M, Scheithauer S, Hengst M, Honnef D, Ritter K, Muhler E, Hausler M: Low to medium WU-virus titers in young children with lower respiratory tract infections. Intervirology. 2008, 51: 444-446. 10.1159/000209673.PubMedView ArticleGoogle Scholar
Copyright
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.