MW polyomavirus and STL polyomavirus present in tonsillar tissues ...

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Sep 10, 2015 - and STL polyomavirus (STLPyV), in tonsillar tissues from Chinese children ... polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV) and.
RESEARCH NOTE

MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease

J. Peng, K. Li, C. Zhang and Q. Jin MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China

Abstract We aimed to explore the frequency of all 13 human polyomaviruses (HPyVs), especially MW polyomavirus (MWPyV) and STL polyomavirus (STLPyV), in tonsillar tissues from Chinese children with chronic tonsillar disease. We examined 99 swabs from mucosal surfaces of palatine tonsils, in which six HPyVs were detected. MWPyV and STLPyV were each detected in two samples. This provides new evidence for the hypothesis that the lymphoid system may play a role in HPyV infection and persistence. We need to define their role in tonsillar disease in the future. Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Keywords: Chronic tonsillar disease, human polyomavirus, MW polyomavirus, STL polyomavirus, tonsillar tissue Original Submission: 28 June 2015; Revised Submission: 18 August 2015; Accepted: 31 August 2015 Editor: D. Raoult Article published online: 10 September 2015

Corresponding author: J. Peng or Q. Jin, No. 6, Rongjing Eastern Street, BDA, Beijing 100176, People’s Republic of China E-mails: [email protected] (J. Peng), [email protected] (Q. Jin)

It is well known that human polyomavirus (HPyV) infection is common in humans and occurs early in life. The seroprevalence rates of several HPyVs range from 40% to 80% in healthy adult blood donors [1]. Until now, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV) and trichodysplasia spinulosa-associated polyomavirus (TSPyV) have

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proved to be aetiological agents of nephropathy, progressive multifocal leukoencephalopathy, Merkel cell carcinoma, and trichodysplasia spinulosa, respectively [2–5]. The pathogenicity of the remaining nine HPyVs is unclear [6–8]. Several groups have detected JCPyV, BKPyV, WU polyomavirus (WUPyV), KI polyomavirus (KIPyV), MCPyV and TSPyV DNA in tonsillar tissue [9–13], which suggests that lymphoid tissue may be a latency site for these HPyVs. Their role in tonsillar disease remains to be defined. It is not known whether MW polyomavirus (MWPyV) and STL polyomavirus (STLPyV) can infect lymphoid tissue [14,15]. Recently, we examined the prevalence of 13 HPyVs in faecal samples from Chinese children by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry [6,7], which is a powerful platform for detecting multiplex PCR products [16,17]. In this study, we aimed to explore the frequency of 13 HPyVs, especially MWPyV and STLPyV, in tonsillar tissues from Chinese children with chronic tonsillar disease. We also sought to identify the genomic characteristics of the MWPyV and STLPyV strains present in tonsillar tissue. All samples used in this study were obtained from our institute collections. This study was approved by the ethics committee of Institute of Pathogen Biology. We obtained informed consent from the parents of all of the children who provided samples. This study enrolled 99 children (65 boys and 34 girls), ranging in age from 2 years to 9 years (mean age, 4.6 ± 2.5 years). A total of 99 tonsillar tissues, which were immediately frozen at –70°C after tonsillectomy, were collected from children who suffered from recurring tonsillitis or tonsillar hypertrophy in Beijing, China, from January 2013 to December 2013. DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. In this study, the samples were screened for all previously reported HPyVs (BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV 6, HPyV 7, TSPyV, HPyV 9, MWPyV, STLPyV, HPyV 12, and New Jersey polyomavirus) with a multiplex PCR–mass spectrometry assay as described in previous reports, with β-globin as a DNA extraction quality control [6,7]. Briefly, the assay is composed of primary PCR, primer extension, and MALDI-TOF MS separation of products on a silicon chip array. These methods can accurately identify 13 HPyVs, and the detection limit was approximately ten copies per reaction. The specific plasmids and sterile water were used as a positive control and a negative control, respectively [6,7]. To our knowledge, this is the first study to screen for all 13 HPyVs in human tonsillar tissue. Six HPyV strains were detected, and seven were not (Table 1). Twenty-six (26.3%) of the 99 specimens contained at least one HPyV. The highest frequency of the HPyVs tested was 13.1%, which was determined for

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TABLE 1. Detection of human polyomaviruses Human polyomavirus

No. of samples

%

Single infection WUPyV KIPyV MCPyV TSPyV MWPyV STLPyV Dual infection WUPyV/MCPyV Total

24 11 2 4 3 2 2 2 2 26

24.2 11.1 2.0 4.0 3.0 2.0 2.0 2.0 2.0 26.3

KIPyV, KI polyomavirus; MCPyV, Merkel cell polyomavirus; MWPyV, MW polyomavirus; STLPyV, STL polyomavirus; TSPyV, trichodysplasia spinulosaassociated polyomavirus; WUPyV, WU polyomavirus.

WUPyV. MCPyV, TSPyV, KIPyV, MWPyV, and STLPyV were detected in six (6.0%), three (3.0%), two (2.0%), two (2.0%) and two (2.0%) of the 99 samples, respectively. A single HPyV infection was found in 24.2% (24/99) of the samples, and dual HPyV infections were found in 2.0% (2/99) of the samples. All positive samples were collected during January–June, 2013 (Fig. S1). In previous studies, MWPyV was detected in stool (2.3%), wart and respiratory samples (1.5% in patients and 9.2% in controls) [14,18,19], whereas STLPyV was only detected in stool (2.2% in patients and 3.0% in controls) and urine (0.3%) samples [7,15]. This is the first study to detect MWPyV and STLPyV in tonsillar tissue. It provides new evidence for the hypothesis that the lymphoid system may play a role in HPyV infection and persistence [9,10,13]. Whole genome sequencing was performed with one STLPyV specimen (Y57) and one MWPyV specimen (Y96). The primers used for the whole genome sequencing of STLPyV have been described previously [7]. The primers used for the whole genome sequencing of MWPyV are summarized in Table S1. PCR amplification and sequence analysis were performed according to a previous study [7]. The obtained whole genome sequences have been submitted to GenBank under the accession numbers KF651951 and KR338953. Each HPyV isolate encoded full-length open reading frames for the predicted proteins (Tables S2 and S3). The results of sequence analysis showed a high level of conservation among all MWPyV and STLPyV sequences (Tables S4 and S5). All whole genome sequences of MWPyV and all large T antigen (partial) sequences of STLPyV were obtained from GenBank (http://www.ncbi.nlm. nih.gov/genbank/) for phylogenetic analysis with the Molecular Evolutionary Genetics Analysis (MEGA) version 6 software packages [20]. In previous studies, we obtained six MWPyV genome sequences (HB040C, HB087C, HB039C, HN037, HB017C, and HN104) and two STLPyV genome sequences (HB124 and HB201) [7] from viruses isolated from faecal samples collected from Chinese children. Phylogenetic analysis

FIG. 1. Neighbour-joining phylogenetic tree of MW polyomavirus and STL polyomavirus generated by MEGA 6. The bootstrap values are shown as percentages of 1000 replicates for the major lineages within the trees. (a) MW polyomavirus tree based on 21 whole genome sequences. Y96 is from children with chronic tonsillar disease in this study. Other sequences were obtained from GenBank (NC_018102, JQ898292, JX262162, JX259273, KC549586–KC549594, KC571700– KC571705, and KC690147). (b) STL polyomavirus tree based on the partial nucleotide sequences of large T antigen (431nt). Y57 is from children with chronic tonsillar disease in this study. Other sequences were obtained from GenBank (JX463183–JX463192, KF525270, KF530304, and KM893862). The isolates labelled with dark triangles, dark squares and dark circles are the original isolates, the isolates obtained in our previous studies, and the isolates in this study, respectively.

showed that there are three genotypes of MWPyVs (Fig. 1a). MWPyV isolates from China belong to three genotypes. Genotype 1 includes Y96, the St Louis isolate WD976, UCMXPyV-1, 10ww, TEDDY_01, and nine Queensland isolates. Genotype 2 includes HB039C, HB040C, HB087C, and HN037, as well as the Malawi isolate MA095. Genotype 3 contains only one member, HN104, which was isolated from a child with diarrhoea. On the basis of the available data, all MWPyVs that were isolated from respiratory specimens belong to genotype

Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 97.e1–97.e3

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Peng et al.

1. On the basis of the results of the phylogenetic analysis, there are two genotypes of STLPyV. Y57 and the original two STLPyV isolates (MA138 and WD972) belong to genotype 1, whereas HB124 and HB201 belong to genotype 2 (Fig. 1b). The detection of MWPyV and STLPyV in different countries indicates that they are geographically widespread. This study provides new evidence supporting the hypothesis that the lymphoid system may play a role in HPyV infection and persistence. In this study, no samples from healthy people were tested. All of these data are preliminary, and will need to be confirmed and extended with a larger sample.

Transparency declaration The authors declare no conflicts of interest.

Acknowledgements J. Peng was funded by grants from the Ministry of Science and Technology of the People’s Republic of China of the People’s Republic of China (2014ZX10004-001), the Programme for Changjiang Scholars and Innovative Research Team in University (IRT13007), the Institute of Pathogen Biology, CAMS&PUMC (2013IPB102), the PUMC Youth Fund, and the Fundamental Research Funds for the Central Universities (3332013118).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cmi.2015.08.028.

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