Detection of TS polyomavirus DNA in tonsillar tissues of children and adults: Evidence for site of viral latency

Journal of Clinical Virology 59 (2014) 55–58

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Short Communication

Detection of TS polyomavirus DNA in tonsillar tissues of children and adults: Evidence for site of viral latency Mohammadreza Sadeghi a,∗ , Leena-Maija Aaltonen b , Lea Hedman a,c , Tingting Chen a , Maria Söderlund-Venermo a , Klaus Hedman a,c a

Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Central Hospital, Helsinki, Finland c Department of Virology and Immunology, Helsinki University Central Hospital Laboratory Division, Helsinki, Finland b

a r t i c l e

i n f o

Article history: Received 2 September 2013 Received in revised form 25 October 2013 Accepted 18 November 2013 Keywords: TSPyV qPCR Serology Tonsil RNase P gene Persistence

a b s t r a c t Background: The trichodysplasia spinulosa-associated polyomavirus (TSPyV), a recently discovered species of the family Polyomaviridae, is associated with development of trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised individuals. The viral seroprevalence in the general population is ∼70%, with little known of its route of transmission, latency, or primary infection site. Objectives: We aimed to determine whether the viral DNA is detectable in tonsillar tissue of constitutionally healthy individuals, and what the corresponding antiviral seroreactivities are. Study design: We tested 229 matched pairs of tonsillar tissue biopsies and serum samples from asymptomatic donors for TSPyV DNA by real-time quantitative PCR with primer pairs and Taq-Man probes targeting the VP1 and LT genes. The sera were studied by enzyme immunoassay (EIA) for TSPyV-VP1-IgG and the PCR-positive individuals also for -IgM and -IgG-avidity. Results: TSPyV DNA was detectable in 8 (3.5%) of 229 tonsillar tissues, and in none of the corresponding sera. TSPyV IgG seroprevalence among children was 39% and among adults 70%. Each of the 8 PCR-positive subjects had antiviral IgG of high avidity but not IgM. Conclusions: TSPyV PCR positivity of tonsillar samples of individuals with long-term immunity provides the first evidence of TSPyV in tonsils and suggests lymphoid tissue as a latency site of this emerging human pathogen. © 2013 Elsevier B.V. All rights reserved.

1. Background The human polyomavirus (HPyV) family today tentatively comprises 12 members [1–13], the infections by which are widespread and initially apparently asymptomatic [14]. The eighth HPyV discovered is the TS polyomavirus (TSPyV), infecting approximately 70% of the world’s population [15–17]. Primary infection generally occurs during childhood, followed by the development of antiviral antibodies [17,18]. Vigorous replication of the virus in the inner root sheath cells of hair follicles accompanies trichodysplasia spinulosa (TS) [7], a rare skin disease of severely immunocompromised hosts including organ transplant recipients [19,20]. Data for TSPyV transmission and prevalence are scarce. TSPyV occurrence in TS lesions at striking abundance, high prevalence, and

specific VP1 expression, imply that TSPyV replication is linked to TS pathogenesis [7,19,20]. The viral DNA has also been encountered at low prevalence and very low copy number in plucked eyebrows [7], skin swabs [19], and nasopharyngeal [21] as well as faecal and urine samples from asymptomatic individuals [8,21]. HPyVs in general establish persistent infections and undergo periodic reactivations, causing disease in susceptible hosts [22]. Evidence suggests that the lymphoid system plays a role in polyomavirus infections and persistence [23–26]. The previously identified HPyVs BKV and JCV or their DNA have occurred in tonsillar tissue in children, and JCV DNA also in the spleen and lymph nodes. We and others [24,25,27–31] have reported MCPyV DNA in tonsillar tissue (Table 1), suggesting lifelong persistence in lymphoid tissue or mucosa [28]. 2. Objectives

Abbreviations: HPyV, human polyomavirus family; TSPyV, trichodysplasia spinulosa-associated polyomavirus; TS, trichodysplasia spinulosa; VLP, virus like particle; qPCR, quantitative PCR. ∗ Corresponding author. Tel.: +358 9 1912 6744; fax: +358 9 1912 6491. E-mail address: reza.sadeghi@helsinki.fi (M. Sadeghi). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.11.008

Despite the ubiquitous distribution of TSPyV, the incidence of TS is very low, and the cases that occur usually have underlying immunosuppression. Our objective was to determine whether TSPyV can infect the lymphoid tissue, as do the BK, JC, KI, WU,

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Table 1 Prevalences of PyV DNA in various cohorts with tonsillar tissue analysed by PCR-based methods. HPyV JCPyV BKPyV KIPyV WUPyV MCPyV TSPyV HPyV6 HPyV7 HPyV9 HPyV10 STLPyV HPyV12

No. investigated samples

Prevalence (%)

Method

References

Country

70 children and adults 32 donors 91 children and adults 80 children 50 children 91 children and adults 29 children and adults 91 children and adults 229 children and adults 50 children 229 children and adults ND ND ND ND ND ND ND

27 (39%) 14 (44%) 5 (5.5%) 4 (5.0%) 3 (6%) 11 (12%) 2 (6.9%) 4 (4.4%) 5 (2.2) 6 (12%) 8 ND ND ND ND ND ND ND

PCR PCR PCR qPCR qPCR PCR qPCR PCR PCR qPCR PCR ND ND ND ND ND ND ND

Monaco et al. [26] Kato et al. [24] Babakir et al. [29] Comar et al. [30] Comar et al. [31] Babakir et al. [29] Astegiano et al. [32] Babakir et al. [29] Kantola et al. [28] Comar et al. [31] Kantola et al. [28] ND ND ND ND ND ND ND

USA Japan Italy Italy Italy Italy Italy Italy Finland Italy Finland ND ND ND ND ND ND ND

and MC polyomaviruses [24–26,28–32]. To this end, we screened archived tonsillar tissue and the corresponding sera from our recent study for TSPyV DNA by qualitative real-time PCR [28]. We also quantified the single-copy gene RNase P, such that the DNA copy numbers could be normalised to cell numbers. We determined the sensitivity, specificity, accuracy, and reproducibility of these assays and also studied the corresponding sera for TSPyV-specific antibodies, by newly established virus-like particle (VLP)-based assays (Chen et al., EID, in revision) [16,17].

3. Study design 3.1. Patients, tissues and sera The clinical material comprised matched pairs of tonsillar tissue and sera from 229 subjects: 80 asymptomatic children and 149 adults (Table 2). Paediatric donors ranged in age from 1.5 to 15 years (average, 7.5), and adult donors from 16 to 72 (average, 30.6). Of the specimens tested, 105 were from male and 124 from female subjects. The tonsillar tissues were from tonsillectomies mostly for chronic tonsillitis or tonsillar hypertrophy.

3.2. DNA extraction from tonsillar tissues and sera Total nucleic acid extraction from the tonsillar tissues was done by the DNA Mini kit (Qiagen, Crawley, UK) according to manufacturer’s instructions, and from the sera by lysis buffer/proteinase K treatment and phenol–chloroform extraction [16,28,33].

Table 2 The tonsillar tissues in this study were obtained during tonsillectomy mostly due to chronic tonsillitis.

1 2 3 4 5 6 7 8 9 10

Diagnosis

No. samples tested

Hyperplastic palatine tonsils Chronic tonsillitis Acute tonsillitis of unknown causes Sleep apnea Peritonsillar abscess Hyperplastic palatine and adenoid tonsils Oral breathing Foul breathe Chronic pansinuitis Unknown

43 108 9 2 19 25 8 1 3 11

Total

229

3.3. DNA screening Samples were first screened for the single-copy human RNase P gene by real-time quantitative PCR (qPCR). The plasmid containing this gene was a generous gift from Dr Janet S. Butel (Baylor College of Medicine, Houston, Texas, USA) [34]. Each sample was subjected to Taq Man PCR for the control RNase P gene using as forward primer 5 -GAGGGAAGCTCATCAGTGGGG-3 , corresponding to nt 9–29, as reverse primer 5 -CTTGGGAAGGTCTGAGACTAGGG3 , corresponding to nt 70–92, and fluorogenic probe 5 FAM-AGTGCGTCCTGTCACTCCACTC-TAMRA-3 corresponding to nt 40–61 of Homo sapiens ribonuclease P RNA component H1 (RPPH1), RNase P RNA (GenBank accession no. NR 002312.1). Standard curves were obtained for the RNase P gene plasmid with serial 10-fold dilutions, and copy numbers ranging from 108 to 100 . PCR amplification reactions were set up in a reaction volume of 17 ␮l using 10 ␮l TaqMan universal PCR master mix (Applied Biosystems), and amplifications were performed with the Stratagene Mx3005p (Stratagene, Foster City, CA, USA). Reactions were considered positive if >10 viral genome copies/reaction were detectable. This strategy would detected potential PCR inhibitors in the DNA preparations and determined the human cell equivalents in each DNA sample, as well as normalised TSPyV viral loads to human cell numbers. 3.4. TSPyV PCR For TSPyV detection, qPCR was performed by previously described LT3 primers and probe [7,16]. All samples were reanalysed with the VP1 PCR primer set [7], and positive samples were sequenced. 3.5. TSPyV serology TSPyV IgG antibodies in 197 serum samples available from the 229 patients were measured by in-house EIA based on VP1 VLPs as described [17]. The PCR-positive individuals were also tested for IgM by ␮-capture EIA and for IgG avidity by the corresponding EIA (Chen et al., EID, in revision). 4. Results 4.1. DNA recoveries DNA was extracted from the specimens and screened for suitability for qPCR analysis by amplification of the cellular RNase P

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qPCR Posive (n=0)

PCR sera (n=229) Serology sera (n=179) EIA Seroposive (n=114) Posive children (39%) Posive adults (70%)

Tonsillectomy paents (n=229) Madian age: 21.0 y Age range: 1.5 -72.0 y

qPCR,PCR Posive (n=8) Posive children (2.2%) Posive adults (1.3%)

Tonsillar ssue biopsies (n=229)

Fig. 1. The novel TS polyomaviruses were detected in 3.5% of the tonsil biopsies of the children and adults with chronic tonsillitis or tonsillar hypertrophy. The TSPyV IgG seroprevalence was 58%.

Table 3 Summary of clinicopathological data and results of quantitative PCR, DNA load, and PCR in TSPyV DNA-positive cases. Case

1 2 3 4 5 6 7 8

Clinical information

Quantitative PCR

PCR

Type

Age (years)

Sex

TSPyV Lt1

TSPyV lt1 DNA load (copies per cell)

TSPyV VP1

Hyperplastic palatine and adenoid tonsils Hyperplastic palatine tonsils Chronic tonsillitis Hyperplastic palatine and adenoid tonsils Chronic tonsillitis Chronic tonsillitis Hyperplastic palatine and adenoid tonsils Hyperplastic palatine tonsils

6 8 5 8 17 32 3 44

F M M M F F F F

+ + + + + + + +

0.85 0.91 0.48 0.9 0.75 0.32 0.44 0.61

+ + + + + + + +

gene. Total DNA yields ranged from 3.7 × 104 to 3.4 × 106 cell equivalents for the tonsillar tissues. 4.2. TSPyV detection from tonsils and sera We tested 229 matched sample pairs (tissues and sera) for TSPyV DNA by qPCR (Fig. 1). The viral DNA was detected in 8 (3.5%) of the tonsillar biopsies from 3 children or adults with chronic tonsillitis and 5 with tonsillar hypertrophy (Table 3). The average age of the PCR-positive subjects was 15.4 years (range 3–44) (Table 3). In sharp contrast, no TSPyV DNA was detectable in the 179 serum samples available from the 229 donors (Fig. 1). Tonsillar tissue contained on average 0.65 (range 0.32–0.91) TSPyV genome copies per cell (Table 3). TSPyV positivity was confirmed by conventional PCR and sequence analysis. The PCR amplicons showed identical sequences, and 100% similarity with the three TSPyV reference sequences available in Genbank (Accession no. NC014361, GU989205, and JQ723730). Of note, two of the cases positive for TSPyV were co-infected; one with KIPyV and the other with WUPyV as determined earlier [28] (Table 3). 4.3. TSPyV antibodies From the 229 subjects we examined by VLP-EIA 77 serum samples from children and 120 from adults for TSPyV IgG antibodies. The TSPyV IgG seroprevalence among children was 39%, and among adults 70%. All of the 8 TSPyV PCR-positive individuals were TSPyV IgG positive and IgM negative, and showed high avidity of TSPyVIgG (values from 32.0 to 71.4%; mean, 53.4% ± 12.8%).

Coinfection

KIPyV WUPyV – – – – – –

polyomaviruses BKV and JCV, the main transmission route is thought to be respiratory [23–26]. We screened tonsillar tissues and sera for the presence of TSPyV DNA, finding that the viral genomes were present exclusively in the tonsillar samples. High loads of TSPyV DNA in lesional samples are significantly associated with TS disease [19]. Furthermore TSPyV DNA has been found at low prevalence and copy number in nasopharyngeal [21,35] and stool samples [8,21], kidney biopsies [36], and urine [8]. It is however not known how these asymptomatic individuals have acquired the TSPyV infection. TSPyV reactivation mechanisms also remain to be determined, although immunosuppression seems to play a major role. We found no TSPyV DNA in serum, in agreement with recent findings [8,16] that plasma or whole blood also from immunocompromised patients lack this virus. The currently examined serum samples have been found PCR-negative also for KIPyV, WUPyV, and MCPyV [28]. Seroepidemiological studies indicate that TSPyV is ubiquitous [15–17]. Furthermore, we have observed the TSPyV VP1 seroprevalence to increase from 34% to 48% at 6–10 years of age to 70% in adulthood, and to be 67% among the elderly [16,17]. The present TSPyV antibody results concord with the existing data. The 8 subjects carrying TSPyV DNA in tonsils had antiviral IgG of high avidity but not IgM, whereby tonsillar tissue appears to be a latency site for this virus. Obviously, such data by no means exclude respiratory transmission of this virus, a possibility that deserves exploration. In conclusion, based on presence of the newly found TSPyV in human tonsils, we suggest that lymphoid tissue is a persistence site for TSPyV. Thereby, shedding from this site during reactivation may play a role in TSPyV transmission.

5. Discussion Funding For TSPyV, as for the other newly discovered human polyomaviruses, little is known of the transmission route(s), tissue tropism, and latency site(s). For the two long-known

This study was supported by Helsinki University Central Hospital Research & Education and Research & Development funds, the

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Sigrid Jusélius Foundation, the Academy of Finland, the Helsinki University funds, and the Medical Society of Finland (FLS). Competing interests None declared. Ethical approval The study was approved by the Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (Dnro 450/13/03/04/08 HUS). Acknowledgments We wish to thank Dr. Janet S. Butel from Baylor College of Medicine, Houston, Texas (USA) for providing the RNase P gene plasmids. We thank Dr. Maria Perdomo for reading the manuscript. M.S. expresses his gratitude to the Ministry of Science, Research and Technology of Iran for a research scholarship as well as to Bu-Ali Sina University, Hamedan for the opportunity to advanced studies. References [1] Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet 1971;1:1257–60. [2] Gardner SD, Field AM, Coleman DV, Hulme B. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1971;1: 1253–7. [3] Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G, Persson MA, et al. Identification of a third human polyomavirus. J Virol 2007;81:4130–6. [4] Gaynor AM, Nissen MD, Whiley DM, Mackay IM, Lambert SB, Wu G, et al. Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS Pathog 2007;3:e64. [5] Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008;319:1096–100. [6] Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB. Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin. Cell Host Microbe 2010;7:509–15. [7] van der Meijden E, Janssens RW, Lauber C, Bouwes Bavinck JN, Gorbalenya AE, Feltkamp MC. Discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient. PLoS Pathog 2010;6:e1001024. [8] Scuda N, Hofmann J, Calvignac-Spencer S, Ruprecht K, Liman P, Kuhn J, et al. A novel human polyomavirus closely related to the African green monkeyderived lymphotropic polyomavirus. J Virol 2011;85:4586–90. [9] Siebrasse EA, Reyes A, Lim ES, Zhao G, Mkakosya RS, Manary MJ, et al. Identification of MW polyomavirus, a novel polyomavirus in human stool. J Virol 2012;86:10321–6. [10] Buck CB, Phan GQ, Raiji MT, Murphy PM, McDermott DH, McBride AA. Complete genome sequence of a tenth human polyomavirus. J Virol 2012;86: 10887–912. [11] Yu G, Greninger AL, Isa P, Phan TG, Martinez MA, de la Luz Sanchez M, et al. Discovery of a novel polyomavirus in acute diarrheal samples from children. PLoS One 2012;7:e49449. [12] Lim ES, Reyes A, Antonio M, Saha D, Ikumapayi UN, Adeyemi M, et al. Discovery of STL polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique T antigen by alternative splicing. Virology 2013;436:295–303. [13] Korup S, Rietscher J, Calvignac-Spencer S, Trusch F, Hofmann J, Moens U, et al. Identification of a novel human polyomavirus in organs of the gastrointestinal tract. PLoS One 2013;8:e58021. [14] Dalianis T, Hirsch HH. Human polyomaviruses in disease and cancer. Virology 2013;437:63–72.

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