Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

High abundance of high-risk Human Papillomavirus genotypes in wastewater in Uruguay.

The aim of this study was to determine the frequency of Human Papillomavirus (HPV) genotypes in wastewater of Salto city, Uruguay, in order to obtain a general overview of the circulating genotypes in their population. HPV was detected in 34% (32/93) of the wastewater samples collected and analyzed during 2020/21 in Salto city, Uruguay. Thirty-three genotypes were observed, of which 16 presented read abundance higher than 1%, including both high-risk (HR) and low-risk (LR) genotypes. HR genotypes 31, 16, 58, 52, 33 and 59 were detected representing 40% (163,220 reads) of the total read abundance, with genotypes 31 (64,365), 16 (39,337) and 58 (36,332) being the most abundant. LR genotypes 72, 6, 11 and 40 were also detected in a high frequency, accounting for 37% (148,359) of the HPV reads. This study highlights the high frequency of HR genotypes of HPV, circulating in the population of Salto city which is a burden in public health mainly due to the devastating impact of cervical cancer in women.

Evaluation of low-cost viral concentration methods in wastewaters: Implications for SARS-CoV-2 pandemic surveillances.

In the pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) many strategies have been performed in order to control viral spread in the population and known the real-time situation about the number of infected persons. In this sense, Wastewater Based Epidemiology (WBE) has been applied as an excellent tool to evaluate the virus circulation in a population. In order to obtain reliable results, three low-cost viral concentration methods were evaluated in this study, polyethylene glycol (PEG) precipitation, skimmed milk flocculation (SM) and Aluminum polychloride flocculation, for Pseudomonas aeruginosa bacteriophage PP7 as a surrogate for non-enveloped viruses and Bovine Coronavirus (BCoV) as a surrogate for enveloped virus, with emphasis for SARS- CoV-2. Our results suggest that PEG precipitation for viral concentration, for both enveloped and non-enveloped virus from wastewater is an appropriate approach since it was more sensitive compared to SM flocculation and Aluminum polychloride flocculation. This methodology can be used for WBE studies in order to follow the epidemiology of the SARS-CoV-2 pandemic, mainly in developing countries where the economic resources are frequently limited.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

What is the state of the art of computer vision-assisted cytology? A Systematic Literature Review.

Cytology is a low-cost and non-invasive diagnostic procedure employed to support the diagnosis of a broad range of pathologies. Cells are harvested from tissues by aspiration or scraping, and it is still predominantly performed manually by medical or laboratory professionals extensively trained for this purpose. It is a time-consuming and repetitive process where many diagnostic criteria are subjective and vulnerable to human interpretation. Computer Vision technologies, by automatically generating quantitative and objective descriptions of examinations' contents, can help minimize the chances of misdiagnoses and shorten the time required for analysis. To identify the state-of-art of computer vision techniques currently applied to cytology, we conducted a Systematic Literature Review, searching for approaches for the segmentation, detection, quantification, and classification of cells and organelles using computer vision on cytology slides. We analyzed papers published in the last 4 years. The initial search was executed in September 2020 and resulted in 431 articles. After applying the inclusion/exclusion criteria, 157 papers remained, which we analyzed to build a picture of the tendencies and problems present in this research area, highlighting the computer vision methods, staining techniques, evaluation metrics, and the availability of the used datasets and computer code. As a result, we identified that the most used methods in the analyzed works are deep learning-based (70 papers), while fewer works employ classic computer vision only (101 papers). The most recurrent metric used for classification and object detection was the accuracy (33 papers and 5 papers), while for segmentation it was the Dice Similarity Coefficient (38 papers). Regarding staining techniques, Papanicolaou was the most employed one (130 papers), followed by H&E (20 papers) and Feulgen (5 papers). Twelve of the datasets used in the papers are publicly available, with the DTU/Herlev dataset being the most used one. We conclude that there still is a lack of high-quality datasets for many types of stains and most of the works are not mature enough to be applied in a daily clinical diagnostic routine. We also identified a growing tendency towards adopting deep learning-based approaches as the methods of choice.

Recurrent Dissemination of SARS-CoV-2 Through the Uruguayan-Brazilian Border.

Uruguay is one of the few countries in the Americas that successfully contained the coronavirus disease 19 (COVID-19) epidemic during the first half of 2020. Nevertheless, the intensive human mobility across the dry border with Brazil is a major challenge for public health authorities. We aimed to investigate the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains detected in Uruguayan localities bordering Brazil as well as to measure the viral flux across this ∼1,100 km uninterrupted dry frontier. Using complete SARS-CoV-2 genomes from the Uruguayan-Brazilian bordering region and phylogeographic analyses, we inferred the virus dissemination frequency between Brazil and Uruguay and characterized local outbreak dynamics during the first months (May-July) of the pandemic. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 Brazilian lineages B.1.1.28 and B.1.1.33 into Uruguayan localities at the bordering region. The most probable sources of viral strains introduced to Uruguay were the Southeast Brazilian region and the state of Rio Grande do Sul. Some of the viral strains introduced in Uruguayan border localities between early May and mid-July were able to locally spread and originated the first outbreaks detected outside the metropolitan region. The viral lineages responsible for Uruguayan urban outbreaks were defined by a set of between four and 11 mutations (synonymous and non-synonymous) with respect to the ancestral B.1.1.28 and B.1.1.33 viruses that arose in Brazil, supporting the notion of a rapid genetic differentiation between SARS-CoV-2 subpopulations spreading in South America. Although Uruguayan borders have remained essentially closed to non-Uruguayan citizens, the inevitable flow of people across the dry border with Brazil allowed the repeated entry of the virus into Uruguay and the subsequent emergence of local outbreaks in Uruguayan border localities. Implementation of coordinated bi-national surveillance systems is crucial to achieve an efficient control of the SARS-CoV-2 spread across this kind of highly permeable borderland regions around the world.

Environmental Surveillance through Next-Generation Sequencing to Unveil the Diversity of Human Enteroviruses beyond the Reported Clinical Cases.

The knowledge about circulation of Human Enteroviruses (EVs) obtained through medical diagnosis in Argentina is scarce. Wastewater samples monthly collected in Córdoba, Argentina during 2011-2012, and then in 2017-2018 were retrospectively studied to assess the diversity of EVs in the community. Partial VP1 gene was amplified by PCR from wastewater concentrates, and amplicons were subject of next-generation sequencing and genetic analyses. There were 41 EVs detected, from which ~50% had not been previously reported in Argentina. Most of the characterized EVs (60%) were detected at both sampling periods, with similar values of intratype nucleotide diversity. Exceptions were enterovirus A71, coxsackievirus B4, echovirus 14, and echovirus 30, which diversified in 2017-2018. There was a predominance of types from EV-C in 2017-2018, evidencing a common circulation of these types throughout the year in the community. Interestingly, high genetic similarity was evidenced among environmental strains of echovirus 30 circulating in 2011-2012 and co-temporal isolates obtained from patients suffering aseptic meningitis in different locations of Argentina. This study provides an updated insight about EVs circulating in an important region of South America, and suggests a valuable role of wastewater-based epidemiology in predicting outbreaks before the onset of cases in the community.

Detection, risk factors and molecular diversity of norovirus GIII in cattle in Uruguay.

Uruguay is a leading exporter of bovine meat and dairy products, and cattle production is one of the principal economic backbones in this country. A main clinical problem faced by livestock farmers is neonatal calf diarrhea (NCD); however, causes of NCD have not been extensively studied in Uruguay. Bovine norovirus (BoNoV) has been proposed as one of the possible etiologies of NCD as experimentally infected calves developed diarrhea and enteropathy, although limited information is available from field surveys. The aims of this study were to determine the frequency of infection, to investigate possible risk factors, and to determine the molecular diversity of BoNoV in Uruguay. A total of 761 samples of feces or intestinal contents from dairy and beef calves were analyzed through RT-qPCR. The overall frequency of detection of BoNoV was 66.1% with higher frequency in dairy (70.5%) than beef (15.9%) calves (p < 0.01). BoNoV was detected similarly in diarrheic (78.8%) and non-diarrheic (76.2%) dairy calves (p = 0.50). Calves ≤2 weeks of age (84%) were infected more often than older (62.7%) calves (p < 0.01). Phylogenetic analysis confirmed the presence of GIII.1 and GIII.2 genotypes. In addition, we reported the circulation of recombinant strains and the detection of a strain with the recently described novel VP1 genotype. This study represents the first report describing the circulation, the associated risk factors, and the molecular diversity of BoNoV in Uruguay.

Phylogenetic Analyses of Rotavirus A from Cattle in Uruguay Reveal the Circulation of Common and Uncommon Genotypes and Suggest Interspecies Transmission.

Uruguay is one of the main exporters of beef and dairy products, and cattle production is one of the main economic sectors in this country. Rotavirus A (RVA) is the main pathogen associated with neonatal calf diarrhea (NCD), a syndrome that leads to significant economic losses to the livestock industry. The aims of this study are to determine the frequency of RVA infections, and to analyze the genetic diversity of RVA strains in calves in Uruguay. A total of 833 samples from dairy and beef calves were analyzed through RT-qPCR and sequencing. RVA was detected in 57.0% of the samples. The frequency of detection was significantly higher in dairy (59.5%) than beef (28.4%) calves (p < 0.001), while it did not differ significantly among calves born in herds that were vaccinated (64.0%) or not vaccinated (66.7%) against NCD. The frequency of RVA detection and the viral load were significantly higher in samples from diarrheic (72.1%, 7.99 log10 genome copies/mL of feces) than non-diarrheic (59.9%, 7.35 log10 genome copies/mL of feces) calves (p < 0.005 and p = 0.007, respectively). The observed G-types (VP7) were G6 (77.6%), G10 (20.7%), and G24 (1.7%), while the P-types were P[5] (28.4%), P[11] (70.7%), and P[33] (0.9%). The G-type and P-type combinations were G6P[11] (40.4%), G6P[5] (38.6%), G10P[11] (19.3%), and the uncommon genotype G24P[33] (1.8%). VP6 and NSP1-5 genotyping were performed to better characterize some strains. The phylogenetic analyses suggested interspecies transmission, including transmission between animals and humans.

Rotavirus contamination of surface waters from the northwest of Argentina.

Fecal pollution of water is a serious concern because it is associated with the transmission of pathogens. The aim of this study was to analyze the occurrence of group A rotavirus (RVA) in surface waters from the Arias-Arenales River in Salta, a northern city in Argentina, and to define possible sources of fecal viral pollution. A total of 116 water samples were analyzed and RVA was detected in 3.4% (95% CI: 0.1-7.0%), with concentrations ranging from 1.9 × 105 to 3.8 × 106 genome copies per liter. RVA strains were characterized as G1P[8], G4P[8] and G9P[8], which are common genotypes circulating in the local population. The Arias-Arenales River presented unusual and sporadic contamination by RVA, originated from stormwater discharges and a variety of non-identified sources, and support the essential need of viral indicators for enhanced monitoring of water quality.

Detection, Quantification, and Microbial Risk Assessment of Group A Rotavirus in Rivers from Uruguay.

The aim of this study was to detect, quantify, and assess the risk of infection and illness for Group A Rotavirus (RVA) in the watersheds of the Santa Lucia and Uruguay rivers in Uruguay. Monthly sampling was carried out for one year in six sites in the watershed of the Santa Lucía River and four in the Uruguay River. All the collection sites are used for recreational activities. Viral concentration was performed with the adsorption-elution method, and detection and quantification of RVA was carried out by TaqMan quantitative PCR (qPCR). Quantitative microbial risk assessment was applied to estimate the daily and annual risk of RVA infection, as well as the daily risk of illness considering direct exposure through recreational activity. RVA was detected in 42% (20/48) of the analyzed samples in the Uruguay River and 40% (29/72) in the Santa Lucía River. The virus was present in all the analyzed points in both watersheds. A pattern of seasonality, characterized by a higher detection frequency of the virus during coldest month of the year, was observed in both basins. The mean concentration for RVA was 1.3 × 105 genomic copies/L. The microbiological risk assessment shows that Santa Lucía watershed presented the highest daily risk of infection (6.41E-01) and illness (3.20E-01) estimated for the point downstream of Florida City; meanwhile for Uruguay River, the highest probabilities of infection (6.82E-01) and illness (3.41E-01) were estimated for the collection site for drinking water intake in Salto city. These results suggest that RVA contamination of these important rivers negatively impact on their microbiological quality since they are used for recreation and drinking water intake, demonstrating that the disposal of waste from cities located in their riverside confers a constant threat of infection for the general population, especially for children.

Hand-foot-and-mouth disease in uruguay: Coxsackievirus A6 identified as causative of an outbreak in a rural childcare center.

To know the epidemiological context of hand-foot-and-mouth disease (HFMD) in a region of Uruguay and to identify the Enterovirus responsible for an outbreak in a rural childcare center in 2018. Swab samples from skin lesions and/or stools samples were collected from children suffering HFMD during an outbreak in a rural childcare center. Samples were subject to viral RNA extraction and reverse-transcription polymerase chain reaction towards VP1 coding segment, to identify the Enterovirus type by sequencing and phylogenetic analysis. Total of 149 cases of HFMD affecting 98 boys and 51 girls were reported in Salto Province-Uruguay in 2018. Total 60% of the cases were originated from outbreaks, which occurred in ten educative and childcare institutions from both urban and rural areas. Coxsackievirus-6 (CV-A6) was identified as responsible for one of the rural outbreaks. Uruguayan strains were more related to strains reported in Russia, Turkey, and Germany (2014-2017) than to strains reported in Brazil and Argentina from 2015 to 2016. This is the first report of CV-A6-associated HFMD in Uruguay, evidencing a wide geographic range of the virus in the Latin American region. Our report also warns about CV-A6-associated HFMD during winter, contrarily to most reports that register HFMD during summer and fall seasons.

Description of a human Bocavirus recombinant strain in the Americas.

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.

Bovine coronavirus in Uruguay: genetic diversity, risk factors and transboundary introductions from neighboring countries.

Bovine coronavirus (BCoV) is a recognized cause of severe neonatal calf diarrhea, with a negative impact on animal welfare, leading to economic losses to the livestock industry. Cattle production is one of the most important economic sectors in Uruguay. The aim of this study was to determine the frequency of BCoV infections and their genetic diversity in Uruguayan calves and to describe the evolutionary history of the virus in South America. The overall detection rate of BCoV in Uruguay was 7.8% (64/824): 7.7% (60/782) in dairy cattle and 9.5% (4/42) in beef cattle. The detection rate of BCoV in samples from deceased and live calves was 10.0% (6/60) and 7.6% (58/763), respectively. Interestingly, there was a lower frequency of BCoV detection in calves born to vaccinated dams (3.3%, 8/240) than in calves born to unvaccinated dams (12.2%, 32/263) (OR: 4.02, 95%CI: 1.81-8.90; p = 0.00026). The frequency of BCoV detection was higher in colder months (11.8%, 44/373) than in warmer months (1.5%, 3/206) (OR: 9.05, 95%CI: 2.77-29.53, p = 0.000013). Uruguayan strains grouped together in two different lineages: one with Argentinean strains and the other with Brazilian strains. Both BCoV lineages were estimated to have entered Uruguay in 2013: one of them from Brazil (95%HPD interval: 2011-2014) and the other from Argentina (95%HPD interval: 2010-2014). The lineages differed by four amino acid changes, and both were divergent from the Mebus reference strain. Surveillance should be maintained to detect possible emerging strains that can clearly diverge at the antigenic level from vaccine strains.

Microbial Source Tracking Analysis Using Viral Indicators in Santa Lucía and Uruguay Rivers, Uruguay.

The aim of this study was to determine the origin (human, bovine or porcine) and the concentration of the fecal sources of contamination in waters from Santa Lucía basin and Uruguay River in Uruguay by using host-specific viral markers (adenoviruses and polyomaviruses) as microbial source tracking (MST). Between June 2015 and May 2016, monthly collections of surface water samples were performed in six sites in Santa Lucía basin and four sites in Uruguay River (n = 120 samples). Viral concentration was carried out using an absorption-elution method. Detection and quantification of human and porcine adenovirus (HAdV and PAdV, respectively) and human and bovine polyomavirus (HPyV and BoPyV, respectively) were performed by quantitative PCR (qPCR). To evaluate the infectivity of circulating HAdV, an integrated cell culture-qPCR (ICC-qPCR) was used. A logistic regression analysis was carried out to estimate the influence of environmental variables on the virus presence in surface waters. Overall, HAdV was the prevalent (18%; 21/120) followed by BoPyV (11%; 13/120) and HPyV (3%; 3/120), whereas PAdV was not detected in this study. The mean concentration ranged from 1.5 × 104 genomic copies/L (gc/L) for HAdV to 1.8 × 102 gc/L for HPyV. Infective HAdVs were observed in two out of ten analyzed samples. A significant effect of environmental temperature (p = 0.001) and river (p = 0.012) on the presence of human viruses was found. These results suggest that fecal contamination could affect the water quality of these rivers, showing deficiencies in the procedure of sewage discharge from regional cities, livestock and dairy farms.

Bovine Astrovirus Surveillance in Uruguay Reveals High Detection Rate of a Novel Mamastrovirus Species.

Viral infections affecting cattle lead to economic losses to the livestock industry worldwide, but little is known about the circulation, pathogenicity and genetic diversity of enteric bovine astrovirus (BoAstV) in America. The aim of this work was to describe the prevalence and genetic diversity of enteric BoAstV in dairy cattle in Uruguay. A total of 457 fecal and 43 intestinal contents from dairy calves were collected between July 2015 and May 2017 and tested by RT-PCR, followed by sequencing and phylogenetic analyses of the polymerase and capsid regions. Twenty-six percent (128/500) of the samples were positive. Three different species within the Mamastrovirus genus were identified, including Mamastrovirus 28, Mamastrovirus 33 (3 samples each) and an unclassified Mamastrovirus species (19 samples). The unclassified species was characterized as a novel Mamastrovirus species. BoAstV circulates in Uruguayan dairy cattle with a high genetic diversity. The eventual clinicopathological significance of enteric BoAstV infection in cattle needs further investigation.

Description of a human Bocavirus recombinant strain in the Americas

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.

An Environmental Surveillance in Uruguay Reveals the Presence of Highly Divergent Types of Human Enterovirus Species C and a High Frequency of Species A and B Types.

Information about Human Enterovirus circulation in Uruguay is scarce. The aim of this study was to generate the first description about their circulation in the country through the study of sewage samples collected before and after the switch from Oral Poliovirus Vaccine to Inactivated Poliovirus Vaccine. Viruses were concentrated by an adsorption-elution to a negatively charged membrane, and real-time quantitative PCR and qualitative PCR methods were used to detect, quantify, and characterize enteroviruses. Positive samples were inoculated in RD cells and two passages were performed. Additionally, RD+ samples were subsequently passed onto L20B cells. Human Enteroviruses were detected in 67.6% of the samples, with concentrations between 4.9 and 6.6 Log10 genomic copies per liter. 10% of positive samples replicated in RD cells, of which none in L20B cells. Molecular characterization of Human Enterovirus strains directly detected from sewage sample concentrates allowed the identification of highly divergent members of species C such as Enterovirus C99 and Coxsackievirus A13, as well as the frequent detection of species A and B members (particularly Coxsackievirus A16 and Echovirus 6, respectively). Other detected types were Coxsackievirus A2, A22, B1, B5, Echovirus 5, and 9. The characterization of viruses isolated in cell culture revealed the presence of Echovirus 6 and Coxsackievirus B3. Despite the absence of poliovirus, a wide circulation of different enterovirus types was evidenced in Uruguayan sewage samples, highlighting that the local populations are exposed to different kinds of diseases originated by several human enterovirus.

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.

Human Bocavirus: Detection, Quantification and Molecular Characterization in Sewage and Surface Waters in Uruguay.

Human bocavirus (HBoV) infections are related to respiratory and gastroenteric diseases. The aim of this study was to investigate the presence of HBoV in both sewage and surface waters in Uruguay. Sixty-eight sewage samples from the cities of Salto, Paysandú, Bella Unión, Fray Bentos, Treinta y Tres and Melo and 36 surface water samples from the cities of Salto, Florida and Santa Lucía were studied. HBoV was screened by multiplex qPCR for the detection of the four subtypes, followed by monoplex qPCRs for the independent quantification of each subtype. A qualitative PCR followed by DNA sequencing and phylogenetic analysis was carried out for molecular characterization of HBoV strains. HBoV was present in a high frequency (69%) in sewage and only one positive sample (3%) was found in surface water. Concerning sewage samples, HBoV1 was detected in 11 (23%) out of the 47 positives samples, with a mean concentration of 8.2 × 104 genomic copies/Liter (gc/L), HBoV3 was detected in 35 (74%) of the positive samples with a mean concentration of 4.1 × 106 gc/L and subtypes 2 and/or 4 were detected in 39 (83%) of the positive samples with a mean concentration of 7.8 × 106 gc/L. After the phylogenetic analysis performed by a Bayesian approach, the four HBoV subtypes were confirmed. This is the first study determining a high frequency of HBoV and the presence of the four HBoV subtypes in aquatic matrices in Latin America, mainly in sewage. Although HBoV was scarcely detected in surface water, a waterborne transmission is likely to occur if people enter in contact with polluted surface waters for recreational activities such as fishing or swimming since an elevated frequency of HBoV was detected in raw sewage which is usually directly discharged into surface waters.