- Email: [email protected]
MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease
Accepted Manuscript MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease Junping Peng, Ke Li, Chi Zhang, Qi Jin PII:
S1198-743X(15)00814-9
DOI:
10.1016/j.cmi.2015.08.028
Reference:
CMI 371
To appear in:
Clinical Microbiology and Infection
Received Date: 28 June 2015 Revised Date:
18 August 2015
Accepted Date: 31 August 2015
Please cite this article as: Peng J, Li K, Zhang C, Jin Q, MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease, Clinical Microbiology and Infection (2015), doi: 10.1016/j.cmi.2015.08.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
MW polyomavirus and STL polyomavirus present in tonsillar
2
tissues from children with chronic tonsillar disease
3 4 5 6 7
Running title: MWPyV and STLPyV present in tonsillar tissues
8
Junping Peng*, Ke Li, Chi Zhang, Qi Jin*
RI PT
Category: Virology
9
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen
11
Biology, Chinese Academy of Medical Sciences & Peking Union Medical College,
12
Beijing, People’s Republic of China.
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M AN U
SC
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* Corresponding author:
15
Mailing address: No. 6 Rongjing Eastern Street, BDA, Beijing 100176, People’s
16
Republic of China.
17
Phone: 86 10 67877735. Fax: 86 10 67877736.
18
E-mail: [email protected] (Junping Peng) or [email protected] (Qi Jin).
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MW polyomavirus and STL polyomavirus present in tonsillar
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tissues from children with chronic tonsillar disease
29
Abstract
31
We aimed to explore the frequency of all 13 human polyomaviruses (HPyVs),
32
especially MWPyV and STLPyV, in tonsillar tissues from Chinese children with
33
chronic tonsillar disease. We examined 99 swabs from mucosal surfaces of palatine
34
tonsils, in which six HPyVs were detected. MWPyV and STLPyV were each detected
35
in two samples. It provides new evidence for the hypothesis that the lymphoid system
36
may play a role in HPyV infection and persistence. We need to define their role in
37
tonsillar disease in the future.
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Key Words: human polyomavirus; tonsillar tissue; MW polyomavirus; STL
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polyomavirus; chronic tonsillar disease
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It is well known that human polyomavirus (HPyV) infection is common in humans and occurs early in life. The seroprevalence of several HPyVs range from
51
40% to 80% in healthy adult blood donors [1]. Until now, BK polyomavirus (BKPyV),
52
JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV), and trichodysplasia
53
spinulosa-associated polyomavirus (TSPyV) have proved to be etiologic agents of
54
nephropathy, progressive multifocal leukoencephalopathy, Merkel cell carcinoma,
55
and trichodysplasia spinulosa, respectively [2-5]. The pathogenicity of the remaining
56
nine HPyVs is unclear [6-8]. Several groups have detected JCPyV, BKPyV, WU
57
polyomavirus (WUPyV), KI polyomavirus (KIPyV), MCPyV, and TSPyV DNA in
58
tonsillar tissue [9-13], which suggests that lymphoid tissue may be a latency site for
59
these HPyVs. Their role in tonsillar disease remains to be defined. It is not known
60
whether MW polyomavirus (MWPyV) and STL polyomavirus (STLPyV) can infect
61
lymphoid tissue [14, 15].
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Recently, we examined the prevalence of 13 HPyVs in fecal samples from Chinese children using matrix-assisted laser desorption ionization-time of flight mass
64
spectrometry (MALDI-TOF MS) [6, 7], which is a powerful platform for detecting
65
multiplex PCR products [16, 17]. In this study we aimed to explore the frequency of
66
13 HPyVs, especially MWPyV and STLPyV, in tonsillar tissues from Chinese
67
children with chronic tonsillar disease. We also sought to identify the genomic
68
characteristics of the MWPyV and STLPyV strains present in tonsillar tissue.
69 70
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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 3
ACCEPTED MANUSCRIPT obtained informed consent from the parents of all of the children who provided
72
samples. This study enrolled 99 children (65 boys and 34 girls), ranging in age from 2
73
to 9 years (mean age 4.6±2.5 years). A total of 99 tonsillar tissues, which was
74
immediately frozen at -70°C after tonsillectomy, were collected from children who
75
suffered from recurring tonsillitis or tonsillar hypertrophy in Beijing, China, from
76
January 2013 to December 2013. DNA was extracted using the QIAamp DNA Mini
77
Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. In this
78
study, the samples were screened for all previously reported HPyVs (BKPyV, JCPyV,
79
KIPyV, WUPyV, MCPyV, Human polyomavirus 6, Human polyomavirus 7, TSPyV,
80
Human polyomavirus 9, MWPyV, STLPyV, Human polyomavirus 12 and New
81
Jersey polyomavirus) according to mPCR-Mass assay as describe in previous reports,
82
using β-globin as a DNA extraction quality control[6, 7]. Briefly, the assay is
83
composed of primary PCR, primer extension, and MALDI-TOF MS separation of
84
products on a silicon chip array. These methods can accurately identify 13 HPyVs and
85
the detection limit was approximately 10 copies per reaction. The specific plasmids
86
and sterile water were used as positive control and negative control, respectively [6,
87
7].
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To our knowledge, this is the first study to screen for all 13 HPyVs in human
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tonsillar tissue. Six HPyV strains were detected, while seven were not (Table 1). 26
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(26.3%) of the 99 specimens contained at least 1 HPyV. The highest frequency of the
91
HPyVs tested was 13.1%, which was determined for WUPyV. MCPyV, TSPyV,
92
KIPyV, MWPyV, and STLPyV were detected in six (6.0%), three (3.0%), two (2.0%), 4
ACCEPTED MANUSCRIPT two (2.0%), and two (2.0%) of the 99 samples, respectively. A single HPyV infection
94
was found in 24.2% (24/99) of the samples, and dual HPyV infections were found in
95
2.0% (2/99) of the samples. All positive samples were collected during January-June,
96
2013 (Fig S1). In previous studies, MWPyV was tested in stool (2.3%), warts, and
97
respiratory samples (1.5% in patients and 9.2% in controls)[14, 18, 19], while
98
STLPyV was only tested in stool (2.2% in patients and 3.0% in controls) and urine
99
(0.3%) samples [7, 15]. It is the first report to detect MWPyV and STLPyV in
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tonsillar tissue. It provides new evidence for the hypothesis that the lymphoid system
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may play a role in HPyV infection and persistence [9, 10, 13].
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Whole genome sequencing was performed using one STLPyV specimen (Y57) and one MWPyV specimen (Y96). The primers used for the whole genome
104
sequencing of STLPyV were described previously [7]. The primers used for the whole
105
genome sequencing of MWPyV are summarized in Table S1. PCR amplification and
106
sequence analysis were performed according to a previous study [7]. The obtained
107
whole genome sequences have been submitted to GenBank under the accession
108
numbers KF651951 and KR338953. Each HPyV isolate encoded full-length open
109
reading frames for the predicted proteins (Table S2-S3). The results of sequence
110
analysis showed a high level of conservation amongst all MWPyV and STLPyV
111
sequences (Table S4-S5). All whole genome sequences of MWPyV and all Large T
112
antigen (partial) sequences of STLPyV were obtained from GenBank
113
(http://www.ncbi.nlm.nih.gov/genbank/) for phylogenetic analysis using the
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Molecular Evolutionary Genetics Analysis (MEGA) version 6 software packages [20].
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ACCEPTED MANUSCRIPT In previous studies, we obtained six MWPyV genome sequences (HB040C, HB087C,
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HB039C, HN037, HB017C and HN104) and two STLPyV genome sequences
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(HB124 and HB201) [7] from viruses isolated from fecal samples collected from
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Chinese children. Phylogenetic analysis showed that there are three genotypes of
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MWPyVs (Fig. 1A). MWPyV isolates from China belong to three genotypes.
120
Genotype 1 included Y96, the St. Louis isolate WD976, UC-MXPyV-1, 10ww,
121
TEDDY_01, and nine Queensland isolates. Genotype 2 included the HB039C,
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HB040C, HB087C, HN037 isolates, as well as the Malawi isolate MA095. Genotype
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3 only contained only one member, HN104, which was isolated from a child with
124
diarrhea. Based on the data available, all MWPyVs that were isolated from respiratory
125
specimens belong to genotype 1. Based on the results of the phylogenetic analysis,
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there are two genotypes of STLPyV. Y57 and the original two STLPyV isolates
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(MA138 and WD972) belong to genotype 1, while HB124 and HB201 belong to
128
genotype 2 (Fig. 1B). The detection of MWPyV and STLPyV in different countries
129
indicate that they are geographically widespread.
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It provides new evidence for the hypothesis that the lymphoid system may play a
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role in HPyV infection and persistence. In this study no samples from healthy people
132
were tested. All these data are preliminary and will need to be confirmed and
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extended to a larger sample.
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Funding Junping Peng was funded by grants from the Ministry of Science and Technology 6
ACCEPTED MANUSCRIPT of the People’s Republic of China (2014ZX10004-001), the Program for Changjiang
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Scholars and Innovative Research Team in University (IRT13007), the Institute of
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Pathogen Biology, CAMS&PUMC (2013IPB102), PUMC Youth Fund，and the
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Fundamental Research Funds for the Central Universities (3332013118).
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Transparency Declaration The authors declare no conflicts of interest.
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References
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Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (b.K.) isolated from urine after renal transplantation. Lancet. 1971; 1: 1253-1257.
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Kean JM, Rao S, Wang M, Garcea RL. Seroepidemiology of human polyomaviruses. PLoS Pathog. 2009; 5: e1000363.
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Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet. 1971; 1:
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van der Meijden E, Janssens RW, Lauber C, Bouwes Bavinck JN, Gorbalenya AE, Feltkamp MC.
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Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human merkel
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Li K, Guo J, Zhao R, et al. The prevalence of 10 human polyomaviruses in fecal samples from children with acute gastroenteritis: A case-control study. J Clin Microbiol. 2013; 51: 7
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children. J Clin Virol. 2015; 66: 19-23. 8
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Li K, Zhang C, Zhao R, et al. The prevalence of stl polyomavirus in stool samples from chinese
DeCaprio JA, Garcea RL. A cornucopia of human polyomaviruses. Nat Rev Microbiol. 2013; 11:
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3107-3109.
264-276. 9
Kato A, Kitamura T, Takasaka T, et al. Detection of the archetypal regulatory region of jc virus from the tonsil tissue of patients with tonsillitis and tonsilar hypertrophy. J Neurovirol. 2004;
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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.
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Kantola K, Sadeghi M, Lahtinen A, et al. Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples: Implications for respiratory transmission and
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latency. J Clin Virol. 2009; 45: 292-295. 12
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polyomaviruses in human tonsils. J Clin Virol. 2009; 46: 75-79. 13
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latency. J Clin Virol. 2014; 59: 55-58.
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Sadeghi M, Aaltonen LM, Hedman L, Chen T, Soderlund-Venermo M, Hedman K. Detection of ts polyomavirus DNA in tonsillar tissues of children and adults: Evidence for site of viral
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Babakir-Mina M, Ciccozzi M, Bonifacio D, et al. Identification of the novel ki and wu
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Siebrasse EA, Reyes A, Lim ES, et al. Identification of mw polyomavirus, a novel polyomavirus
in human stool. J Virol. 2012; 86: 10321-10326. 15
Lim ES, Reyes A, Antonio M, et al. Discovery of stl polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique t antigen by alternative splicing. Virology. 2013; 8
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by genotyping both e6 and l1 genes. J Clin Microbiol. 2013; 51: 402-408.
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simultaneous detection of 21 common respiratory viruses. J Clin Microbiol. 2015. 18
187 188
Zhang C, Xiao Y, Du J, et al. Application of multiplex pcr couple with maldi-tof analysis for
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Buck CB, Phan GQ, Raiji MT, Murphy PM, McDermott DH, McBride AA. Complete genome sequence of a tenth human polyomavirus. J Virol. 2012; 86: 10887.
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Peng J, Gao L, Guo J, et al. Type-specific detection of 30 oncogenic human papillomaviruses
Rockett RJ, Sloots TP, Bowes S, et al. Detection of novel polyomaviruses, tspyv, hpyv6, hpyv7,
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436: 295-303.
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hpyv9 and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid. PLoS One.
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2013; 8: e62764.
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Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. Mega6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013; 30: 2725-2729.
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Figure legends
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Fig 1. Neighbor-joining phylogenetic tree of MW polyomavirus and STL
196
polyomavirus generated by MEGA 6, respectively. The bootstrap values are shown as
197
percentages of 1,000 replicates for the major lineages within the trees. A: MW
198
polyomavirus tree base on 21 whole genome sequences. Y96 is from children with
199
chronic tonsillar disease in this study. Other sequences were obtained from GenBank
200
(NC_018102,
201
KC571700-KC571705 and KC690147). B: STL polyomavirus tree based on the part
202
nucleotide sequences of Large T antigen (431nt). Y57 is from children with chronic
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JQ898292,
JX262162,
9
JX259273,
KC549586-KC549594,
ACCEPTED MANUSCRIPT tonsillar disease in this study. Other sequences were obtained from GenBank
204
(JX463183-JX463192, KF525270, KF530304 and KM893862). The isolates which
205
were labeled with dark triangle, dark square and dark circle means the original
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isolates, isolates obtained in our previous studies and isolate in this study,
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respectively.
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ACCEPTED MANUSCRIPT Human polyomavirus* Single infection WUPyV KIPyV MCPyV TSPyV MWPyV STLPyV Dual infection WUPyV/MCPyV Total
Number of samples 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
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Table 1 Detection of human polyomaviruses
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* WUPyV, WU polyomavirus; KIPyV, KI polyomavirus; MCPyV, Merkel cell polyomavirus; TSPyV, trichodysplasia spinulosa associated polyomavirus; MWPyV, MW polyomavirus; STLPyV, STL
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polyomavirus.
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ACCEPTED MANUSCRIPT Table S1 Primers for whole genome amplification and sequencing of MW polyomavirus
Name*
Sequence
Position
Product (bp)
CATTTCATCACGAGCCCTTT
300-319
MWPyV_1631R
TACATCATTGCCCATCCTTG
1628-1609
MWPyV_1576F
CGGACACCACAATGACAGTT
1573-1592
MWPyV_3142R
TGCAATTGATGAATATATGGTTG
3139-3117
MWPyV_3015F
CACAACCATCYAAATGATCC
MWPyV_4489R
GCTTTTTGCTTTGGTTTGGA
4485-4466
MWPyV_4394F
GCAAGCRAAGCAGATCAAAT
4390-4409
MWPyV_470R
GAATAACAGAGATTACAGCACCCATA
467-442
1329
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MWPyV_303F
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3012-3031
1567
1474
1017
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* Position based on nucleotide sequence of Merkel cell polyomavirus strain TKS (FJ464337) and MW
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polyomavirus strain WD976 (JQ898292), respectively.
ACCEPTED MANUSCRIPT Table S2 Putative proteins encoded by MWPyV_Y96 Putative coding region(s)
Predicted size (aa)
VP2
443-1375
310
VP3
773-1375
200
VP1
1365-2576
403
LTAg
4939-4700,4344-2578
668
STAg
4939-4319
206
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Protein
ACCEPTED MANUSCRIPT Table S3 Putative proteins encoded by STLPyV_Y57 Putative coding region(s)
Predicted size (aa)
VP2
352-1263
303
VP3
676-1263
195
VP1
1241-2446
401
LTAg
4775-4533,4187-2451
660
190T
4775-4206
190
STAg
4775-4188
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Protein
ACCEPTED MANUSCRIPT Table S4 Identity shared between Y96 and published MWPyV genomes Identity (%)
Country
Source
WD976
97
USA
fecal specimen
MA095
94
Malawi
fecal specimen
10ww
97
USA
peri-anal condyloma
UC-MXPyV-1
97
Mexico
TEDDY_01
97
USA
QLDMW02
97
Australia
QLDMW03
96
Australia
QLDMW04
97
Australia
fecal specimen
QLDMW05
97
Australia
respiratory specimen
QLDMW09
97
Australia
respiratory specimen
QLDMW10
97
Australia
respiratory specimen
QLDMW11
97
Australia
respiratory specimen
QLDMW12
96
Australia
respiratory specimen
QLDMW13
96
Australia
respiratory specimen
96
China
fecal specimen
94
China
fecal specimen
95
China
fecal specimen
95
China
fecal specimen
94
China
fecal specimen
93
China
fecal specimen
HB040C HB087C
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HB037 HN104
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HB039C
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HB017C
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Name
fecal specimen
fecal specimen fecal specimen fecal specimen
ACCEPTED MANUSCRIPT Table S5 Identity shared between Y57 and published STLPyV genomes Identity (%)
Country
Source
WD972
98
USA
fecal specimen
MA138
94
Malawi
fecal specimen
11ww
92
USA
HB124
92
China
HB201
92
China
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Name
peri-anal wart
fecal specimen fecal specimen
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ACCEPTED MANUSCRIPT
Fig S1. Seasonal distribution of human polyomavirus in Chinese children with chronic tonsillar disease. WUPyV: WU
polyomavirus;
KIPyV:
KI
polyomavirus;
MCPyV:
Merkel
cell
polyomavirus;
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spinulosa-associated polyomavirus; MWPyV: MW polyomavirus; STLPyV: STL polyomavirus.
TSPyV:
trichodysplasia
S1198-743X(15)00814-9
DOI:
10.1016/j.cmi.2015.08.028
Reference:
CMI 371
To appear in:
Clinical Microbiology and Infection
Received Date: 28 June 2015 Revised Date:
18 August 2015
Accepted Date: 31 August 2015
Please cite this article as: Peng J, Li K, Zhang C, Jin Q, MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease, Clinical Microbiology and Infection (2015), doi: 10.1016/j.cmi.2015.08.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
MW polyomavirus and STL polyomavirus present in tonsillar
2
tissues from children with chronic tonsillar disease
3 4 5 6 7
Running title: MWPyV and STLPyV present in tonsillar tissues
8
Junping Peng*, Ke Li, Chi Zhang, Qi Jin*
RI PT
Category: Virology
9
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen
11
Biology, Chinese Academy of Medical Sciences & Peking Union Medical College,
12
Beijing, People’s Republic of China.
13
M AN U
SC
10
* Corresponding author:
15
Mailing address: No. 6 Rongjing Eastern Street, BDA, Beijing 100176, People’s
16
Republic of China.
17
Phone: 86 10 67877735. Fax: 86 10 67877736.
18
E-mail: [email protected] (Junping Peng) or [email protected] (Qi Jin).
20 21 22
EP AC C
19
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23 24 25 26 1
ACCEPTED MANUSCRIPT 27
MW polyomavirus and STL polyomavirus present in tonsillar
28
tissues from children with chronic tonsillar disease
29
Abstract
31
We aimed to explore the frequency of all 13 human polyomaviruses (HPyVs),
32
especially MWPyV and STLPyV, in tonsillar tissues from Chinese children with
33
chronic tonsillar disease. We examined 99 swabs from mucosal surfaces of palatine
34
tonsils, in which six HPyVs were detected. MWPyV and STLPyV were each detected
35
in two samples. It provides new evidence for the hypothesis that the lymphoid system
36
may play a role in HPyV infection and persistence. We need to define their role in
37
tonsillar disease in the future.
TE D
38
M AN U
SC
RI PT
30
Key Words: human polyomavirus; tonsillar tissue; MW polyomavirus; STL
40
polyomavirus; chronic tonsillar disease
42 43 44 45
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46 47 48 2
ACCEPTED MANUSCRIPT 49
It is well known that human polyomavirus (HPyV) infection is common in humans and occurs early in life. The seroprevalence of several HPyVs range from
51
40% to 80% in healthy adult blood donors [1]. Until now, BK polyomavirus (BKPyV),
52
JC polyomavirus (JCPyV), Merkel cell polyomavirus (MCPyV), and trichodysplasia
53
spinulosa-associated polyomavirus (TSPyV) have proved to be etiologic agents of
54
nephropathy, progressive multifocal leukoencephalopathy, Merkel cell carcinoma,
55
and trichodysplasia spinulosa, respectively [2-5]. The pathogenicity of the remaining
56
nine HPyVs is unclear [6-8]. Several groups have detected JCPyV, BKPyV, WU
57
polyomavirus (WUPyV), KI polyomavirus (KIPyV), MCPyV, and TSPyV DNA in
58
tonsillar tissue [9-13], which suggests that lymphoid tissue may be a latency site for
59
these HPyVs. Their role in tonsillar disease remains to be defined. It is not known
60
whether MW polyomavirus (MWPyV) and STL polyomavirus (STLPyV) can infect
61
lymphoid tissue [14, 15].
SC
M AN U
TE D
62
RI PT
50
Recently, we examined the prevalence of 13 HPyVs in fecal samples from Chinese children using matrix-assisted laser desorption ionization-time of flight mass
64
spectrometry (MALDI-TOF MS) [6, 7], which is a powerful platform for detecting
65
multiplex PCR products [16, 17]. In this study we aimed to explore the frequency of
66
13 HPyVs, especially MWPyV and STLPyV, in tonsillar tissues from Chinese
67
children with chronic tonsillar disease. We also sought to identify the genomic
68
characteristics of the MWPyV and STLPyV strains present in tonsillar tissue.
69 70
AC C
EP
63
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 3
ACCEPTED MANUSCRIPT obtained informed consent from the parents of all of the children who provided
72
samples. This study enrolled 99 children (65 boys and 34 girls), ranging in age from 2
73
to 9 years (mean age 4.6±2.5 years). A total of 99 tonsillar tissues, which was
74
immediately frozen at -70°C after tonsillectomy, were collected from children who
75
suffered from recurring tonsillitis or tonsillar hypertrophy in Beijing, China, from
76
January 2013 to December 2013. DNA was extracted using the QIAamp DNA Mini
77
Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. In this
78
study, the samples were screened for all previously reported HPyVs (BKPyV, JCPyV,
79
KIPyV, WUPyV, MCPyV, Human polyomavirus 6, Human polyomavirus 7, TSPyV,
80
Human polyomavirus 9, MWPyV, STLPyV, Human polyomavirus 12 and New
81
Jersey polyomavirus) according to mPCR-Mass assay as describe in previous reports,
82
using β-globin as a DNA extraction quality control[6, 7]. Briefly, the assay is
83
composed of primary PCR, primer extension, and MALDI-TOF MS separation of
84
products on a silicon chip array. These methods can accurately identify 13 HPyVs and
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the detection limit was approximately 10 copies per reaction. The specific plasmids
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and sterile water were used as positive control and negative control, respectively [6,
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7].
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To our knowledge, this is the first study to screen for all 13 HPyVs in human
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tonsillar tissue. Six HPyV strains were detected, while seven were not (Table 1). 26
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(26.3%) of the 99 specimens contained at least 1 HPyV. The highest frequency of the
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HPyVs tested was 13.1%, which was determined for WUPyV. MCPyV, TSPyV,
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KIPyV, MWPyV, and STLPyV were detected in six (6.0%), three (3.0%), two (2.0%), 4
ACCEPTED MANUSCRIPT two (2.0%), and two (2.0%) of the 99 samples, respectively. A single HPyV infection
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was found in 24.2% (24/99) of the samples, and dual HPyV infections were found in
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2.0% (2/99) of the samples. All positive samples were collected during January-June,
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2013 (Fig S1). In previous studies, MWPyV was tested in stool (2.3%), warts, and
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respiratory samples (1.5% in patients and 9.2% in controls)[14, 18, 19], while
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STLPyV was only tested in stool (2.2% in patients and 3.0% in controls) and urine
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(0.3%) samples [7, 15]. It is the first report to detect MWPyV and STLPyV in
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tonsillar tissue. It provides new evidence for the hypothesis that the lymphoid system
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may play a role in HPyV infection and persistence [9, 10, 13].
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Whole genome sequencing was performed using one STLPyV specimen (Y57) and one MWPyV specimen (Y96). The primers used for the whole genome
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sequencing of STLPyV were described previously [7]. The primers used for the whole
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genome sequencing of MWPyV are summarized in Table S1. PCR amplification and
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sequence analysis were performed according to a previous study [7]. The obtained
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whole genome sequences have been submitted to GenBank under the accession
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numbers KF651951 and KR338953. Each HPyV isolate encoded full-length open
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reading frames for the predicted proteins (Table S2-S3). The results of sequence
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analysis showed a high level of conservation amongst all MWPyV and STLPyV
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sequences (Table S4-S5). All whole genome sequences of MWPyV and all Large T
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antigen (partial) sequences of STLPyV were obtained from GenBank
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(http://www.ncbi.nlm.nih.gov/genbank/) for phylogenetic analysis using the
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Molecular Evolutionary Genetics Analysis (MEGA) version 6 software packages [20].
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HB039C, HN037, HB017C and HN104) and two STLPyV genome sequences
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(HB124 and HB201) [7] from viruses isolated from fecal samples collected from
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Chinese children. Phylogenetic analysis showed that there are three genotypes of
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MWPyVs (Fig. 1A). MWPyV isolates from China belong to three genotypes.
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Genotype 1 included Y96, the St. Louis isolate WD976, UC-MXPyV-1, 10ww,
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TEDDY_01, and nine Queensland isolates. Genotype 2 included the HB039C,
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HB040C, HB087C, HN037 isolates, as well as the Malawi isolate MA095. Genotype
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3 only contained only one member, HN104, which was isolated from a child with
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diarrhea. Based on the data available, all MWPyVs that were isolated from respiratory
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specimens belong to genotype 1. Based on the results of the phylogenetic analysis,
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there are two genotypes of STLPyV. Y57 and the original two STLPyV isolates
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(MA138 and WD972) belong to genotype 1, while HB124 and HB201 belong to
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genotype 2 (Fig. 1B). The detection of MWPyV and STLPyV in different countries
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indicate that they are geographically widespread.
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It provides new evidence for the hypothesis that the lymphoid system may play a
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role in HPyV infection and persistence. In this study no samples from healthy people
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were tested. All these data are preliminary and will need to be confirmed and
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extended to a larger sample.
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Funding Junping Peng was funded by grants from the Ministry of Science and Technology 6
ACCEPTED MANUSCRIPT of the People’s Republic of China (2014ZX10004-001), the Program for Changjiang
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Scholars and Innovative Research Team in University (IRT13007), the Institute of
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Pathogen Biology, CAMS&PUMC (2013IPB102), PUMC Youth Fund，and the
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Fundamental Research Funds for the Central Universities (3332013118).
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Transparency Declaration The authors declare no conflicts of interest.
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References
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polyomaviruses in human tonsils. J Clin Virol. 2009; 46: 75-79. 13
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Sadeghi M, Aaltonen LM, Hedman L, Chen T, Soderlund-Venermo M, Hedman K. Detection of ts polyomavirus DNA in tonsillar tissues of children and adults: Evidence for site of viral
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Babakir-Mina M, Ciccozzi M, Bonifacio D, et al. Identification of the novel ki and wu
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Siebrasse EA, Reyes A, Lim ES, et al. Identification of mw polyomavirus, a novel polyomavirus
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Lim ES, Reyes A, Antonio M, et al. Discovery of stl polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique t antigen by alternative splicing. Virology. 2013; 8
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by genotyping both e6 and l1 genes. J Clin Microbiol. 2013; 51: 402-408.
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simultaneous detection of 21 common respiratory viruses. J Clin Microbiol. 2015. 18
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Zhang C, Xiao Y, Du J, et al. Application of multiplex pcr couple with maldi-tof analysis for
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Buck CB, Phan GQ, Raiji MT, Murphy PM, McDermott DH, McBride AA. Complete genome sequence of a tenth human polyomavirus. J Virol. 2012; 86: 10887.
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Rockett RJ, Sloots TP, Bowes S, et al. Detection of novel polyomaviruses, tspyv, hpyv6, hpyv7,
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436: 295-303.
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hpyv9 and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid. PLoS One.
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Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. Mega6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013; 30: 2725-2729.
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Figure legends
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Fig 1. Neighbor-joining phylogenetic tree of MW polyomavirus and STL
196
polyomavirus generated by MEGA 6, respectively. The bootstrap values are shown as
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percentages of 1,000 replicates for the major lineages within the trees. A: MW
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polyomavirus tree base on 21 whole genome sequences. Y96 is from children with
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chronic tonsillar disease in this study. Other sequences were obtained from GenBank
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(NC_018102,
201
KC571700-KC571705 and KC690147). B: STL polyomavirus tree based on the part
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nucleotide sequences of Large T antigen (431nt). Y57 is from children with chronic
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JQ898292,
JX262162,
9
JX259273,
KC549586-KC549594,
ACCEPTED MANUSCRIPT tonsillar disease in this study. Other sequences were obtained from GenBank
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(JX463183-JX463192, KF525270, KF530304 and KM893862). The isolates which
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were labeled with dark triangle, dark square and dark circle means the original
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isolates, isolates obtained in our previous studies and isolate in this study,
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respectively.
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10
ACCEPTED MANUSCRIPT Human polyomavirus* Single infection WUPyV KIPyV MCPyV TSPyV MWPyV STLPyV Dual infection WUPyV/MCPyV Total
Number of samples 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
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Table 1 Detection of human polyomaviruses
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* WUPyV, WU polyomavirus; KIPyV, KI polyomavirus; MCPyV, Merkel cell polyomavirus; TSPyV, trichodysplasia spinulosa associated polyomavirus; MWPyV, MW polyomavirus; STLPyV, STL
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polyomavirus.
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ACCEPTED MANUSCRIPT Table S1 Primers for whole genome amplification and sequencing of MW polyomavirus
Name*
Sequence
Position
Product (bp)
CATTTCATCACGAGCCCTTT
300-319
MWPyV_1631R
TACATCATTGCCCATCCTTG
1628-1609
MWPyV_1576F
CGGACACCACAATGACAGTT
1573-1592
MWPyV_3142R
TGCAATTGATGAATATATGGTTG
3139-3117
MWPyV_3015F
CACAACCATCYAAATGATCC
MWPyV_4489R
GCTTTTTGCTTTGGTTTGGA
4485-4466
MWPyV_4394F
GCAAGCRAAGCAGATCAAAT
4390-4409
MWPyV_470R
GAATAACAGAGATTACAGCACCCATA
467-442
1329
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MWPyV_303F
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1567
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* Position based on nucleotide sequence of Merkel cell polyomavirus strain TKS (FJ464337) and MW
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polyomavirus strain WD976 (JQ898292), respectively.
ACCEPTED MANUSCRIPT Table S2 Putative proteins encoded by MWPyV_Y96 Putative coding region(s)
Predicted size (aa)
VP2
443-1375
310
VP3
773-1375
200
VP1
1365-2576
403
LTAg
4939-4700,4344-2578
668
STAg
4939-4319
206
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Protein
ACCEPTED MANUSCRIPT Table S3 Putative proteins encoded by STLPyV_Y57 Putative coding region(s)
Predicted size (aa)
VP2
352-1263
303
VP3
676-1263
195
VP1
1241-2446
401
LTAg
4775-4533,4187-2451
660
190T
4775-4206
190
STAg
4775-4188
195
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Protein
ACCEPTED MANUSCRIPT Table S4 Identity shared between Y96 and published MWPyV genomes Identity (%)
Country
Source
WD976
97
USA
fecal specimen
MA095
94
Malawi
fecal specimen
10ww
97
USA
peri-anal condyloma
UC-MXPyV-1
97
Mexico
TEDDY_01
97
USA
QLDMW02
97
Australia
QLDMW03
96
Australia
QLDMW04
97
Australia
fecal specimen
QLDMW05
97
Australia
respiratory specimen
QLDMW09
97
Australia
respiratory specimen
QLDMW10
97
Australia
respiratory specimen
QLDMW11
97
Australia
respiratory specimen
QLDMW12
96
Australia
respiratory specimen
QLDMW13
96
Australia
respiratory specimen
96
China
fecal specimen
94
China
fecal specimen
95
China
fecal specimen
95
China
fecal specimen
94
China
fecal specimen
93
China
fecal specimen
HB040C HB087C
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HB037 HN104
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HB039C
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HB017C
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Name
fecal specimen
fecal specimen fecal specimen fecal specimen
ACCEPTED MANUSCRIPT Table S5 Identity shared between Y57 and published STLPyV genomes Identity (%)
Country
Source
WD972
98
USA
fecal specimen
MA138
94
Malawi
fecal specimen
11ww
92
USA
HB124
92
China
HB201
92
China
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Name
peri-anal wart
fecal specimen fecal specimen
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Fig S1. Seasonal distribution of human polyomavirus in Chinese children with chronic tonsillar disease. WUPyV: WU
polyomavirus;
KIPyV:
KI
polyomavirus;
MCPyV:
Merkel
cell
polyomavirus;
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spinulosa-associated polyomavirus; MWPyV: MW polyomavirus; STLPyV: STL polyomavirus.
TSPyV:
trichodysplasia