Molecular evolution pattern of Merkel cell polyomavirus identified by viral metagenomics in plasma of high-risk blood donors from the Brazilian Amazon.

Merkel cell polyomavirus (MCPyV) is a common human skin pathogen, shows high seroprevalence and is considered the etiologic agent of Merkel cell carcinoma. However, studies which detect MCPyV DNA in blood products may reveal the importance of this virus for the transfusion medicine. In this study we analyzed by viral metagenomics 36 plasma samples obtained from blood donors positive for the common blood transmitted infections from the city of Macapá (Brazilian Amazon). The generated raw data were were analyzed through a specific bioinformatics pipeline aimed at discovery of emerging viruses. The genomes of interest were analyzed phylogeographically and phylogenetically. MCPyV complete genome was recovered from one HBV-positive pool with high coverage (~ 223×) indicating acute viremia or reactivated infection. Interestingly, the phylogeographic position of the identified strain suggests its ancestry compared to MCPyV isolate from Colombian Amazon which hypothesizes that viral dissemination in the Amazon may have originated from Brazil. In conclusion, this study brings information for the genetic relationships of MCPyV isolated from blood donors from the Brazilian Amazon and demonstrates the possible phylogeographic behavior of our strain in relation to the other findings. We also demonstrated a strong evidence of viremic MCPyV phase in blood donations, however, more studies are necessary in order to understand the MCPyV impact on transfusion therapy.

Merkel cell polyomavirus is implicated in a subset of Merkel cell carcinomas, in the Indian subcontinent.

Merkel cell carcinoma is a rare, lethal cancer histopathologically composed of cells showing similarity with mechanoreceptor Merkel cells. Merkel cell tumors manifest in two distinct forms. While a virus called Merkel cell polyomavirus is involved in the pathogenesis of one form of Merkel tumors, the other is driven by ultraviolet (UV)-linked mutations. In this study we investigated 18 cases, from the Indian population, of Merkel cell carcinoma for immunohistochemical (IHC) expression of Merkel cell polyomavirus (MCV) T antigen, including 12 cases tested by PCR, to identify viral etiopathology. We tested the tumors with two sensitive antibodies (CM2B4 and Ab3), targeting the viral large T antigen protein and with PCR primers targeting the N terminus of T antigen. Overall, we observed 38.8% (7/18) tumors displaying positive IHC expression of Merkel cell polyomavirus T antigen and 25% (3/12) tumors showing positive results, by both, immunohistochemistry and PCR. This constitutes the first report from India showing implication of MCV in Merkel cell carcinomas. Moreover, this is one of the larger series of Merkel cell carcinomas, tested for MCV, by both immunohistochemistry and PCR, in this part of the world. These results further indicate that a slightly more number of such cases in India are likely to be caused by UV-linked damage, as opposed to Merkel cell polyomavirus mediated tumorigenesis, which is definitely implicated in a subset of cases.

Phylodynamics of Merkel-cell polyomavirus and human polyomavirus 6: A long-term history with humans.

New human polyomaviruses have been described in the last years, including the Merkel-cell polyomavirus (MCPyV; Human polyomavirus 5) and the Human polyomavirus 6 (HPyV6). Although their infection is usually asymptomatic, in immunocompromised host can cause life-threatening pathologies, such as the Merkel cell carcinoma, an aggressive skin neoplasia associated to the MCPyV. Despite being prevalent viruses in population, epidemiological data from South America are scarce, as well as the characterization of the viral types circulating and their origin. The aims of this work were to describe MCPyV and HPyV6 from environmental samples with different geographical origin and to analyze their phylogenetic and evolutionary histories, particularly for MCPyV. Partial and complete genome sequences were obtained from sewage samples from Argentina, Uruguay and Spain. A total number of 87 sequences were obtained for MCPyV and 33 for HPyV6. Phylogenetic analysis showed that MCPyV sequences distributed according to their geographic origin in Europe/North America, Africa, Asia, South America and Oceania groups, suggesting that viral diversification might have followed human migrations across the globe. In particular, viruses from Argentina associated with Europe/North America and South America genotypes, whereas those from Uruguay and Spain also grouped with Africa genotype, reflecting the origin of the current population in each country, which could arrive not only during ancient human migration but also during recent migratory events. In addition, the South American group presented a high level of clusterization, showing internal clusters that could be related to specific locations, such as French Guiana and Brazil or the Southern region into South America, such as Argentina and Uruguay, suggesting a long term evolutionary process in the region. Additionally, in this work, we carried out the first analysis about the evolutionary history of MCPyV trough the integration of phylogenetic, epidemiological and historical data. Since a strong association is observed between the phylogenetic relationships and the origin of the sampled population, this analysis was based on the hypothesis of co-divergence between the virus and human populations. This analysis resulted in a substitution rate of 5.1 × 10-8 s/s/y (∼5.1% of divergence per million years) for the complete genome of MCPyV, which is in the range of those estimated for other double-stranded DNA viruses. Regarding HPyV6, a South American group with clusterization was observed (sequences from Uruguay). Meanwhile, sequences from Argentina grouped with European ones (France and Spain) and remained separated from those isolated in China, USA or Australia. The analysis of viruses from the environment allowed us to deep characterize prevalent infections in different geographic regions, reveling that viruses circulating in each population reflected its origin and that there are specific lineages associated with South America.

Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes.

UNLABELLED: It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts. IMPORTANCE: The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years.

Phylogenetic and structural analysis of merkel cell polyomavirus VP1 in Brazilian samples.

Our understanding of the phylogenetic and structural characteristics of the Merkel Cell Polyomavirus (MCPyV) is increasing but still scarce, especially in samples originating from South America. In order to investigate the properties of MCPyV circulating in the continent in more detail, MCPyV Viral Protein 1 (VP1) sequences from five basal cell carcinoma (BCC) and four saliva samples from Brazilian individuals were evaluated from the phylogenetic and structural standpoint, along with all complete MCPyV VP1 sequences available at Genbank database so far. The VP1 phylogenetic analysis confirmed the previously reported pattern of geographic distribution of MCPyV genotypes and the complexity of the South-American clade. The nine Brazilian samples were equally distributed in the South-American (3 saliva samples); North American/European (2 BCC and 1 saliva sample); and in the African clades (3 BCC). The classification of mutations according to the functional regions of VP1 protein revealed a differentiated pattern for South-American sequences, with higher number of mutations on the neutralizing epitope loops and lower on the region of C-terminus, responsible for capsid formation, when compared to other continents. In conclusion, the phylogenetic analysis showed that the distribution of Brazilian VP1 sequences agrees with the ethnic composition of the country, indicating that VP1 can be successfully used for MCPyV phylogenetic studies. Finally, the structural analysis suggests that some mutations could have impact on the protein folding, membrane binding or antibody escape, and therefore they should be further studied.

Ecology of Merkel Cell Polyomavirus in Healthy Skin Among Individuals in an Asian Cohort.

BACKGROUND: Despite the oncogenic potential of Merkel cell polyomavirus (MCPyV), it has been found in the normal skin of healthy individuals; however, little is known about geographical variations in the ecology of MCPyV in this tissue. METHODS: This study included 284 Japanese participants. Sun-unexposed arm and sun-exposed forehead skin swab samples were obtained and analyzed for MCPyV infection, using quantitative polymerase chain reaction. Phylogenetic analyses were also conducted, based on the full-length genes encoding MCPyV large T antigen and viral protein 1. RESULTS: This study provides the first analyses of the age-specific prevalence and levels of MCPyV infection in normal skin. Steep increases in prevalence and viral load were observed in individuals aged >40 years. MCPyV infections with a high viral load were predominantly observed in the foreheads of subjects aged >60 years, among whom a high burden of MCPyV tended to persist. Phylogenetic analyses showed that all of the gene sequences obtained in this study clustered in a major clade, suggesting the existence of an Asian/Japanese genotype. CONCLUSIONS: This large study suggests that MCPyV infection with high viral loads is prevalent in the sun-exposed skin of elderly adults, making it necessary to follow up this cohort for possible transformation of MCPyV to a pathogenetic form.

Frequent and abundant Merkel cell polyomavirus detection in urban wastewaters in Italy.

Viruses strongly associated with human cancer have recently been detected in urban sewages and other water environments worldwide. The aim of the present study was to assess the presence of Merkel cell polyomavirus (MCPyV), a newly discovered, potentially oncogenic human virus, in urban sewage samples collected at wastewater treatment plants (WTPs) in Italy. A total of 131 raw sewage samples were collected from 21 WTPs in nine Italian regions and analyzed by both qualitative (PCR/nested) and quantitative (Real-Time qRT-PCR) methods. Of these, 66 samples (50.3 %) were positive for MCPyV by the qualitative assay. Quantitative data showed high viral loads in wastewaters (mean, 1.5E + 05 genome copies/liter). High concentrations of MCPyV were found in all WTPs under study, suggesting a wide circulation of the virus and thus the need for further studies to assess possible waterborne MCPyV transmission.

Phylogenetic analysis of Merkel cell polyomavirus based on full-length LT and VP1 gene sequences derived from neoplastic tumours in Japanese patients.

Most Merkel cell polyomavirus (MCPyV) gene sequences have been reported from Western countries and few data are available for the virus sequences from other geographical areas, especially Asia. Thus, we performed phylogenetic analyses based on the nucleotide sequences of the full-length large T-antigen (LT) and viral protein 1 (VP1) genes derived from a variety of cancers in Japanese patients and compared them with sequences from Caucasians. The LT and VP1 gene-based phylogenetic trees identified two main genetic clades. One clade comprised strains isolated from Caucasians, whereas all of the Japanese tumour-derived MCPyV strains belonged to another clade. These findings confirm that most of the MCPyV strains present in Japan form a clade, distinct from Caucasian strains.

Merkel cell polyomavirus (MCPyV) strains in Japanese merkel cell carcinomas (MCC) are distinct from Caucasian type MCPyVs: genetic variability and phylogeny of MCPyV genomes obtained from Japanese MCPyV-infected MCCs.

Most of merkel cell carcinomas (MCC), a rare, aggressive skin cancer with neuroendocrine features, harbor merkel cell polyomavirus (MCPyV). Seroepidemiological studies suggested high prevalence of MCPyV in the human population. More than ten sequence data on MCPyV strains in Japan have been available, whereas most sequence data were detected from patients living in Europe or European ancestry. Analysis of nine almost complete and 19 partial sequences from two oncogenes, small T antigen (ST) and large T antigen (LT) genomes obtained from 32 Japanese MCPyV-infected MCC revealed that each Japanese MCPyV-infected MCC harbored a specific MCPyV strain with some synonymous or, silent mutations and stop codons or deletions, but functional domains of T antigen had no amino acid changes. All stop codons were localized after retinoblastoma protein-binding domain. These Japanese MCPyV strains were very closely interrelated to themselves and a consensus sequence of Japanese strain was generated. Phylogenetic analysis of our nine sequences and 70 other sequences for ST and LT gene registered in GenBank indicated that Japanese or Asian MCPyV strains formed distinct clades from Caucasian clade, and phylogenetic tree of our nine and 75 other sequences for ST gene formed characteristic three clades and showed that all Japanese or Asian strains were included in the dominant clade. These suggested the possibility of geographically related genotypes of MCPyV. The genomic characterization of MCPyV variants will provide an important database and insights for illuminating their evolutional and biological differences.

The novel human polyomaviruses HPyV6, 7, 9 and beyond.

Since the discovery of Merkel cell polyomavirus and its causative association with Merkel cell carcinoma (MCC), six human polyomaviruses (HPyVs) have been identified that, so far, lack any disease association, which include the human polyomaviruses (HPyV) 6, 7, 9, 10 and 12 as well as the Saint Louis polyomavirus (STLPyV). PCR studies revealed that HPyV6 and HPyV7 are shed from the skin of healthy subjects and of patients suffering from various skin tumours. HPyV6, 7 and 9 were sporadically detected in body fluids and excretions of immunocompromised patients and healthy subjects. HPyV10 was identified in papillomavirus-induced anal condylomas, and variants of HPyV10, named MWPyV and MX polyomavirus (human) (MXPyV), as well as STLPyV were detected in faeces of diarrheal and healthy children. HPyV12 was discovered in organs of the digestive tract of patients suffering from various malignant diseases. Serological studies using capsomer-based or virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) revealed that HPyV6, 7, 9 and 12 are circulating in the human population. As all other HPyVs, the novel polyomaviruses encode small and large T antigens and thus are potentially oncogenic. However, several studies have revealed a lack of association of HPyV6, 7 and 9 with numerous human tumours. In the future, it will be important to unravel the cell types and body compartments of the novel HPyVs' reservoir and to search for possible associations with cancer and non-malignant diseases.

Novel polyomaviruses in South American bats and their relationship to other members of the family Polyomaviridae.

Bats are the natural reservoir of a variety of viruses, including a polyomavirus (PyV) from a North American brown bat. We investigated 163 spleen samples from 22 bat species from French Guiana for the presence of PyVs. In total, we detected 25 PyV-positive animals belonging to nine different bat species. Phylogenetic analysis was performed on the genomes of eight representative PyVs, and showed that the bat PyVs form three distinct lineages within the genus Orthopolyomavirus and are genetically different from the previously described North American bat virus. Interestingly, two lineages cluster with PyVs found in chimpanzees, orangutans and gorillas. In addition, one group of bat PyVs is genetically related to the human Merkel cell polyomavirus.

Detection of Merkel cell polyomavirus in cervical squamous cell carcinomas and adenocarcinomas from Japanese patients.

BACKGROUND: Merkel cell polyomavirus (MCPyV) was identified originally in Merkel cell carcinoma (MCC), a rare form of human skin neuroendocrine carcinoma. Evidence of MCPyV existence in other forms of malignancy such as cutaneous squamous cell carcinomas (SCCs) is growing. Cervical cancers became the focus of our interest in searching for potentially MCPyV-related tumors because: (i) the major histological type of cervical cancer is the SCC; (ii) the uterine cervix is a common site of neuroendocrine carcinomas histologically similar to MCCs; and (iii) MCPyV might be transmitted during sexual interaction as demonstrated for human papillomavirus (HPV). In this study, we aimed to clarify the possible presence of MCPyV in cervical SCCs from Japanese patients. Cervical adenocarcinomas (ACs) were also studied. RESULTS: Formalin-fixed paraffin-embedded tissue samples from 48 cervical SCCs and 16 cervical ACs were examined for the presence of the MCPyV genome by polymerase chain reaction (PCR) and sequencing analyses. PCR analysis revealed that 9/48 cervical SCCs (19%) and 4/16 cervical ACs (25%) were positive for MCPyV DNA. MCPyV-specific PCR products were sequenced to compare them with reference sequences. The nucleotide sequences in the MCPyV large T (LT)-sequenced region were the same among MCPyV-positive cervical SCCs and AC. Conversely, in the MCPyV viral protein 1 (VP1)-sequenced region, two cervical SCCs and three cervical ACs showed several nucleotide substitutions, of which three caused amino acid substitutions. These sequencing results suggested that three MCPyV variants of the VP1 were identified in our cases. Immunohistochemistry showed that the LT antigen was expressed in tumor cells in MCPyV-positive samples. Genotyping of human HPV in the MCPyV-positive samples revealed that infected HPVs were HPV types 16, 31 and 58 for SCCs and HPV types 16 and 18 for ACs. CONCLUSIONS: This study provides the first observation that MCPyV coexists in a subset of HPV-associated cervical cancers from Japanese patients. The prevalence of MCPyV in these lesions was close to that observed in the cutaneous SCCs. Further worldwide epidemiological surveys are warranted to determine the possible association of MCPyV with pathogenesis of cervical cancers.

Merkel cell polyomavirus DNA in immunocompetent and immunocompromised patients with respiratory disease.

Merkel cell polyomavirus (MCPyV) was identified originally in association with a rare but aggressive skin cancer, Merkel cell carcinoma. The virus has since been found in the respiratory tract of some patients with respiratory disease. However, the role of MCPyV in the causation of respiratory disease has not been established. To determine the prevalence of MCPyV in 305 respiratory samples from immunocompetent and immunocompromised patients and evaluate their contribution to respiratory diseases, specimens were screened for MCPyV using single, multiplex, or real-time PCR; co-infection with other viruses was examined. Of the 305 samples tested, 10 (3.27%) were positive for MCPyV. The virus was found in two groups of patients: in 6 (2%) nasopharyngeal aspirate samples from children aged 26 days to 7 months who were immunocompetent; and in 4 (1.3%) of nasopharyngeal aspirate samples taken from patients aged 41 to 69 years who were severely immunosuppressed from leukemia or transplant therapy. Both groups had upper or lower respiratory tract infection. Co-infections with other viruses were found in 30% of the MCPyV positive samples. The data present a pattern of infection similar to that seen with the polyomaviruses JC and BK in which the virus is acquired during childhood, probably by the respiratory route. The viruses then establish latency and become reactivated in the event of immunosuppression.