High rates of anal Merkel Cell Polyomavirus and HPV co-infection among people living with HIV.

Knowledge of Human Polyomavirus (HPyV) infection in the anal area and its association with sexually transmitted infections such as Human Papillomavirus (HPV) and Human Immunodeficiency Virus (HIV) remains limited. Therefore, anal specimens from 150 individuals of both sexes were analyzed for screening purposes. HPV DNA was found in 50.7% of cases, with a predominance of high-risk (HR) genotypes. HPyV DNA was found in 39.3% of samples, with Merkel Cell Polyomavirus (MCPyV) being the most common, with a higher viral load than JCPyV and BKPyV. In addition, MCPyV viral load increased in people living with HIV (PLWH) with HPV infection (p < 0.0001).

Mapping of Human Polyomavirus in Renal Cell Carcinoma Tissues.

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

Progressive multifocal leukoencephalopathy in multiple myeloma: a case report of a patient with SARS-CoV-2 infection and an updated systematic literature review.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the human polyomavirus 2 (HPyV-2, previously known as JCV) in immunosuppressed individuals. Few cases of PML have been described in multiple myeloma (MM) patients. METHODS: We described a case of PML in a patient with MM with fatal worsening that occurred during SARS-CoV-2 infection. We also performed a literature review to update the 16 cases series of MM patients with PML already collected until April 2020. RESULTS: A 79-year-old female patient with refractory IgA lambda MM in Pomalidomide- Cyclophosphamide-Dexamethasone regimen developed gradual lower limbs and left arm paresis along with a decreased consciousness 3.5 years after the MM diagnosis. Symptoms developed shortly after the recognition of hypogammaglobulinemia. After SARS-CoV-2 infection, her neurological status quickly worsened until she deceased. MRI features and JCV-positive PCR on CSF confirmed the PML diagnosis. Our literature review adds sixteen clinical cases of PML in MM published between May 2020 and March 2023 to the 16 cases already collected in the previously published review by Koutsavlis. DISCUSSION: PML has been increasingly described in MM patients. It remains questionable if the HPyV-2 reactivation is determined by the severity of MM itself, by the effect of drugs or by a combination of both. SARS-CoV-2 infection may have a role in worsening PML in affected patients.

Antivirals against human polyomaviruses: Leaving no stone unturned.

Human polyomaviruses (HPyVs) encompass more than 10 species infecting 30%-90% of the human population without significant illness. Proven HPyV diseases with documented histopathology affect primarily immunocompromised hosts with manifestations in brain, skin and renourinary tract such as polyomavirus-associated nephropathy (PyVAN), polyomavirus-associated haemorrhagic cystitis (PyVHC), polyomavirus-associated urothelial cancer (PyVUC), progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma (MCC), Trichodysplasia spinulosa (TS) and pruritic hyperproliferative keratinopathy. Although virus-specific immune control is the eventual goal of therapy and lasting cure, antiviral treatments are urgently needed in order to reduce or prevent HPyV diseases and thereby bridging the time needed to establish virus-specific immunity. However, the small dsDNA genome of only 5 kb of the non-enveloped HPyVs only encodes 5-7 viral proteins. Thus, HPyV replication relies heavily on host cell factors, thereby limiting both, number and type of specific virus-encoded antiviral targets. Lack of cost-effective high-throughput screening systems and relevant small animal models complicates the preclinical development. Current clinical studies are limited by small case numbers, poorly efficacious compounds and absence of proper randomized trial design. Here, we review preclinical and clinical studies that evaluated small molecules with presumed antiviral activity against HPyVs and provide an outlook regarding potential new antiviral strategies.

Detection of Human polyomavirus 2 (HPyV2) in oyster samples in northern Brazil.

BACKGROUND: Human polyomavirus 2 (HPyV2 or JCPyV) is persistent in the environment due to its excretion in urine and feces; it is detected in samples of wastewater, surface water and drinking water. A lack of basic sanitation and sewage collection results in the presence of this virus in food, especially in oysters, since they are bioaccumulators and are consumed in their natural form, thus posing a risk to human health. METHODS: This study investigated the frequency of HPyV2 in samples of oysters marketed in northeastern Pará State, Brazil, and optimized a real-time PCR (qPCR) protocol for the detection of an endogenous oyster control. A total of 217 oysters in 22 pools from five municipalities in the state of Pará were analyzed. Samples underwent dissection and total maceration of oyster tissue using a viral concentration technique, followed by DNA extraction with phenol-chloroform and amplification of the VP1 region for molecular detection via qPCR. RESULTS: HPyV2 was detected in 18.2% (4/22) of the pooled samples, with frequencies of 25, 20, 20 and 16% in the municipalities of Salinópolis, Augusto Corrêa, São Caetano de Odivelas and Curuçá, respectively. Notably, the sample pool from the municipality of Bragança did not have detectable HPyV2 and this was the only sampled location with a water treatment station. In this study, Crassostrea genus-specific primers (AFL52 ribosomal RNA gene) of oyster were developed for use as an endogenous control in the qPCR analysis, which will be useful for future studies. CONCLUSIONS: The detection of HPyV2 in oyster samples commercialized in the state of Pará shows the circulation of this virus in the studied municipalities. Thus, it is necessary to implement measures for improving sewage collection and basic sanitation to avoid contamination of water and food with HPyV2.

Prevalence and Viral Loads of Cutaneous Human Polyomaviruses in the Skin of Patients With Chronic Inflammatory Skin Diseases.

BACKGROUND: Human skin microorganisms have been associated with various skin diseases. However, most studies have focused on bacterial communities, and little is known about normally resident skin viruses such as the Polyomaviridae and their association with cutaneous disorders. METHODS: We investigated the infection levels of Merkel cell polyomavirus (MCPyV), human polyomavirus 6 (HPyV6), and human polyomavirus 7 (HPyV7), using triplet skin swabs collected from lesional and nonlesional skins of 86 Japanese patients with inflammatory skin diseases and mycosis fungoides, and from 149 healthy control individuals. RESULTS: This age-matched case-control study provides the first analyses of the loads of polyomaviruses in association with various skin diseases. The viral loads were significantly higher for HPyV6/HPyV7 and lower for MCPyV in patients with psoriasis. The viral load variation was observed not only at lesion sites, but also at clinically unaffected skin sites in most of the patients. The viral strains tested were all of the Asian/Japanese genotype. CONCLUSIONS: Our findings suggest a covariation in the infection levels of cutaneous polyomaviruses in certain inflammatory skin conditions. Worldwide prospective longitudinal studies are warranted to understand the influence of such alterations on the pathogenesis of inflammatory skin disorders.

Prevalence and Genetic Variability of Human Polyomaviruses 6 and 7 in Healthy Skin Among Asymptomatic Individuals.

Background: Despite the pathogenetic potential of human polyomavirus 6 (HPyV6) and human polyomavirus 7 (HPyV7), they have been found in the normal skin of healthy individuals. However, little is known about the prevalence, infection levels, and geographical variations of these polyomaviruses in the skin. Methods: Using skin swabs from 470 participants aged 2-98 years, we estimated the prevalence of copy numbers of HPyV6 and HPyV7 with respect to age and ethnicity. Phylogenetic analyses were conducted based on viral sequences obtained from Asian and white populations. Results: This study provides the first analyses of the age-specific prevalence and levels of HPyV6 and HPyV7 infections in normal skin. Comparisons of age groups revealed that the prevalence and viral loads were significantly higher in elderly persons. Phylogenetic analyses demonstrated the existence of Asian/Japanese-specific strains genetically distinct from strains prevalent in the skin of the white population studied. Conclusions: This large study suggests that HPyV6 and HPyV7 infections in the skin are highly prevalent in elderly adults. Further research is warranted to understand whether persistent infection with high viral loads in the skin could be a risk factor for the development of HPyV6- and HPyV7-associated skin disorders.

Molecular mechanisms supporting a pathogenic role for human polyomavirus 6 small T antigen: Protein phosphatase 2A targeting and MAPK cascade activation.

BRAF inhibitors are highly effective therapies in treating a subset of melanomas but are associated with induction of secondary cutaneous squamous cell carcinoma (cSCC). Recently, Human Polyomavirus 6 (HPyV6) was found to actively express viral proteins in BRAF inhibitor-induced cSCCs; however, the specific cellular mechanisms by which HPyV6 may facilitate neoplastic cell growth require further investigation. The current study describes a novel pathogenic mechanism of action for HPyV6 small tumor (sT) antigen which involves binding to protein phosphatase 2A (PP2A) via its WFG motif and zinc binding sites. Our findings demonstrate an important role of HPyV6 sT for activation of PP2A's downstream oncogenic pathways (MEK/ERK/c-Jun), which may underlie the pathogenesis of BRAF inhibitor-induced neoplasms. J. Med. Virol. 89:742-747, 2017. © 2016 Wiley Periodicals, Inc.

Multiplex detection in tonsillar tissue of all known human polyomaviruses.

BACKGROUND: In the past few years, eleven new human viruses have joined the two previously known members JCPyV and BKPyV of the Polyomaviridae family, by virtue of molecular methods. Serology data suggest that infections with human polyomaviruses (HPyVs) occur since childhood and the viruses are widespread in the general population. However, the viral persistence sites and transmission routes are by and large unknown. Our previous studies demonstrated that the four new HPyVs - KIPyV, WUPyV, MCPyV and TSPyV - were present in the tonsils, and suggested lymphoid tissue as a persistent site of these emerging human viruses. We developed a Luminex-based multiplex assay for simultaneous detection of all 13 HPyVs known, and explored their occurrence in tonsillar tissues of children and adults mostly with tonsillitis or tonsillar hypertrophy. METHODS: We set up and validated a new Luminex-based multiplex assay by using primer pairs and probes targeting the respective HPyV viral protein 1 (VP1) genes. With this assay we tested 78 tonsillar tissues for DNAs of 13 HPyVs. RESULTS: The multiplex assay allowed for simultaneous detection of 13 HPyVs with high analytical sensitivity and specificity, with detection limits of 100-102 copies per microliter, and identified correctly all 13 target sequences with no cross reactions. HPyV DNA altogether was found in 14 (17.9%) of 78 tonsils. The most prevalent HPyVs were HPyV6 (7.7%), TSPyV (3.8%) and WUPyV (3.8%). Mixed infection of two HPyVs occurred in one sample. CONCLUSIONS: The Luminex-based HPyV multiplex assay appears highly suitable for clinical diagnostic purposes and large-scale epidemiological studies. Additional evidence was acquired that the lymphoid system plays a role in HPyV infection and persistence. Thereby, shedding from this site during reactivation might take part in transmission of the newly found HPyVs.

Studies of human polyomaviruses, with HPyV7, BKPyV, and JCPyV present in urine of allogeneic hematopoietic stem cell transplanted patients with or without hemorrhagic cystitis.

BACKGROUND: BK polyomavirus (BKPyV) can cause hemorrhagic cystitis (HC) in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients and polyomavirus-associated nephritis in renal transplant patients, while JC polyomavirus (JCPyV) can generate progressive multifocal leukoencephalopathy in immunocompromised individuals. Since 2007, additional human polyomaviruses (HPyVs) have been identified. In this study, we examined the urines of allo-HSCT patients for possible presence of polyomaviruses BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and HPyV10 (MWPyV). METHODS: A total of 185 urinary samples obtained 2002-2007 from 105 allo-HSCT patients, 32/105 with HC, were tested for the above-listed HPyVs by a bead-based multiplex assay. Of these, 142 urine samples had previously been tested for BKPyV and JCPyV by nested polymerase chain reaction (PCR). RESULTS: Aside from BKPyV and JCPyV, which dominated, HPyV7 was detected in 5 BKPyV-positive urinary samples from 1 patient. The multiplex assay was more sensitive and specific than the nested PCR. BKPyV and/or JCPyV were found in all but 1 of the previously BKPyV- or JCPyV-positive samples, although 6 previously BKPyV-positive cases were now JCPyV-positive or the reverse. Furthermore, 18/79 previously negative samples were found to be BKPyV and/or JCPyV positive, and a total of 21 double infections were found. Lastly, in 1/29 HC patients, only JCPyV was detected. CONCLUSION: HPyV7 was found for the first time in urine of an allo-HSCT patient, and BKPyV and JCPyV were more commonly found in urine samples using the bead-based assay compared to testing by nested PCR. Finally, only JCPyV was detected in the urine of 1 HC patient.

Human polyomavirus 7-associated pruritic rash and viremia in transplant recipients.

Human polyomavirus 7 (HPyV7) is one of 11 HPyVs recently discovered through genomic sequencing technologies. Two lung transplant recipients receiving immunosuppressive therapy developed pruritic, brown plaques on the trunk and extremities showing a distinctive epidermal hyperplasia with virus-laden keratinocytes containing densely packed 36-45-nm icosahedral capsids. Rolling circle amplification and gradient centrifugation testing were positive for encapsidated HPyV7 DNA in skin and peripheral blood specimens from both patients, and HPyV7 early and capsid proteins were abundantly expressed in affected tissues. We describe for the first time that HPyV7 is associated with novel pathogenicity in some immunosuppressed individuals.

Human polyomavirus 9 infection in kidney transplant patients.

Several human polyomaviruses of unknown prevalence and pathogenicity have been identified, including human polyomavirus 9 (HPyV9). To determine rates of HPyV9 infection among immunosuppressed patients, we screened serum samples from 101 kidney transplant patients in the Netherlands for HPyV9 DNA and seroreactivity. A total of 21 patients had positive results for HPyV9 DNA; positivity rates peaked at 3 months after transplantation, but the highest viral loads were measured just after transplantation. During 18 months of follow-up, HPyV9 seroprevalence increased from 33% to 46% among transplant patients; seroprevalence remained stable at ≈30% in a control group of healthy blood donors in whom no HPyV9 DNA was detected. Further analysis revealed an association between detection of HPyV9 and detection of BK polyomavirus but not of cytomegalovirus. Our data indicate that HPyV9 infection is frequent in kidney transplant patients, but the nature of infection-endogenous or donor-derived-and pathogenic potential of this virus remain unknown.

A multivalent polyomavirus vaccine elicits durable neutralizing antibody responses in macaques.

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of ≥ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.

Human polyomaviruses JCPyV and MCPyV in urothelial cell carcinoma: a single institution experience.

Objective: Urothelial cell carcinoma (UCC) is the most common type of urinary bladder. JCPyV and BKPyV have been detected in the urine and tissue of urothelial cell carcinomas (UCC) in immunocompetent patients. Here, we investigated the presence of several HPyVs in UCC samples using diverse molecular techniques to study the prevalence of HPyVs in UCC. Methods: A large single-institution database of urine cytology specimens (UCS; n = 22.867 UCS) has previously been searched for decoy cells (n = 30), suggesting polyomavirus infection. The available urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC patients were tested for the presence of JCPyV-LTAg expression by immunohistochemistry (IHC) labeled with SV40-LTAg antibody (clone: PAb416) and subsequent PCR followed by sequencing. In addition, the presence of the oncogenic Merkel cell polyomavirus (MCPyV) and the presence of human polyomavirus 6 (HPyV6) and 7 (HPyV7) DNA were tested with DNA PCR or IHC. Results: Of the 30 patients harboring decoy cells, 14 were diagnosed with UCC of the urinary bladder (14/30; 46.6%) before presenting with decoy cells in the urine. The SV40-LTAg IHC was positive in all 14 UCC urine sediments and negative in the FFPE tissues. JCPyV-DNA was identified in all five available UCS and in three FFPE samples of UCC (three of 14; 21.4%). Two UCC cases were positive for MCPyV-DNA (two of 14; 14.3%), and one of them showed protein expression by IHC (one of 14; 7.1%). All specimens were HPyV6 and HPyV7 negative. Conclusion: Our findings show the presence of JCPyV in the urine and UCC of immunocompetent patients. Moreover, MCPyV was detected in two UCC cases. In total, five UCC cases showed the presence of either JCPyV or MCPyV. The evidence here supports the hypothesis that these viruses might sporadically be associated with UCC. Further studies are needed to confirm the relevance of JCPyV or MCPyV as a possible risk factor for UCC development.

Seroprevalence of Four Polyomaviruses Linked to Dermatological Diseases: New Findings and a Comprehensive Analysis.

Our aim was to study the seroprevalence of human polyomaviruses (HPyV) linked to skin diseases. A total of 552 serum samples were analysed by the enzyme-linked immunosorbent assay to detect IgG antibodies against Merkel cell polyomavirus (MCPyV), HPyV6, HPyV7 and Trichodysplasia spinulosa-associated polyomavirus (TSPyV) using recombinant major capsid proteins of these viruses. The individuals (age 0.8−85 years, median 33) were sorted into seven age groups: <6, 6−10, 10−14, 14−21, 21−40, 40−60 and >60 years. The adulthood seroprevalence was 69.3%, 87.7%, 83.8% and 85% for MCPyV, HPyV6, HPyV7 and TSPyV, respectively. For all four polyomaviruses, there was increasing seropositivity with age until reaching the adulthood level. There was a significant increase in seroreactivity for those age groups in which the rate of already-infected individuals also showed significant differences. The adulthood seropositvity was relatively stable with ageing, except for TSPyV, for which elevated seropositivity was observed for the elderly (>60 years) age group. Since seroepidemiological data have been published with wide ranges for all the viruses studied, we performed a comprehensive analysis comparing the published age-specific seropositivities to our data. Although the cohorts, methods and even the antigens were variable among the studies, there were similar results for all studied polyomaviruses. For MCPyV, geographically distinct genotypes might exist, which might also result in the differences in the seroprevalence data. Additional studies with comparable study groups and methods are required to clarify whether there are geographical differences.

Prevalence of 13 polyomaviruses in actinic keratosis and matched healthy skin samples of immunocompetent individuals.

BACKGROUND: Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). UV radiation is the major risk factor for AK, but certain human papillomaviruses (HPVs) of the beta genus are also involved in its development. Differently, the role of polyomaviruses (PyVs) in skin carcinogenesis is still debated. Fiftheen PyVs have been isolated from human tissues so far, including Merkel cell polyomavirus (MCPyV), the aetiological agent of Merkel cell carcinoma. METHODS: The presence of 13 PyVs was assessed in skin samples from AK patients (n = 342). Matched fresh-frozen scrapings from healthy skin (HS) and AK lesions from 242 patients, and formalin-fixed paraffin-embedded AK biopsies from a different cohort of 100 patients were analyzed by multiplex PyVs genotyping assay. RESULTS: The most frequent lesion site was the scalp in men (27.3%), and the cheek area in women (29.0%). Differences between men and women were significant for the scalp, the cheek area and the lips. Almost all the scrapings were PyV-positive (HS: 89.7%, AK: 94.6%; p = 0.04). The three most frequent PyVs were MCPyV, HPyV6 and JCPyV (HS: 87.2%, 58.7%, 6.6%, respectively; AK: 88.8%, 51.2%, 9.9%, respectively). HPyV9, TSPyV, BKPyV, HPyV7, LIPyV and SV40 were detected in < 2% of the scrapings. In most cases, matched HS and AK scrapings were both positive (MCPyV: 78.1%, HPyV6: 41.7%), or both negative for the individual genotypes (for the remaining PyVs). PyV prevalence in AK biopsies was 22.0%. Only MCPyV (21.0%) and HPyV6 (3.0%) were detected in these samples. CONCLUSIONS: PyV prevalence in HS and AK scrapings was high, but detection of PyVs exclusively in AK scrapings was rare. PyV positivity rate in AK biopsies was modest. Further research is need to reach firm conclusions regarding the role of these viruses in AK development.

Emerging role of human polyomaviruses 6 and 7 in human cancers.

BACKGROUND: Currently 12 human polyomaviruses (HPyVs) have been identified, 6 of which have been associated with human diseases, including cancer. The discovery of the Merkel cell polyomavirus and its role in the etiopathogenesis in the majority of Merkel cell carcinomas has drawn significant attention, also to other novel HPyVs. In 2010, HPyV6 and HPyV7 were identified in healthy skin swabs. Ever since it has been speculated that they might contribute to the etiopathogenesis of skin and non-cutaneous human cancers. MAIN BODY: Here we comprehensively reviewed and summarized the current evidence potentially indicating an involvement of HPyV6 and HPyV7 in the etiopathogenesis of neoplastic human diseases. The seroprevalence of both HPyV6 and 7 is high in a normal population and increases with age. In skin cancer tissues, HPyV6- DNA was far more often prevalent than HPyV7 in contrast to cancers of other anatomic sites, in which HPyV7 DNA was more frequently detected. CONCLUSION: It is remarkable to find that the detection rate of HPyV6-DNA in tissues of skin malignancies is higher than HPyV7-DNA and may indicate a role of HPyV6 in the etiopathogenesis of the respected skin cancers. However, the sheer presence of viral DNA is not enough to prove a role in the etiopathogenesis of these cancers.

Natural History of Cutaneous Human Polyomavirus Infection in Healthy Individuals.

Several human polyomaviruses (HPyVs) were recently discovered. Merkel cell polyomavirus (MCPyV) induces Merkel cell carcinoma. HPyV6, HPyV7, and TSPyV have been associated with rare skin lesions in immunosuppressed patients. HPyV9, HPyV10, and Saint Louis Polyomavirus (STLPyV) have not been convincingly associated with any disease. The aim of this prospective study was to evaluate the cutaneous prevalence, persistence and viral load of HPyVs in healthy individuals. Eight hundred seventy forehead and hand swabs were collected from 109 volunteers 4-6 weeks apart (collection period-1). Fifty-nine participants were available for follow-up a decade later (collection period-2). HPyV-DNA prevalence and viral loads of MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, and STLPyV were determined by virus-specific real-time PCRs. Risk factors for HPyV prevalence, short- and long-term persistence were explored by logistic regression analyses. Baseline prevalence rates were similar for forehead and hand: MCPyV 67.9/67.0%, HPyV6 31.2/25.7%, HPyV7 13.8/11.0%, HPyV10 11.9/15.6%, STLPyV 7.3/8.3%, TSPyV 0.9/0.9%, and HPyV9 0.9/0.9%. Short-term persistence in period-1 was found in 59.6% (MCPyV), 23.9% (HPyV6), 10.1% (HPyV7), 6.4% (HPyV10), 5.5% (STLPyV), and 0% (TSPyV and HPyV9) on the forehead, with similar values for the hand. Long-term persistence for 9-12 years occurred only for MCPyV (forehead/hand 39.0%/44.1% of volunteers), HPyV6 (16.9%/11.9%), and HPyV7 (3.4%/5.1%). Individuals with short-term persistence had significantly higher viral loads at baseline compared to those with transient DNA-positivity (p < 0.001 for MCPyV, HPyV6, HPyV7, and HPyV10, respectively). This was also true for median viral loads in period-1 of MCPyV, HPyV6, and HPyV7 of volunteers with long-term persistence. Multiplicity (two or more different HPyVs) was a risk factor for prevalence and persistence for most HPyVs. Further risk factors were older age for HPyV6 and male sex for MCPyV on the forehead. Smoking was not a risk factor. In contrast to MCPyV, HPyV6, HPyV7, and rarely STLPyV, polyomaviruses TSPyV, HPyV9, and HPyV10 do not seem to be long-term constituents of the human skin virome of healthy individuals. Furthermore, this study showed that higher viral loads are associated with both short- and long-term persistence of HPyVs on the skin. HPyV multiplicity is a risk factor for prevalence, short-term and/or long-term persistence of MCPyV, HPyV6, HPyV7, and HPyV10.

Human polyomaviruses 10 and 11 in faecal samples from Brazilian children.

The human polyomaviruses (HPyVs) 10 and 11 have been detected in faecal material and are tentatively associated with diarrhoeal disease. However, to date, there are insufficient data to confirm or rule out this association, or even to provide basic information about these viruses, such as how they are distributed in the population, the persistence sites and their pathogenesis. In this study, we analysed stool specimens from Brazilian children with and without acute diarrhoea to investigate the excretion of HPyV10 and HPyV11 as well as their possible associations with diarrhoea. A total of 460 stool specimens were obtained from children with acute diarrhoea of unknown aetiology, and 106 stool specimens were obtained from healthy asymptomatic children under 10 years old. Samples were collected during the periods of 1999-2006, 2010-2012 and 2016-2017, and found previously to be negative for other enteric viruses and bacteria. The specimens were screened for HPyV10 and HPyV11 DNA by the polymerase chain reaction (PCR). Randomly selected positive samples were sequenced to confirm the presence of HPyV10 and HPyV11. The sequenced strains showed a percent of nucleotide identity of 93.4-99.6% and 85.5-98.9% with the reference HPyV10 and HPyV11 strains, respectively, confirming the PCR results. HPyV10 and HPyV11 were detected in 7.2% and 4.7% of the stool specimens from children with and without diarrhoea, respectively. The prevalence of both viruses was the same among children with diarrhoea and healthy children. There was also no difference between boys and girls or the degree of disease (severe, moderate or mild) among groups. Phylogenetic analysis showed that all of the genotypes described so far for HPyV10 and HPyV11 circulate in Rio de Janeiro. Our results do not support an association between HPyV10 and HPyV11 in stool samples and paediatric gastroenteritis. Nevertheless, the excretion of HPyV10 and HPyV11 in faeces indicates that faecal-oral transmission is possible.

High Prevalence of Human Polyomavirus 7 in Cholangiocarcinomas and Adjacent Peritumoral Hepatocytes: Preliminary Findings.

Cholangiocarcinoma (CCA) is a rare biliary-duct malignancy with poor prognosis. Recently, the presence of the human polyomavirus 6 (HPyV6) has been reported in the bile of diverse hepatobiliary diseases, particularly in the bile of CCA patients. Here, we investigated the presence of novel HPyVs in CCA tissues using diverse molecular techniques to assess a possible role of HPyVs in CCA. Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 42 CCA patients were included in this study. PCR-based screening for HPyVs was conducted using degenerated and HPyV-specific primers. Following that, we performed FISH, RNA in situ hybridization (RNA-ISH), and immunohistochemistry (IHC) to assess the presence of HPyVs in selected tissues. Of all 42 CCAs, 25 (59%) were positive for one HPyV, while 10 (24%) CCAs were positive for 2 HPyVs simultaneously, and 7 (17%) were negative for HPyVs. Of the total 35 positive CCAs, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 as confirmed by sequencing. The presence of viral nucleic acids was confirmed by specific FISH, while the RNA-ISH confirmed the presence of HPyV6 on the single-cell level. In addition, expression of HPyV7, HPyV6, and MCPyV proteins were confirmed by IHC. Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect bile duct epithelium, hepatocytes, and CCA cells, which possibly suggest an indirect role of these viruses in the etiopathogenesis of CCA. Furthermore, the observed hepatotropism of these novel HPyV, in particular HPyV7, might implicate a role of these viruses in other hepatobiliary diseases.