Recent Developments in Studying Human Polyomaviruses

Journal of General Virology (2013), 94, 482–496 DOI 10.1099/vir.0.048462-0

Review From Stockholm to Malawi: recent developments in studying human polyomaviruses

Mariet C. W. Feltkamp,1 Siamaque Kazem,1 Els van der Meijden,1 Chris Lauber1 and Alexander E. Gorbalenya1,2

Correspondence 1Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands Mariet Feltkamp 2Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, [email protected] 119899 Moscow, Russia

Until a few years ago the polyomavirus family (Polyomaviridae) included a dozen viruses identified in avian and mammalian hosts. Two of these, the JC and BK-polyomaviruses isolated a long time ago, are known to infect humans and cause severe illness in immunocompromised hosts. Since 2007 an unprecedented number of eight novel polyomaviruses were discovered in humans. Among them are the KI- and WU-polyomaviruses identified in respiratory samples, the Merkel cell polyomavirus found in skin carcinomas and the polyomavirus associated with trichodysplasia spinulosa, a skin disease of transplant patients. Another four novel human polyomaviruses were identified, HPyV6, HPyV7, HPyV9 and the Malawi polyomavirus, so far not associated with any disease. In the same period several novel mammalian polyomaviruses were described. This review summarizes the recent developments in studying the novel human polyomaviruses, and touches upon several aspects of polyomavirus virology, pathogenicity, epidemiology and phylogeny.

Introduction polyomaviruses were identified in rodents, cattle and birds, but not in humans until this century. The polyomaviruses have been recognized as a separate virus family (Polyomaviridae) consisting of several species From 2005 on, eight novel human polyomaviruses (HPyV) since 1999. Before that time they formed the genus were identified. Two of those are involved in disease Polyomavirus in the family Papovaviridae that contained development in immunosuppressed and/or elderly patients, the papillomaviruses, polyomaviruses and the simian and therefore seem to fit the ‘opportunistic’ polyomavirus vacuolating agent 40 (SV40). Nowadays, the latter virus profile characterized by symptomatic reactivations after a forms the namesake species that is designated the period of persistent latent infection. For the other novel polyomavirus type species (prototype) as listed in the HPyVs pathogenicity is still unknown. In this review, the ICTV 9th Report (Norkin et al., 2012). novel HPyVs will be discussed with respect to virological and pathogenic properties as far as they are known, addressing The first members of the polyomavirus family, mouse some epidemiological and phylogenetic aspects of poly- polyomavirus (MPyV) and SV40, were identified halfway omavirus in general. through the last century as filterable agents that caused tumours in newborn mice and hamsters (Eddy et al., 1962; Fig. 1 shows a timeline of polyomavirus (putative) species Girardi et al., 1962; Gross, 1953). SV40 contaminated many identifications at the time of writing (October 2012), animal lots of human poliovirus vaccine produced in monkey and human. A list of polyomaviruses that prototype the kidney cells. This raised a serious concern, but so far SV40 (tentative) namesake species, name abbreviations and has never been shown to be oncogenic in humans (Strickler RefSeq/GenBank numbers of genome sequences are shown et al., 1998). The first two human polyomaviruses were in Table 1. In this review, the polyomaviruses will be termed discovered in 1971. The JC-polyomavirus (JCPyV) was according to their species names and abbreviations men- identified in a brain tissue extract from a progressive tioned in the pending 2011 ICTV taxonomy proposal multifocal leukoencephalopathy (PML) patient that bore the regarding the family Polyomaviridae, as presented in a recent initials J. C. (Padgett et al., 1971). The first BK-polyomavirus publication on the subject (Johne et al., 2011). The (BKPyV) reported was isolated from the urine of a phylogenic tree composed by Johne, Norkin and collabora- nephropathic kidney transplant patient B. K. (Gardner tors is shown in Fig. 2. The most recent polyomavirus et al., 1971). Both JCPyV and BKPyV were not oncogenic discoveries are not mentioned in the pending ICTV in humans despite being closely related phylogenetically proposal. These will be termed according to their pro- with SV40 (Johne et al., 2011). Subsequently, more visional names given in their first publication, respectively.

482 048462 G 2013 SGM Printed in Great Britain Review of the new human polyomaviruses

TSPyV HPyV6 HPyV7 HPyV9 OraPyV2 MasPyV OraPyV1 MWPyV MCPyV WUPyV FPyV GHPyV 2010 LPyV 2011 BKPyV 2012 2009

SA12 2008 2006 2007

MPyV 2000 1979 1971 1963 1950 1954 2012 1953 1960 1967 1971 MptV SV40 1976 HaPyV 1981 2012 2006 2007

JCPyV 2008 2009

BPyV 2011 APyV CPyV 2010 KIPyV EPyV SqPyV GggPyV1 BatPyV PtvPyV CaPyV ChPyV SLPyV

Fig. 1. A time line that indicates 32 (putative) polyomavirus species discovered at the time of writing (October 2012). The year of discovery refers to the first description of the virus, not necessarily to the year that the full genome DNA sequence was obtained. The virus name abbreviations are explained in Table 1 where also the citations to the first descriptions can be found.

New human polyomaviruses and methods of approach were published in a previous virus discovery paper discovery describing the identification of the human bocavirus (Allander et al., 2005). With the steady advancement of nucleic acid amplification and detection techniques, the need for cytopathic changes For the identification of WUPyV a ‘shotgun sequencing’ observed in cell culture to detect the presence of a virus was strategy was used on a single nasopharyngeal aspirate that bypassed. Recent versions of these molecular techniques proved negative for 17 known viral respiratory pathogens allow sensitive and high-throughput analyses of large in diagnostic PCR. Total nucleic acid isolated from the numbers of clinical samples that led to a revolution in virus sample was randomly amplified and PCR products were discovery. Nucleic acid isolated from both diseased and cloned and sequenced. The reads were analysed in a healthy tissues can be used as ‘a template’ for such techniques comparable fashion to the above, which revealed several and therefore the identified viruses are not necessarily sequences that displayed significant homology with JCPyV pathogenic. These techniques are frequently combined with and BKPyV (Gaynor et al., 2007). Subsequent analyses of strategies to enrich the original sample for viral DNA or the compiled WUPyV genome showed highest sequence RNA, by reducing the content of host genomic DNA. identity with the KI-polyomavirus just isolated from respiratory samples, described above. KI-polyomavirus (KIPyV) and WU-polyomavirus (WUPyV) Merkel cell polyomavirus (MCPyV) In 2007 almost coincidentally, two novel HPyV were Exciting and worrisome at the same time has been the reported, the Karolinska Institute polyomavirus (KIPyV) discovery of the MCPyV, which finally proved assumptions identified in Stockholm, Sweden and the Washington of polyomavirus involvement in human oncogenesis true. University polyomavirus (WUPyV) identified in St Louis, Merkel cell carcinomas (MCC) are rare but aggressive USA. Both were discovered in respiratory tract samples from cutaneous tumours of neuroendocrine origin that typically individuals with (acute) respiratory tract infections, but by develop in the elderly and immunocompromised patients. totally different methods. Using a technique called digital transcriptome subtraction KIPyV was identified by pooling centrifuged and filtered (Feng et al., 2007), MCPyV was identified in MCC (Feng supernatants from randomly selected DNase-treated naso- et al., 2008). This technique compares (subtracts) cDNA pharyngeal aspirates, from which DNA and RNA were libraries generated with random RT-PCR from diseased extracted (Allander et al., 2007). The nucleic acids served as a and healthy tissues and thereby identifies mRNA tran- template for random-PCR, and the products were separated scripts potentially unique for either or both. In this case by gel electrophoresis, cloned and sequenced. This resulted with the help of sophisticated software, unique sequences in several sequence reads that were subsequently trimmed, were further analysed and some of them displayed clustered and sorted using dedicated software. Finally, BLAST homology to the monkey B-lymphotrophic polyomavirus searches were performed to look for similarities with known (LPyV) and BKPyV. Through primer-genome-walking the (viral) sequences listed in GenBank. Details of the technical complete genome was sequenced. New to HPyVs, but http://vir.sgmjournals.org 483 M. C. W. Feltkamp and others

Table 1. List of 32 polyomavirus names identified up to October 2012, which prototype putative species

Host Polyomavirus (putative) species name First publication* RefSeq/GenBank accession no.D

Human JC polyomavirus (JCPyV) Padgett et al. (1971) NC_001699 BK polyomavirus (BKPyV) Gardner et al. (1971) NC_001538 KI polyomavirus (KIPyV) Allander et al. (2007) NC_009238 WU Polyomavirus (WUPyV) Gaynor et al. (2007) NC_009539 Merkel cell polyomavirus (MCPyV) Feng et al. (2008) NC_010277 Trichodysplasia spinulosa-associated van der Meijden et al. (2010) NC_014361 polyomavirus (TSPyV) Human polyomavirus 6 (HPyV6) Schowalter et al. (2010) NC_014406 Human polyomavirus 7 (HPyV7) Schowalter et al. (2010) NC_014407 Human polyomavirus 9 (HPyV9) Scuda et al. (2011) NC_015150 Malawi polyomavirus (MWPyV) Siebrasse et al. (2012) JQ898291 Ape Bornean orangutan polyomavirus Groenewoud et al. (2010) NC_013439 (OraPyV1) Sumatran orangutan polyomavirus Groenewoud et al. (2010) FN356901 (OraPyV2) Chimpanzee polyomavirus (ChPyV) Deuzing et al. (2010) NC_014743 Gorilla gorilla gorilla polyomavirus Leendertz et al. (2011) HQ385752 (GggPyV1) Pan troglodytes verus polyomavirus Leendertz et al. (2011) HQ385746 (PtvPyV) Monkey Simian virus 40 (SV40) Eddy et al. (1962) NC_001669 Baboon polyomavirus 1 (SA12) Malherbe & Harwin (1963) NC_007611 B-lymphotropic polyomavirus (LPyV) zur Hausen & Gissmann (1979) NC_004763 Squirrel monkey polyomavirus (SqPyV) Verschoor et al. (2008) NC_009951 Rodent Murine pneumotropic virus (MptV) Kilham & Murphy (1953) NC_001505 Murine polyomavirus (MPyV) Gross (1953) NC_001515 Hamster polyomavirus (HaPyV) Graffi et al. (1967) NC_001663 Bat polyomavirus (BatPyV) Misra et al. (2009) NC_011310 Mastomys polyomavirus (MasPyV) Orba et al. (2011) AB588640 Mammal Bovine polyomavirus (BPyV) Reissig et al. (1976) NC_001442 (other) California sea lion polyomavirus (SLPyV) Colegrove et al. (2010) NC_013796 Equine polyomavirus (EPyV) Renshaw et al. (2012) JQ412134 Bird Avian polyomavirus (APyV) Bozeman et al. (1981) NC_004764 Goose hemorrhagic polyomavirus (GHPyV) Guerin et al. (2000) NC_004800 Finch polyomavirus (FPyV) Johne et al. (2006) NC_007923 Crow polyomavirus (CPyV) Johne et al. (2006) NC_007922 Canary polyomavirus isolate (CaPyV) Halami et al. (2010) GU345044

*Year of first description, not necessarily the year of first complete genome sequencing. DGenome sequence according RefSeq/GenBank number can be found in http://www.ncbi.nlm.nih.gov/RefSeq/ known for oncogenic animal polyomaviruses detected in amplification (RCA) (Johne et al., 2009). This technique tumours, the MCPyV genome was found integrated into exploits random hexamer primers and a DNA polymerase the host genome (Feng et al., 2008). (Phi-29) with proof-reading and strand-displacement properties. If a sample contains a relative excess of a particular small circular dsDNA molecule, for instance a plasmid or a Trichodysplasia spinulosa-associated polyomavirus (small) viral genome, RCA will produce long, high-molecular (TSPyV) DNA molecules with concatenated (linearized) stretches of Trichodysplasia spinulosa is a rare follicular skin disease of DNA of potential interest. These stretches will appear as immunocompromised patients that will be described further sharp bands on gel when the RCA-product is cut with below. In 1999, Haycox and coworkers proposed a viral specific restriction enzymes and can be cloned and sequenced origin, because affected hair follicle cells contained clusters of subsequently. This approach already proved successful in the virus particles (Haycox et al., 1999). It took until 2010 before past to identify a number of animal papillomaviruses and the identity of this virus was uncovered (van der Meijden polyomaviruses and finally revealed the genome of TSPyV et al., 2010), with the help of a technique called rolling-circle (Johne et al., 2009; van der Meijden et al.,2010).

484 Journal of General Virology 94 Review of the new human polyomaviruses

Wukipolyomavirus WUPyV Avipolyomavirus KIPyV HPyV6

HPyV7 GHPyV CPyV CaPyV

FPyV

APyV

BKPyV MCPyV SA12 OraPyV2 MPyV JCPyV HaPyV SV40 LPyV

TSPyV OraPyV1

SLPyV BPyV SqPyV Fig. 2. Phylogenetic tree of 25 polyomaviruses Orthopolyomavirus based on whole genomic nucleotide sequences, BatPyV MPtV as published before by Johne, Norkin and others 0.2 (Johne et al.,2011).

Human polyomaviruses 6 and 7 (HPyV6 and HPyV7) and HPyV7, as well as many human papillomaviruses. In order to detect free, non-integrated, MCPyV genomes Some contigs showed the highest homology with LPyV on the skin, RCA was performed on forehead swab samples and after filling the contig sequence gaps by designing of healthy individuals. With some adjustments to increase additional primers, a virus genome was found that RCA sensitivity, such as DNA exonuclease treatment to coincided with HPyV9 (Sauvage et al., 2011). digest cellular (linear) DNA, this approach revealed the identity of a new polyomavirus, HPyV6, on the skin of a healthy individual (Schowalter et al., 2010). With the use of Malawi polyomavirus (MWPyV/HPyV10) a new degenerate PCR primer set to detect HPyV6- and Recently, another novel human polyomavirus was iden- WUPyV-like sequences, an additional novel polyomavirus tified in stool of a 1-year-old healthy child from Malawi was found in these healthy skin samples that was called (Siebrasse et al., 2012). This virus was discovered by HPyV7 (Schowalter et al., 2010). suspending a small amount of faeces that subsequently was centrifuged and filtered through 0.45 and 0.22 mm pores. After chloroform extraction-free-DNA was removed by Human polyomaviruses 9 (HPyV9) DNase treatment. The solution that still contained Several serological studies already showed the presence of encapsidated DNA was SDS and Proteinase K-treated, ‘cross-reactive’ antibodies in a percentage of human sera and DNA was isolated. Finally, this DNA was subjected to that recognized LPyV (Kean et al., 2009; Viscidi & RCA and pyrosequencing. Hundreds of reads were Clayman, 2006), which was isolated from an African obtained, sequenced and aligned to sequences within the green monkey (Pawlita et al.,1985).Furthermore,LPyV- GenBank database. This revealed several polyomavirus-like derived sequences were obtained from human blood, reads and with the use of a primer walking strategy the indicating the occurrence of LPyV(-like) infections in complete MWPyV genome was fulfilled (Siebrasse et al., humans (Delbue et al.,2008).In2011,anovel 2012). Very recently an almost identical virus called polyomavirus which closely resembled LPyV was iden- HPyV10 was identified in an anal wart of an immuno- tified independently by two research groups in human compromised patient with WHIM syndrome with the help serum and skin swabs, respectively, and tentatively called of RCA (Buck et al., 2012). MWPyV and HPyV10 probably HPyV9. For screening of the serum samples a degenerate represent a single species. polyomavirus PCR primer set was used (Leendertz et al., 2011), which generated an unknown, putative HPyV Genome and (putative) proteins sequence next to many known other HPyV sequences. With primer-walking the rest of the HPyV9 sequence was Genome organization and transcription uncovered (Scuda et al., 2011). In the other report, DNA Viruses of the family Polyomaviridae, including the recently isolated from healthy skin swabs was subjected to high- identified human viruses, are among the smallest known to throughput sequencing, which resulted in millions of infect humans, in relation to both particle size and genome reads and several contigs that matched MCPyV, HPyV6 length (Imperiale & Major, 2007). The 45 nm non-enveloped http://vir.sgmjournals.org 485 M. C. W. Feltkamp and others

Ori agnoprotein and VP4 expressed by JCPyV, BKPyV and several animal polyomaviruses including birds seem not to NCCR be encoded by the novel HPyVs (Johne & Mu¨ller, 2007), Agno but for most of the novel viruses the transcription-patterns Small T VP2 have not been revealed yet. In addition to a number of transcripts coding for the complete small and large T- antigens, MCPyV and SV40 may use a 17kT-like transcript 1 called T4 or 57kT that directs the synthesis of large T- antigen with a large central splice (Shuda et al., 2008; VP3 Zerrahn et al., 1993). 4000 1000 HPyV Besides transcription regulation by the NCCR, T-antigen ~5000 bp expression was shown to be modulated post-transcription- ally by microRNAs in SV40 (Sullivan et al., 2005). For Large T 3000 2000 JCPyV and BKPyV a viral microRNA was described that targets the stress-induced ligand ULBP3 and thereby inhibits (antiviral) NK T-cell activity (Bauman et al., 2011). For VP1 MCPyV, a microRNA was detected that potentially regulates early gene expression (Seo et al., 2009). Moreover in MCC, MCPyV-encoded microRNAs were identified that potentially regulate T- and B-cell receptor signalling hampering viral (tumour) immune recognition (Lee et al., 2011).

Fig. 3. Schematic representation of a human polyomavirus genome made up of circular dsDNA. Regions that code for the T-antigens indicated gene products are shown in colour. The agnoprotein- The T-antigens play essential roles in viral transcription encoding gene is not found in every HPyV genome. and replication, as well as in host cell tuning to enable virus replication (Imperiale & Major, 2007; Sullivan & Pipas, 2002). As polyomaviruses are completely dependent on the host cell replication machinery, a significant part of this virion harbours a circular dsDNA genome of approximately tuning is aimed at inducing S-phase and bypassing cell 5000 bp that is divided into three regions, the non-coding cycle-control measures. In some cases, these features can control region (NCCR) and the early and late coding regions result in uncontrolled cell growth and eventually tumour (Fig. 3). formation. So far, MCPyV is the only (new) HPyV for The NCCR contains the origin of replication flanked which these phenomena have been noted (Chang & Moore, by several large T-antigen-binding sites, as well as the 2012). As a consequence, the T-antigens of MCPyV have transcription promoters and regulatory elements. The been studied already quite extensively (see below, and the novel human polyomaviruses display a similar NCCR MCPyV Pathogenicity paragraph), whereas for the other organization. In JCPyV- or BKPyV-diseased individuals new HPyVs this knowledge is almost entirely based on with viral DNA detectable in different body compartments, nucleotide and predicted amino acid sequence properties. NCCRs of these isolates can vary from what is called the The small and large T-antigens share their N terminus, while ‘archetype’ NCCR that is thought to be the NCCR found in having C-terminal regions of different sizes encoded in transmissible virus found in urine. It is believed that the different reading frames that are expressed by alternative NCCR mutations, deletions and repeats increase T-antigen splicing. The novel human polyomaviruses seem no transcription and the replication rate, as was shown for exception to this rule. This shared region occupied by the instance for JCPyV (Gosert et al., 2010). Systematic mutual so-called J-domain contains important motifs for virus analyses of the NCCR regions among different isolates replication and cellular transformation, such as CR1 and within individuals have not been reported yet for the novel DnaJ (Sullivan & Pipas, 2002). Downstream, the small T- human polyomaviruses. antigen contains a PP2A subunit-binding motif probably to The early coding region contains the genes for the small activate the Akt-mTOR pathway related to (tumour) cell and large T-antigens that are transcribed through altern- survival (Andrabi et al., 2011; Buchkovich et al., 2008). The ative splicing. The late coding region found on the opposite large T-antigen further includes a number of functionally strand codes for the VP1, VP2 and VP3 structural capsid important sequence signatures: a nuclear-localization signal, proteins (Fig. 3). At the 39 ends, the early and late regions an origin-binding domain and an ATPase domain-contain- are separated by a small intergenic region. Analogous to ing helicase domain (Arora et al., 2012; Sullivan & Pipas, JCPyV and BKPyV, so far no ORFs have been found in the 2002), as well as a pRB-binding motif and p53-interaction novel human polyomaviruses to indicate the presence of a domain that probably play a role in cellular transformation. middle T-antigen as found in rodent polyomaviruses. The Apart from MCPyV (Shuda et al., 2008), the presence of

486 Journal of General Virology 94 Review of the new human polyomaviruses

(most of) these motifs was identified by bioinformatics in UV-radiation on MCC development, but this has not been TSPyV and HPyV9 (Scuda et al., 2011; van der Meijden et al., confirmed experimentally. In 2008, the presence of MCPyV 2010), but they remain to be characterized functionally. in MCC was shown for the first time (Feng et al., 2008), a finding which has been confirmed by many researchers worldwide showing that at least 75 % of all MCC are VP capsid proteins MCPyV-positive (Arora et al., 2012). The late VP1, VP2 and VP3 structural proteins together Several findings indicate direct involvement of MCPyV make up the 40–45 nm polyomavirus capsid (Baker et al., in Merkel cell carcinogenesis. For instance, in almost all 1989). Five VP1 molecules form a capsomer that interacts cancer cases MCPyV is found linearized and clonally with either a VP2 or VP3 molecule to autoassemble into a integrated in the host cell genome, although the site of capsid composed of 72 VP1 pentamers (Imperiale & integration appears random (Feng et al., 2008; Laude et al., Major, 2007). Similar sequences probably encoding VP1, 2010; Martel-Jantin et al., 2012). Furthermore, the MCPyV VP2 and VP3 have been identified in all of the novel large T-gene was found to carry ‘signature’ mutations polyomaviruses. So far there is no reason to believe that specific for the carcinomas. Their expression results in a the novel viruses will behave differently with respect to truncated large T-protein that lacks the C-terminal helicase capsid formation, although there is debate whether domain, the putative p53-interaction domain and, some- MCPyV VP3 is functional (Pastrana et al.,2009).VP4, times, also the origin-binding domain (Feng et al., 2008; noted for SV40 and expressed very late in infection Sastre-Garau et al., 2009; Shuda et al., 2008). As a probably serving as a porin in the cellular membrane consequence, the function of large T in viral replication facilitating viral release (Daniels et al., 2007; Raghava et al., is selectively impaired. Small T-antigen is unaffected by 2011), has not been reported or predicted for any of the these mutations. So in an apparent parallel to other virus- novel HPyVs. associated cancers, e.g. human papillomavirus (HPV)- How the novel polyomaviruses recognize and infect their associated cervical cancer, also in MCPyV-positive MCC host target cells is largely unknown. Most of the known there seems to be a strong association with non-productive polyomaviruses interact by linkage of the major capsid MCPyV infection. Probably the strongest evidence of protein VP1 with terminal sialic acids (alpha2,3 or pathogenicity (oncogenicity) of MCPyV is provided by alpha2,6) of mainly gangliosides on the host cell membrane studies showing that both large and small T-antigen (Neu et al., 2009), after which they are internalized using expression are indispensable to maintain cellular trans- different entry pathways. For MCPyV, it was recently formation. If T-antigen expression was inhibited experi- shown that this virus uses glycosaminoglycans for initial mentally by short interfering (hairpin) RNAs, MCC cell attachment, whereas later in the encounter sialic acids may lines underwent growth arrest or died (Houben et al., 2010; still be important (Schowalter et al., 2011). Future research Shuda et al., 2011). has to reveal whether the other novel human polyoma- The presence of MCPyV-negative (20–25 %) and MCPyV- viruses use similar strategies for host cell recognition. positive (75–80 %) MCC underscores the existence of at least two genetic forms of this cancer, possibly with a Virus pathogenicity, associated diseases and different prognosis and different demands for treatment. clinical aspects When comparing the prognosis of MCPyV-positive and -negative MCC, there seems to be a trend that MCPyV- For two of the novel HPyVs a causal relationship with a positive ones do better and carry less genomic mutations, for specific clinical condition is highly probable. In both cases the instance in TP53, than the MCPyV-negative ones (Bhatia disease/cancer has lent its name to the virus, trichodysplasia et al., 2010; Sihto et al., 2009, 2011). Serological studies point spinulosa to TSPyV and MCC to MCPyV. For MCPyV, an in the same direction (see below). Other studies, however, additional association with chronic lymphocytic leukaemia is report no difference in clinical outcome between MCPyV- suggested, and for TSPyV an association has been suspected positive and -negative cancers (Hall et al., 2012; Schrama with pilomatricoma. Both will be addressed here as well. For et al., 2011), underscoring the necessity of further study in KIPyV and WUPyV, the evidence for involvement in this regard. Also, the role of MCPyV-directed cellular respiratory tract infections is weak and for the other human immunity in MCC development and progression, as well as polyomaviruses such associations are absent to date. tumour immune-escape needs further clarification (Iyer et al., 2011; Kumar et al., 2011). MCPyV and MCC MCC is a rare aggressive neuro-endocrine tumour of MCPyV and chronic lymphocytic leukaemia epidermal origin with an increasing incidence of approxi- It has been noted for some time that MCC, although mately 0.2–0.5 per 100 000 depending on age, skin type, rare, are more frequently seen in patients with chronic (cumulative) sun exposure and immune status (Schrama lymphocytic leukaemia (CLL) and vice versa, suggesting a et al., 2012; Van Keymeulen et al., 2009). The association common nature (Howard et al., 2006; Kaae et al., 2010; with fair skin and sun exposure suggests a direct effect of Tadmor et al., 2011). Although very few MCC from CLL http://vir.sgmjournals.org 487 M. C. W. Feltkamp and others patients have been analysed, all of them harboured MCPyV Kazem et al., 2012; Matthews et al., 2011; Wanat et al., (Koljonen et al., 2009; Tolstov et al., 2010). Based on this 2012). In a small case-control study, we showed the information and the putative lymphotropism of MCPyV presence of TSPyV DNA in all of the tested trichodysplasia (Toracchio et al., 2010), studies were conducted to look lesions and only in a small number of healthy control for MCPyV in peripheral blood mononuclear cells of samples (P,0.001) (Kazem et al., 2012). TSPyV DNA loads CLL patients. The first study in that regard could detect measured in these lesions were at least 4 logs higher those MCPyV DNA and T-antigen in a small minority of the in healthy skin(-derived) tissues. Furthermore, we showed CLL cases (Shuda et al., 2009; Tolstov et al., 2010). The that TSPyV VP1-antigen was detected only in the affected calculated viral loads, however, were considered too low to hair bulbs, confined to trichohyalin-overexpressing inner suspect a significant contribution of MCPyV to the CLL root sheath cells. Taken together, these data indicate an phenotype. aetiological relationship between active TSPyV infection and trichodysplasia spinulosa. In one patient for whom this Other groups have detected MCPyV DNA in 25–35 % of analysis was performed, a decline in TSPyV load was CLL samples (Imajoh et al., 2012; Pantulu et al., 2010; observed with successful antiviral (cidofovir) treatment of Teman et al., 2011). Also in these cases, the measured viral trichodysplasia spinulosa (van der Meijden et al., 2010). loads were several logs lower than the ~1 copy per cell Whether trichodysplasia spinulosa concerns a symptomatic shown for the MCC, thereby excluding viral involvement reactivation of a persistent TSPyV infection or a symp- in maintenance of the transformed tumour state. tomatic primary TSPyV infection in the window of (very) Nevertheless, a third of the MCPyV-positive samples poor immunity is unclear at the moment. The rarity of the carried a specific large T-antigen mutation potentially condition in the background of the high seroprevalence in blocking the viral helicase activity and suggestive of a role the general population seems to favour the latter for the virus in oncogenesis (Pantulu et al., 2010). possibility, as primary infections at higher age may be Integration of the virus in CLL was not demonstrated, considered a relatively rare event. In both scenarios, next to but fluorescence in situ hybridization patterns of MCPyV- immunity, additional (host) factors are likely to influence positive samples were suggestive in this regard (Haugg the occurrence of clinically relevant TSPyV-infection. et al., 2011). The finding that MCPyV DNA is also present in buffy coats from healthy blood donors further obscures the issue of MCPyV involvement in CLL (Pancaldi et al., TSPyV and pilomatricoma 2011). Because of the tropism of TSPyV for the hair follicle area, and the oncogenic properties of some other polyoma- TSPyV and trichodysplasia spinulosa viruses, a relation was suspected between TSPyV infection and pilomatricoma, a benign skin tumour originating from Trichodysplasia spinulosa is a rare skin disease exclusively proliferating hair-matrix cells. Previously, it was shown found in severely immunocompromised hosts, especially that the murine MPyV induces hair follicle tumours in solid organ transplant recipients (Kazem et al., 2012; mice (Dawe et al., 1987; Freund et al., 1992). Using a rabbit Matthews et al., 2011). It is characterized by gradual antiserum raised against MPyV VP1 (Leavitt et al., 1985), development of papules and spicules (spines) on the face, 40 % of tested human pilomatricomas stained positive sometimes accompanied by alopecia of the eyebrows and (Sanjua´n et al., 2010). Attempts to detect TSPyV in these lashes (Wanat et al., 2012). Worldwide some 30 cases have tumours however failed; both TSPyV DNA and VP1- been described, the first as a cutaneous side effect of antigen could not be detected in ten pilomatricoma cyclosporin-A use (Chastain & Millikan, 2000; Izakovic samples (Kanitakis et al., 2011). So, despite experimental et al., 1995). Haycox and coworkers proposed for the first evidence that shows polyomavirus tropism for the hair time that trichodysplasia spinulosa concerns a viral disease follicle cells, no correlation between TSPyV and piloma- and showed the presence of intranuclear clusters of tricoma could be established. honeycomb-arranged viral particles in the distended and dismorphic hair follicles, especially in proliferating inner root sheath cells that over expressed trichohyalin (Haycox KIPyV/WUPyV and respiratory tract infections et al., 1999). Subsequent reports confirmed the typical Both KIPyV and WUPyV were identified in respiratory histological findings of hyperplastic hair bulbs and the samples. Ever since, several researchers have tried to presence of polyomavirus or papillomavirus-like particles establish an association between upper and/or lower (Lee et al., 2008; Osswald et al., 2007; Sadler et al., 2007; respiratory tract infections and the presence of these Sperling et al., 2004; Wyatt et al., 2005). Attempts to viruses. To summarize, both viruses were regularly detected identify the nature of this virus failed until TSPyV was in respiratory materials (Bialasiewicz et al., 2008), but none identified with the help of RCA in 2010 (van der Meijden of these studies revealed a clear correlation between KIPyV et al., 2010). and WUPyV and respiratory disease (Babakir-Mina et al., To date the presence of TSPyV in trichodysplasia spinulosa 2011). Often KIPyV and WUPyV were detected together lesions has been confirmed in patients from Western with other known (viral) pathogens and not in convin- Europe, North America and Australia (Fischer et al., 2012; cingly high loads to suspect a causal relationship. The

488 Journal of General Virology 94 Review of the new human polyomaviruses influence of host immune status on this possible asso- correlated with progressive disease and a decline with ciation is also not clear at the moment (Csoma et al., 2011; remission. Rao et al., 2011). Polyomavirus persistence and reactivation and HPyV6, HPyV7, HPyV9 and MWPyV/HPyV10 spread of infection So far, for HPyV6, HPyV7 and HPyV9 no disease Persistence and reactivation association was reported or suggested. Future studies It is generally believed that primary infection of human should resolve whether MWPyV/HPyV10 can be consid- polyomaviruses is followed by a persistent, asymptomatic ered a cutaneotropic virus contributing to anal wart (latent) infection with very low levels of replication that development, as suggested in the WHIM patient (Buck remains in the body lifelong. For JCPyV and BKPyV et al., 2012), or rather as a faecally transmitted pathogen or reactivation from the latent state and manifestation of contaminant (Siebrasse et al., 2012). symptomatic infection is observed only in the case of reduced immunity, for instance in long-term immunosuppressed solid organ transplant recipients (BKPyV, nephro- Serology and seroprevalence pathy) and in AIDS patients (JCPyV, PML) (Imperiale & Cross-sectional studies of the known human polyoma- Major, 2007). Nowadays JCPyV-caused PML is increasingly viruses JCPyV and BKPyV have shown that both infections observed in Natalizumab-treated patients, which are not detected via the presence of VP1-directed seroresponses are overall immunocompromised, but carry targeted adhesion prevalent in the general population. Depending on the molecules that prevent T-lymphocyte homing, for instance study, the seroprevalence of JCPyV is approximately 50 % to the brain (Bloomgren et al., 2012; Major, 2010). and BKPyV reaches almost 90 % (Kean et al., 2009). In healthy individuals, the detection rate of JCPyV and Serological studies of the novel human polyomaviruses BKPyV DNA is generally much lower than their seropre- revealed a considerable variation in the presence of serum valence. In urine of healthy blood donors for example, the antibodies (Carter et al., 2009; Chen et al., 2011b; Kantola detection rate of JCPyV and BKPyV DNA was approxi- ´ et al., 2010; Kean et al., 2009; Schowalter et al., 2010; Touze mately 3- and 10-fold lower than the seroprevalence et al., 2010; Trusch et al., 2012; van der Meijden et al., 2011; measured within the same group, respectively (Egli et al., Viscidi et al., 2011). The lowest seroprevalence was 2009). In immunosuppressed solid organ transplant detected for HPyV9 varying between 15 and 45 % (Nicol patients and haematopoietic stem cell recipients, however, et al., 2012; Trusch et al., 2012; E. Van der Meijden and M. BKPyV can be found in urine in the majority of C. W. Feltkamp, unpublished results), the highest for (seropositive) cases, often in very high amounts indicative WUPyV, TSPyV and HPyV6 60–95 % (Chen et al., 2011b; of massive reactivation of infection (Bennett et al., 2012; Kean et al., 2009; Nguyen et al., 2009; Schowalter et al., Hirsch et al., 2009). 2010; van der Meijden et al., 2011). In all serological studies, the strongest increase in seroprevalence is observed For the novel HPyVs the association between diminished at a young age, indicating that primary infections generally immunity and reactivation is less clear. Only for TSPyV, with occur in (early) childhood (Chen et al., 2011a, b; Kean a seroprevalence of 70 % (Chen et al., 2011b; van der Meijden et al., 2009; van der Meijden et al., 2011; Viscidi et al., et al., 2011), are clinical consequences exclusively seen in 2011). So far, the seroprevalences of the novel HPyVs severely immunocompromised hosts (Kazem et al.,2012; measured with VLPs and GST–VP1 fusion proteins appear Matthews et al., 2011). However, the TSPyV DNA detection similar, as was shown for instance for TSPyV (Chen et al., rate, on the skin at least, hardly varies among immuno- 2011b; van der Meijden et al., 2011). competents (2 %) and immunocompromised hosts (4 %) (Kazem et al., 2012; van der Meijden et al., 2010). For For MCPyV, case-control studies were performed to (MCPyV-positive) MCC, a weaker association with immu- investigate the relation between MCPyV-specific serore- nosuppression is found, and also the elderly are at increased sponses and MCC. In all reports, statistically significant risk of developing this tumour, possibly explained by associations were found between the VP1-directed seror- immunosenescence. Also for MCPyV, the DNA detection esponses, measured against VP1–GST-fusion proteins and rates on the skin do not differ much between immunocom- VLPs, and MCC (Carter et al., 2009; Pastrana et al., 2009; petents and immunocompromised, albeit they are generally Touze´ et al., 2011). Furthermore, high-titre MCPyV VP1 much higher than for TSPyV and resemble the seropreva- responses were almost exclusively found in the carcinoma lence of 50 % (Wieland et al., 2011). In general, it should be cases and correlated with better survival (Pastrana et al., noted that both MCC and trichodysplasia spinulosa are very 2009; Touze´ et al., 2011). The best correlation for MCPyV rare conditions implying that (host) factors other than serology and MCC were obtained when the T-antigen is immunity also play a role in controlling infections of these used as the target antigen. Seroresponses against the T- viruses. For KIPyV, WUPyV and HPyV6, HPyV7, HPyV9 antigens are virtually absent in healthy individuals, whereas and MWPyV the relation with immunity is either not known almost half of the MCC patients responded to this antigen or poorly described at the moment. This is largely explained (Paulson et al., 2010). Moreover, a T-antigen titre rise was by the lack of an established clinical condition that could be http://vir.sgmjournals.org 489 M. C. W. Feltkamp and others attributed to one of these novel viruses and the unawareness Scuda et al., 2011), but if this provides a clue regarding of the organ where these viruses latently persist. transmission remains to be seen.

Spread of infection Phylogeny and evolutionary trends: how many From studies of JCPyV and BKPyV it is known that they HPyVs are around? are found in urine, faeces and saliva. The excreted numbers The ten known HPyVs comprise the largest host-specific of virus are so high that these polyomaviruses even serve as subset of the polyomaviruses described so far. This markers of sewage pollution of for instance recreational probably reflects a bias of the virus discovery effort that waters (Korajkic et al., 2011). Which route of virus increasingly focuses on hunting for human viruses, excretion primarily drives human infection is not precisely particularly during the past several years as was detailed known, but the efficiency of infection seems clear from the above. Pairwise divergence between HPyVs varies as much high seroprevalence numbers, for both the known and as in the entire polyomavirus dataset. If measured for the novel human polyomaviruses. combined VP1, VP2 and LT proteins it ranges from 15 Regarding possible routes of excretion and transmission of (for BKPyV/JCPyV pair) to 61 % (BKPyV/HPyV6), or, if cutaneous MCPyV, a recent study showed the presence of repeated substitutions accounted, from 0.17 (BKPyV/ viral DNA on 75 % of samples from environmental surfaces JCPyV) to 1.52 (TSPyV/HPyV6) substitutions per position on average (Fig. 4). Apart from BKPyV/JCPyV, also the (door handles, ticket machines etc.). DNase sample KIPyV/WUPyV and HPyV6/HPyV7 pairs are among the treatment and viral load measurement suggested that most closely related, which correlates with putative shared about 5 % of the detected MCPyV DNA was protected, tropism for the respiratory tract and the skin, respectively. probably encapsidated and therefore potentially infectious (Foulongne et al., 2011). TSPyV DNA has been occasion- In Fig. 5 a novel tentative polyomavirus phylogenetic tree ally detected in urine and kidney tissue (Fischer et al., 2012; is shown. In addition to the Johne tree in which the

KIPyV WUPyV HPyV6 HPyV7 BKPyV JCPyV TSPyV MCPyV HPyV9 MWPyV

KIPyV 00.220.54 0.50 0.54 0.56 0.59 0.58 0.59 0.59 0–0.49

WUPyV 0.2800.53 0.51 0.53 0.55 0.59 0.58 0.60 0.60 0.50–0.99

HPyV6 1.161.140 0.24 0.61 0.60 0.60 0.57 0.60 0.60 1.00–2.00

HPyV7 1.041.060.30 0 0.59 0.59 0.58 0.58 0.59 0.59

BKPyV 1.221.131.55 1.46 0 0.15 0.43 0.47 0.47 0.47

JCPyV 1.301.19 1.48 1.45 0.17 0 0.43 0.48 0.47 0.47

TSPyV 1.411.38 1.52 1.40 0.72 0.73 0 0.38 0.35 0.41

MCPyV 1.361.341.42 1.43 0.85 0.88 0.59 0 0.40 0.48

HPyV9 1.421.471.40 1.45 0.84 0.85 0.51 0.68 0 0.45

MWPyV 1.431.431.51 1.48 0.85 0.84 0.69 0.89 0.81 0

Fig. 4. Genetic comparison between ten known human polyomaviruses. Two measures of genetic distance are shown for the combined polyomavirus protein set comprising VP1, VP2 and LT. The upper-right triangle shows uncorrected dissimilarity percentages (1 corresponds to 100 % dissimilarity; 0 means identical sequences); the lower-left triangle shows evolutionary distances that correct for multiple substitutions at the same site (the scale is mean substitutions per sequence position) under the WAG amino acid substitution model and rate heterogeneity among sites.

490 Journal of General Virology 94 Review of the new human polyomaviruses

Malawipolyomavirus

I MWPyV Avipolyomavirus

HaPyV MPyV OraPyV1TSPyV CaPyV PtvPyVChPyV MCPyV APyV

0.9 FPyV GggPyV OraPyV2 HPyV9 GHPyV 0.5 0.9 0.9 LPyV CPyV 0.7

EPyV II SV40 KIPyV SA12 0.8 WUPyV BKPyV SLPyVSqPyV JCPyV

MasPyV HPyV6 BPyV 0.1 HPyV7 MptV

BatPyV

Orthopolyomavirus Wukipolyomavirus

Fig. 5. Unrooted phylogenetic tree of 32 (putative) polyomavirus species based on LT, VP1 and VP2 protein comparisons. The tree is based on the alignment of concatenated VP1, VP2 and LT amino acid sequences. Regarding viruses found in both datasets, the obtained branching pattern (topology) of basal nodes in the tree matches that proposed by Johne and colleagues (Johne et al., 2011). One distinct clade designated the Avipolyomaviruses contains only the bird PyV types. The Wukipolyomavirus lineage supports four HPyV types (shown in red), the Malawipolyomavirus lineage only one. Other HPyVs are clustered in the proposed Orthopolyomavirus-I and -II lineages (see text). Bar indicates number of substitutions per site. Numbers at branching events represent probability support values ranging from 0 (no support) to 1 (best support). Only probability support values lower than 1 are shown. The full polyomavirus name can be found in Table 1.

polyomavirus genera have been proposed (Fig. 2) (Johne of human polyomaviruses emerged as result of cross-species et al., 2011), the new tree includes seven animal and human jumps of zoonotic viruses. For instance, TSPyV might have viruses, among which HPyV9 and MWPyV/HPyV10, that originated from an ancestral virus of orangutans that represent additional putative polyomavirus species. From harbour the closely related OraPyV1 polyomavirus and Fig. 5 it is clear that the HPyVs do not form a monophyletic MCPyV from a virus of gorillas, host to the related GggPyV cluster. Rather they are distributed unevenly among four out virus (Fig. 5). Since the genetic diversity accommodated by of five distant lineages. Two lineages that include human the genus Wukipolyomavirus is expected to be on a par with polyomaviruses belong to the genus Orthopolyomavirus those in the Orthopolyomavirus-I and -II clusters, the (Johne et al. 2011), and are called Orthopolyomavirus-I and former genus is also likely to include non-human viruses -II, respectively, for the sake of discussion. The third lineage that either are yet to be discovered or have gone extinct. belongs to the genus Wukipolyomavirus and the fourth Alternatively, polyomaviruses might have diverged by comprises MWPyV alone. virus–host co-evolution, which was the most popular model of evolution for these viruses until a few years ago when The Malawipolyomavirus and Wukipolyomavirus lineages novel human polyomaviruses started to come to light with one and four viruses, respectively, are least populated (Shadan & Villarreal, 1995; Pe´rez-Losada et al., 2006). and include exclusively HPyVs. In contrast, each Ortho- Recently, the applicability of this model to two species polyomavirus-I and -II include 11 putative species from of best-sampled human viruses, BKPyV and JCPyV which only two and three, respectively, are composed of (Shackelton et al., 2006; Krumbholz et al., 2008; Zheng viruses that seem to infect humans. The fifth lineage, et al., 2007), as well as the entire family (Krumbholz et al., comprising no HPyVs, belongs to the genus Avipolyomavirus. 2009) was questioned. Particularly, the intrahost substi- Because of the observed phyletic distribution of HPyVs tution rate of the two human polyomaviruses was revised by among different lineages, it may be envisaged that a number approximately two orders of magnitude upward from that http://vir.sgmjournals.org 491 M. C. W. Feltkamp and others expected upon the divergence by virus–host co-evolution References (Chen et al., 2004; Firth et al., 2010; Shackelton et al., 2006). 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(2010). Classification of papillomaviruses (PVs) over the past few years (Bernard et al., 2010; Forslund, based on 189 PV types and proposal of taxonomic amendments. 2007) remains to be seen. Virology 401, 70–79. Regarding pathogenicity, the majority of the novel Bhatia, K., Goedert, J. J., Modali, R., Preiss, L. & Ayers, L. W. (2010). HPyVs are so far without a (possibly) associated disease. Merkel cell carcinoma subgroups by Merkel cell polyomavirus DNA More studies are needed to identify and correlate the relative abundance and oncogene expression. Int J Cancer 126, 2240–2246. presence of the novel viral sequences with specific Bialasiewicz, S., Whiley, D. M., Lambert, S. B., Jacob, K., Bletchly, C., diseases. Because of the nature of polyomaviruses, next Wang, D., Nissen, M. D. & Sloots, T. P. (2008). 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