Since 1971, 10 PyVs that infect humans have been identified, including JC virus (JCV) and BK virus (BKV) found over >40 years ago, as well as the recently isolated MCPyV, KI PyV (KIPyV), WU PyV (WUPyV), human PyV (HPyV)6, HPyV7, trichodysplasia spinulosa PyV (TSPyV), HPyV9 and MWPyV (37).

PyVs are obiquitous worldwide, with seroprevalence rates ranging from 35 to 90% (34,37). Apart from a productive life cycle with resultant cell lysis, often associated with minimal symptoms, PyVs can also establish a persistent infection; significant disease though, is limited to patients with immune system disfunction (34,37,38). More specifically, JCV is associated with progressive multifocal leukoencephalopathy in HIV-AIDS, autoimmune diseases treated with certain lymphocyte-specific antibodies and hematological diseases; BKV with hemorrhagic cystitis following allogeneic hematopoietic stem cell transplantation and post-kidney transplantation PyV-associated nephropathy (37). TSPyV is linked to trichodysplasia spinulosa, a skin disease found in immune-impaired transplant patients that is characterized by virus-induced lytic and proliferative tumor-like features (37). KIPyV and WUPyV have been isolated from the respiratory tract, HPyV6 and HPyV7 from the skin, HPyV9 from the serum and skin, and MWPyV from stools and skin; However, none of these viruses have been associated with specific human pathology (37). MCPyV is the only PyV with a robust correlation with human cancer, more specifically Merkel cell carcinoma, a rare skin tumor found in elderly, chronically immunosupressed patients (37).

All PyVs have a similar morphological and functional pattern (39), with their virions consisting of non-enveloped icosahedral particles 40–45 nm in diameter, and a genome of ~5 kb organized as circular double-stranded DNA wrapped around host cell-derived histones (37). This genome is divided into 3 functional parts: i) the non-coding control region (NCCR), which contains the origin of replication, the transcription start sites and promoter/enhancer elements, and regulates the expression of the early and late viral genes; ii) the early gene region, encoding the large T-antigen (LTag) and the small Tag which derives from a major transcript by alternative splicing and facilitate viral genome replication and transformation; and iii) the late gene region, encoding capsid proteins VP-1, VP-2 and VP-3, produced by alternative splicing of a primary transcript, and assembled in the nucleus to form the viral capsid (37).

Two possible outcomes may follow cell infection by PyV: i) in the case of a permissive host, viral entry results in viral DNA replication, followed by progeny virion production, and cell lysis; ii) in the case of a non-permissive host, an abortive infection or cell transformation, known as oncogenesis, ensues, characterized by the continued expression of the viral early genes (40–43). The starting point of this latter process, is the genetic and functional uncoupling of the early gene expression from the later steps of the viral life cycle, namely viral DNA replication, late gene expression, virion assembly and host cell lysis. Cell cycle control is subverted by the Tags through the inactivation of signal transduction pathways and the tumor suppressor proteins, pRB and p53, eventually leading to neoplastic transformation (37).

To the best of our knowledge, few studies to date have investigated the association of PyVs with thyroid cancer (44–47), as shown in Table I. Three of these studies (44–46) detected sequences of simian vacuolating virus 40 (SV40), a virus that infected rhesus monkey kidney cells used for polio vaccine production (48), in thyroid gland specimens. Since SV40 has proven to be oncogenic in rodents (37,38,44) and has transforming capacity in human cells in vitro (44), the wide distribution of SV40 contaminated vaccines in the early 1960s, led to a concern for potential tumor induction in humans (37). A single study investigated the presence of BKV in post-operative thyroid specimens (47).

Most of our knowledge regarding the association of SV40 with thyroid cancer, is based on 3 studies examining the presence of SV40 DNA in thyroid specimens (44–46). The first study investigated the presence of SV40 sequences in 69 patients with PTCs using Southern blotting and polymerase chain reaction (PCR) (44). Seven normal peritumoral thyroid specimens, 4 blood specimens from patients with PTCs, 1 Hashimoto's thyroiditis, 5 toxic diffuse goiters, 3 medullary carcinomas and 9 breast carcinomas, were also studied. Southern blot analysis yielded positive results for 3 of the cases of PTC (4.3%). PCR amplification followed by sequence analysis, confirmed the presence of SV40 sequences, including the 203 bp fragment of the aminoterminus of LTag, the 294 bp fragment of the VP1 gene, as well as the 483 bp entire regulatory region, integrated in the tumoral DNA of the aforementioned samples. All the other samples scored negative for SV40 (44). Immunohistochemistry (IHC), performed for the LTag, revealed positive cytoplasmic staining in the 3 SV40-positive cases, not found in the negative controls (44). Another study (45), enroling 109 patients (80 females/29 males), performed by the same group, investigated the presence of SV40 DNA in a larger variety of thyroid pathologies, including 29 specimens of papillary cancer, 20 myeloid cancers, 20 anaplastic cancers and 20 specimens of Graves' disease. Specimens of normal thyroid tissue, 10 adjacent to papillary cancer, 10 adjacent to anaplastic cancer, and 20 from patients affected by multinodular goiter, were also included. Additionally, 20 peripheral blood mononuclear cell samples from relatives of patients with sporadic myeloid cancer, were analyzed. PCR amplification of the 172 bp N-terminal SV40 Tag and filter hibridization confirmed the presence of viral sequences in a percentage ranging from 66% in papillary cancers (P=0.02), to 100% in anaplastic cancers (P=0.01), while 90% of medullary cancer samples were positive (P=0.01). SV40 sequences were detected in a similar percentage, ranging from 60 to 100%, in corresponding normal thyroid tissues next to the tumors, while the corresponding detection percentages in specimens taken from patients with multinodular goiter and Graves' disease, were 10 and 20%, respectively. A total of 25% of blood samples were positive for SV40 Tag DNA (45). Positive samples were further investigated for the presence of the 314 and 294 bp sequences of the SV40 regulatory and VP1 regions, respectively (45). SV40 sequence specificity, was confirmed by DNA sequencing. Subsequent RT-PCR, that was performed in the 24 thyroid cancer specimens yielded positive results for SV40 DNA, revealing mRNAs specific for SV40 Tag in 69% (9/13) of PTCs, and 73% (8/11) of anaplastic carcinomas. None of the 30 samples were found to be SV40-negative, used as negative controls, was positive. IHC was finally performed in the samples found positive by RT-PCR, while the 30 SV40-negative thyroid tissues were used as controls. A total of 33% (3/9) of the PTCs and 100% (8/8) of the anaplastic carcinomas were immunoreactive, and showed mainly cytoplasmic staining. The third study included 99 patients (21 males/78 females) with thyroid nodules, 8 of whom (8.08%), were diagnosed with PTCs, while 91 (91.02%) had benign thyroid nodules, 8 of which, harboured Hashimoto's thyroiditis (46). Both nodular and normal thyroid tissue was obtained from each patient. All 198 specimens were investigated for the presence of SV40, through the amplification of Tag coding sequences, using PCR (46). Sequences of SV40 were detected in 4 out of 99 nodules, 2 of which were PTCs, while the remaining were benign thyroid nodules. The presence of SV40 DNA in the thyroid nodules was confirmed by sequence analysis using the SV40P1 primer, and by cloning of the 243 bp PCR product into the Topo XL cloning vector (46).

A 95 bp sequence of the BKV VP1 gene was investigated in frozen thyroid samples obtained from 30 patients with thyroid nodules (6 males/24 females) using PCR (47). A total of 14 out of the 30 patients (46.7%) suffered from PTC, while 16/30 (53.3%) had multinodular hyperplasia. Nodular, as well as adjacent normal tissues, were obtained from each patient. Taken as a whole, 18/30 (60.0%) nodular tissue samples harboured BKV DNA, while the corresponding percentage in adjacent normal tissue was 43,3% (13/30). More specificaly, the VP1 sequence was detected in 8/14 malignant specimens (57.1%), compared to 6/14 (42.8%) of adjacent normal tissue. The corresponding percentages for multinodular hyperplasia were 62.5% (10/16) and 43.7% (7/16), respectively (47).

Overview. Eight identified viruses from the herpesviridae family infect humans, categorized in 3 subfamilies, namely α, β and γ. The α subfamily includes herpes simplex viruses (HSV) type 1 and 2, as well as varicella zoster virus (VZV or HHV-3). Cytomegalovirus (CMV or HHV-5), and roseola viruses (HHV) 6 and 7 constitute the β subfamily, while EBV (HHV-4) and KSHV/HHV8, belong to the γ subfamily (49,50). Human herpesviruses are large (100–200 nm), enveloped, that contain double stranded DNA, enclosed in an icosahedral protein capsid (49).

As with PyVs, herpesviruses are widespread infectious agents, and almost 100% of adults have been infected in their lifetime (49). Lifelong infection is established, as the viral genome latently persists in the host cell nucleus (50). Although all herpesviruses infect epithelial cells (49), thus gaining access to the host, long-term residency and latency is strictly dictated by the specific herpes subfamily (49,50), determined by the presence of cell-surface receptors, as well as intracellular conditions (49), α herpesviruses establish latency in neurons, β herpesviruses in macrophages and lymphocytes, and γ herpesviruses in lymphocytes alone (49,50).

HSV-1 is associated with blisters on the lips, known as herpes labialis, while HSV-2 causes genital blisters (49). Both viruses may be associated with either lesion though (49). HSV may additionaly reach the eyes causing keratitis, potentially leading to blindness if left untreated (51), or may attack the brain, with resultant encephalitis or meningitis (52). Primary infection with VZV causes chickenpox, associated with a vesicular itchy rash primarily affecting the trunk and the head, while later reactivation is known as herpes zoster or shingles, limited to a specific body area (49). HHVs-6 and −7, known as roseola viruses, form skin rashes referred to as exanthema subitum (53). Congenital CMV infection may lead to birth defects (54), while mononucleosis, characterized by fever, sore throat, fatigue, as well as swolen lymph nodes, is the main disease associated with CMV in adolescents, though it is more commonly caused by EBV (49). Both γ subfamily herpesviruses are tumorigenic agents (49). HHV-8 is mainly the etiologic agent associated with Kaposi's sarcoma, a cancer most frequently diagnosed in patients with AIDS (55). EBV is mainly associated with nasopharyngeal carcinoma, as well as with Burkitt, Hodgkin's, immune-suppression-related non-Hodgkin, and extranodal NK/T-cell lymphomas (56).

EBV represents the typical example of herpesviral strategy (49). Upon infection of a cell, the two ends of the initially linear viral genome, bind to each other, persisting in an episomal state, facilitating latency establishment (57,58), as opposed to lytic activation, which requires genome linearization (59). No virions are produced during the latent state, allowing for the expression of only a few viral genes, which affect B-lymphocyte growth mechanisms, causing their immortalization (60). Six nuclear antigens, namely Epstein-Barr nuclear antigen (EBNA)1, −2, −3A, −3B, −3C, as well as the protein EBNA-LP; 3 membrane proteins, EBV latent membrane protein (LMP)1, −2A, −2B; 2 small nuclear RNAs, EBV-encoded small RNA (EBER)1 and −2, as well as BART region trancripts, encoding most of EBV's microRNAs, are associated with this latent infection of immortalized B cells (61). Conversely, the key role for the transition from the latent to the lytic cycle, inducing viral replication, is played by the BZLF1 and BRLF1 proteins (62).

Similar to PyVs, the association of members of the herpes family with thyroid cancer is based on detection methods of the presence of viral genes and gene products in thyroid tumor tissue (29,47,63). The studies that investigate this association are presented in Table II. The majority of these studies refer to EBV (47,63–69), while 4 other members of the family have also been studied, although less extensively (29,69).

The detection of EBV in thyroid cancer has proven controversial (47,63–69). The EBV persisting capacity in B-lymphocytes and its contribution to lymphoma formation (56), prompted the initial investigation of EBV in thyroid lymphomas (63–65), while subsequent studies investigated the presence and contribution of EBV in other types of thyroid malignancies (66–69).

Three studies examined the presence of EBV in thyroid lymphomas (63–65). EBV-related mRNA, as well as the associated proteins were investigated in 32 cases of thyroid lymphoma and 30 cases of Hashimoto's thyroiditis by in situ hybridization (ISH) and IHC on routinely processed tissue sections (63). Three cases of thyroid lymphoma demonstrated the presence of EBER. Gene products, BHLF1, LMP, and BZLF1 proteins were detected in 2 of the EBER-positive cases (63). The second study enrolled 30 patients with thyroid lymphoma and 28 with chronic lymphocytic thyroiditis (10 males/48 females) (64). Only 24 and 16 cases of thyroid lymphoma and chronic thyroiditis, respectively, were finally used, as the rest of the samples revealed poorly preserved DNA, based on the presence of the β-globin PCR band, and were thus excluded from further analysis (64). PCR amplification revealed positive EBV products in 1 case of chronic lymphocytic thyroiditis and 2 cases of thyroid lymphoma, while ISH yielded positive signals in the nucleus of tumor cells of only 1 of the lymhomas found positive by PCR, while LMP-1 was also expressed in the cytoplasm of lymphoma cells (64). The third and most recent study, analyzed the clinicopathological characteristics of primary and secondary thyroid lymphomas affecting the Hong Kong Chinese population over a period of 3 decades, and investigated the expression of EBV genes, using ISH and IHC (65). A total of 23 patients with primary disease, 15 of whom had Hashimoto's thyroiditis, as well as 9 patients with secondary disease were enroled. EBV mRNAs were detected in 1 primary and 1 secondary thyroid lymphoma (65).

EBV has been suggested to contribute to thyroid tumor progression, defined as the pathological dedifferentiation of tumor cells, leading to a more undifferentiated tumor type (66). To examine the potential involvement of EBV expression in the progression of thyroid cancer, 10 PTCs, normal tissue specimens at the peripheral region of 4 of the PTCs, 11 undiferentiated carcinomas, 1 thyroid squamous cell carcinoma (SCC), as well as negative controls including 2 thyroid nodular hyperplasia and 2 Graves' disease specimens, all taken from Japanese patients were used. Specimens were subjected to PCR, RT-PCR, mRNA ISH and indirect immunofluorescence staining. PCR was performed in 13 of the samples, including 9 PTCs, 3 undifferentiated carcinomas and 1 SCC, all of which showed amplified EBV DNA in the region of BamHIW. ISH for the detection of EBV mRNAs expression was performed using BamHIW, EBER1 and EBNA2 probes, while immunofluorescence for the detection of EBV proteins was performed using 4 monoclonal antibodies, namely anti-EBNA2, anti-LMP1, anti-BZLF1 and anti-CD21. Despite the fact that EBV infection, and mRNA and protein expression was detected in all carcinoma specimens, irrespective of the degree of histological differentiation, both mRNA and protein exression was much more prominent in the undifferentiated carcinomas. Both the normal thyroid tissue specimens, as well as the nodular hyperplasia and Graves' disease specimens used as controls, showed no, or very few signals during ISH with any of the probes (66).

A similarly high EBV detection percentage in thyroid nodules consisting of PTCs, and multinodular hyperplasia specimens, as well as adjacent normal thyroid tissues, was recently reported in a previously mentioned study (47). The investigation included the detection of 161, 168 and 118 bp sequences of the LMP1, EBNA2 and EBER1 genes, respectively. As regards LMP1, the sequence was detected in 50% (15/30) of the nodules, and in 46.7% (14/30) of the adjacent normal tissues. More specifically, 57.1% (8/14) of the PTCs and 43.8% (7/16) of multinodular hyperplasia specimens harboured the sequence, while the percentages of the adjacent normal tissues were 35.7% (5/14) and 56.2% (9/16), respectively. Much higher percentages considering EBNA2 sequence detection, were found in the study, although similar between nodular (90%-27/30) and adjacent normal (90–27/30) tissue. The vast majority of both malignant (92.9%, 13/14) as well as multinodular hyperplasia (87.5%, 14/16) specimens were EBNA2-positive, while the corresponding percentages for the adjacent normal tissues were 85.7% (12/14) and 93.8% (15/16), respectively. Sequence analysis confirmed the results. Interestingly, LMP1 sequence frequency in nodular and adjacent normal thyroid tissue presented significant differences compared to EBNA2 sequence frequency (P=0.0015 for nodular tissue, and P=0.0006 for normal tissue). None of the samples contained the EBER1 sequence (47).

In contrast to the studies mentioned above, negative results regarding the association between thyroid tumors and EBV have been reported (67–69). None of the 45 PTCs resected from patients in the southern part of Kyushu, Japan, and subjected to EBER1 ISH tested positive for EBV, despite the fact that a few infiltrating lymhoid cells detected in 1 (2.2%) of the specimens, revealed EBER1 positivity (67). Additionaly, no significant positive signal for EBV was revealed by PCR, ISH for EBER, and IHC, performed in 12 cases (11 females/1 male) of oncocytic PTC with lymphoid stroma (Warthin-like tumor) (68). Positive PCR results of 1 sample, were not confirmed from repeat PCR, or ISH, thus the sample was considered negative for EBV (68). Finally, all 16 benign thyroid tumor samples taken from 34 to 56 year-old female patients from Taiwan, used as controls in a study associating non-familial breast cancer with viral factors, scored negative for EBV using PCR and Southern hybridization (69).

The hypothesis of HSV1 and HSV2 association with thyroid tumors, was investigated analyzing the detection of viral DNA in both benign and malignant thyroid lesions, as well as the expression of nectin-1, a herpesvirus entry mediator, in thyroid tissues and cancer cell lines (29). Thyroid cancer cell susceptibility to HSV, and its associated molecular mechanisms, were explored in vitro (29). Thyroid samples from 109 patients (44 benign/65 malignant), including 43 PTCs, 16 FTCs, 6 anaplastic carcinomas, 30 follicular adenomas and 14 autoimmune thyroid disease specimens were examined (29). The presence of thyroid oncogene mutations was also sought in 73 thyroid tumors. Initialy, HSV DNA was amplified with PCR, followed by sequencing. Subsequent investigation included the detection of HSV proteins by immunohistochemical staining, and the detection of nectin-1 by in-cell western blot analysis. Collectively, HSV DNA was detected in 43/109 (39.4%) of the samples. Considering benign lesions, HSV1 DNA was detected in 11/44 (25%), while HSV2 DNA was detected less frequently (1/44–2%). HSV DNA was much more frequently detected in malignant lesions (31/65, 47.7%; P=0.0454), although HSV1 sequences had the same prevalence in benign and malignant lesions. PTCs and lymph node metastases harboured mostly HSV2 DNA, while HSV1 DNA was detected predominantly in FTCs. Oncogenic mutations were revealed in 70% (21/30) of the HSV-positive tumors, compared with 27.2% (12/44) of the HSV-negative tumors. Immunoreactivity was detected in 21/25 (84%) of the HSV-positive samples investigated, restricted to epithelial cells, unlike stromal fibroblasts, endothelial cells and infiltrating lymphocytes, which were negative. As regards nectin-1, its expression has been shown to be increased in thyroid tumors, particularly papillary cancers compared to normal specimens, as well as in all thyroid cancer cell lines, and correlated with cancer cell susceptibility to HSV infection (29).

Unlike the results of the above-mentioned study, neither HSV1 nor HSV2 were detected in any of the 16 benign thyroid tumor samples, in the previously mentioned study reported by Tsai et al (69). The results were also negative for HHV8, while CMV was related to thyroid tumors (P<0.05), and its DNA was detected in 4/16 (25%) of the specimens.