Occurrence of Polyomaviruses in Recreational Freshwaters from Southern Brazil

There is little information about the transmission pathways of polyomaviruses. Since many of them are regularly found in urine and feces, the possibility that the waterborne route could transmit these viruses has not been explored. The aim of this study was to evaluate the presence of human polyomaviruses JCPyV, BKPyV, MCPyV, STLPyV and TSPyV along the Belo River (Caxias do Sul-Rio Grande do Sul-RS, Brazil). Four points were investigated, with 13 monthly samplings (March/2015 - March/2016). A total of 52 samples were concentrated by ultracentrifugation, total genomic material was extracted using commercial extraction kit and human polyomaviruses were investigated through real time polymerase chain reaction. A total of 32/52 (61.5%) positive samples were found. Only JCPyV and BKPyV were identified, and they were present in all sampling points. The average viral load of JCPyV was 7E+05 copies/mL and of BKPyV was 5E+04 copies/mL after particle concentration process. Finding such polyomaviruses in this environment suggests contamination through human waste and reinforce the notion that fecal-oral route may represent an important transmission mode for these viruses.


Introduction
The Polyomaviridae family consists of 100 viruses described so far, of which 13 can infect humans and 87 infect mainly fish, birds and other mammals [1][2][3]. Their genomes have relatively high similarity to each other and are homologous to SV40 polyomavirus [4]. Polyomaviruses have DNA genomes with approximately 5k bases that is divided into 3 regions named early, late and noncoding control region [1,2]. The first polyomavirus described was BK (BKPyV), which causes persistent subclinical infection in the kidneys of healthy individuals. In immunocompromised individuals, especially those undergoing renal transplantation, reactivation of BKPyV may cause clinical complications such as BKPyV-associated nephropathy and loss of the graft in the transplanted individual [5]. JC polyomavirus (JCPyV), discovered almost at the same time as BKPyV, is the etiologic agent of an extremely severe disease affecting also immunocompromised individuals, the progressive multifocal leukoencephalopathy (PML) [1,3]. MCPyV (Merkel-cell carcinoma polyomavirus), described in 2008 is the etiologic agent of an aggressive neuroendocrine carcinoma (Merkel Cell Carcinoma -MCC) that targets Merkel cells [6]. TSPyV was identified and sequenced from fragments of injured skin of an immunocompromised individual after cardiac transplantation with clinical evidence of a very rare skin cancer named Tricodisplasia spinulosa [7]. And, one of the most recent

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The mode of transmission of polyomaviruses is still poorly understood, and multiple routes of infection including the contact with urine, feces, saliva and blood are likely [9][10][11]. Therefore, finding such viruses in water effluents may add a piece to the understanding the polyomaviruses transmission routes. Another concern regarding the presence of these viruses in waters regards to its oncogenic potential [12]. Waterborne diseases represent a serious public health problem in developing and undeveloped countries [13]. Secondary data from health reporting systems highlight viruses as as responsible for more than 7% of the cases of pathologies associated with water infection [14]. The frequency of these pathogens in water matrices and the negative effects on public health indicate the need to dispose of the wastewater before it is discharged into the environment [15]. Recreational water activities are common practice in countries with tropical climate as Brazil and contamination of water bodies can occur by polluting sources as domestic, agricultural and industrial effluents. In fact, recreational use is the main responsible for the onset of illnesses caused by contact with water, leading to disease outbreaks mainly during holidays and periods of warm weather [16,17].
In Brazil, the quality of water for recreational activities must Belo River is one of the tributaries of the Caí River, located in the southwest of the city of Caxias do Sul and contributes to the formation of the Guaíba basin, responsible for the public supply of the capital Porto Alegre. This river has its sources in the urbanized and industrialized areas of Caxias do Sul, thus contributing with domestic and industrial effluents. During its journey, the river is also impacted with effluents from agricultural activities and livestock and close to these activities the waterway is used for leisure [26].
The aim of the present study was to evaluate the presence of human polyomaviruses in samples of the water source collected at distal points along the river flow.

Materials and Methods
The Belo River in Caxias do Sul, RS drains 21% of the city's ur-  Table 1. Concentration of viral particles was performed by the ultracentrifugation protocol described by Ruskowski [27]. Briefly, a 36mL aliquot was separated from each sample and centrifuged at 41,000Xg at 8oC for three hours. The pellet was re-suspended in 2 mL TE buffer (Tris-EDTA pH 8.0). To allow the material to be used in further studies, total genetic material (RNA and DNA) was extracted using the BioPur® kit from an initial volume of 200μL according to manufacturer instruction. cDNA was synthesized and template containing both DNA + cDNA was used for further experiments. TSPyV molecular detection was performed [28] using TaqMan method. Detection of JCPyV and BKPyV was performed according to Pal et al. [29] also using TaqMan methodology.

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The MCPyV primers were described by Goh et al. [30]  sented a VL of 5E+04 copies/mL (8E+00 to 1.8E+03 copies/mL) after particle concentration process. The average annual temperature of the municipality is 16.3°C. These findings agree to the high prevalence of polyomavirus shedding in Brazil, ranging between 62% and 80% in immunocompetent and immunocompromised individuals respectively [36][37][38].
Besides the urine shed, it was already demonstrated that polyomaviruses are also found in feces of children and adults, with BKPyV being the most frequent in young children [10,39].
Therefore, intense human activity due to the agricultural, urban and livestock runoffs at or near these collection points would explain such level of contamination since in addition to recreation, the river is used as a resource for survival and work [40]. Besides the present study, several groups have shown high prevalence of HPyVs in sewage and other water environments worldwide [41][42][43]. Polyomaviruses have also been described as being very resistance over time and is also resistant to treatments at low pH [41,44]. This would explain why polyomaviruses are resistant to most sewage treatments protocols, in particular to chlorine treatment as JCPyV and BKPyV were also found in P4, after a sewage plant. Rivers located in the Mediterranean area in Barcelona (Spain) and Rio de Janeiro (Brazil), which receives domestic sewage from urbanized areas also were subject of analysis, revealing 100% positivity for JCPyV [45]. The presence of the JCPyV in water from the metropolitan area of Berlin was also demonstrated and agres to that JCPyV is the most prevalent polyomavirus in natural environments due to contamination with human wastes [46]. In agreement to our results, it was recently described the presence of JCPyV and BKPyV in water samples from a nearby region in Porto Alegre [47].
We did not observe MCPyV, TSPyV, and STLPyV at any sampling point. MCPyV virus was found in 50.3% of samples from urban sewage collected at wastewater treatment plants in several regions in Italy [43] and in 50% in Barcelona (Spain) and Rio de Janeiro (Brazil) [45], suggesting that it may also be transmitted by fecal oral route. Although there is no report regarding to the MCPyV shedding in Brazil, approximately 20% of excretion was observed in the immunocompetent population (Urbano et al., unpublished data), which is much lower than the observed to BKPyV and JCPyV.
Nevertheless, no positivity to MCPyV at all in our samples was an unexpected finding. It is possible that these viruses were present

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in the samples but at such low level that our methods were unable to detected them. To our knowledge, there is no data regarding to the rate of STLPyV in urine or TSPyV urine and fecal excretion in healthy individuals. Since these viruses also have high prevalence in general population [48][49][50], one may expect to find them at least in a proportion of the samples if these viruses would also use the fecal-oral route. In fact, it has been proposed that the respiratory tract may be the main route for TSPyV infection [51] and not the fecal-oral as it is suggested for JCPyV, BKPyV and MCPyV.
Freshwater is considered appropriate for use when it is in accordance with the bacterial levels accepted by CONAMA [52]. However, there is no treatment for viral removal in WWTP suggested by CONAMA and the presence of viruses can represent a public health problem [52][53][54]. As an example, RVA, NoV and HAdV were detected in the surface waters of the Rodrigo de Freitas Lagoon, which is considerate suitable for recreation according to the microbiological level's evaluation [53]. Some authors have suggested that the quality of river water (in terms of viruses presence) may be closely linked to the health status of the local population [54][55][56].
However, studies that also estimated viral load could not establish any relationship between viral load and probability of transmission [53,56]. In sum, as already seen in other regions, polyomaviruses are common in water matrices and we found them also in Belo Riv-