Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration

Waterborne diseases, such as gastroenteritis, represent a serious public health problem in developing countries. Several studies in literature confirm that there is a relation between the lack of basic sanitation and the dissemination of viral waterborne diseases, such as the ones caused by adenoviruses (AdV). The poorer the sanitation systems, the greater the impact of these illnesses. In addition, the lack or ineffectiveness of sewage treatment systems aggravates and leads to the deterioration of aquatic ecosystems (Heller et al. 2003; Prado & Miagostovich 2014; Spilki 2015).

The use of water for recreation purposes is a common practice in tropical climate countries. The contamination of these water bodies can be generated by several polluting sources, such as domestic, agricultural, and industrial effluents, the first being the main factor responsible for diseases caused by contact with water. During the summer, waterborne disease outbreaks increase due to recreational activities and contact with water contaminated by domestic sewage. Therefore, the quality of the recreational waters should be analyzed more thoroughly, due to the risk offered to human health by direct and prolonged exposure to pathogenic organisms, such as AdV (Bosch et al. 2008; Sinclair et al. 2009; Lodder et al. 2015).

Human adenoviruses (HAdV) are non-enveloped viruses with icosahedral capsid 60–100 nm in diameter and DNA double-stranded genome, and they belong to the family Adenoviridae. This family is composed of five genera; among them is the genus Mastadenovirus, which includes 57 serotypes of HAdV that are subdivided into seven species that go from A to G. In addition to HAdV, bat, bovine (BAdVs), canine, equine, murine (MAdVs), sheep, swine, simian and viruses from other hosts are included in this genus. HAdVs may have different tropisms, causing a wide variety of diseases that include acute febrile pharyngitis, respiratory infections, acute conjunctivitis, cystitis, gastroenteritis, and systemic infections in immunocompromised patients. HAdV-A, B, C, and E affect the respiratory system, while species D, F and G are more prone to cause gastrointestinal infections (Ghebremedhin 2014; Santos & Soares 2015).

Species classification and AdV serotypes found in aquatic environments help to identify different sources of contamination, as well as to have a better knowledge of their occurrence in water, thus improving the assessment of infectious risk for humans exposed to specific types of HAdV (Kidd et al. 1990; García 2006; Robinson et al. 2013). AdVs are stable in the aquatic environment and may remain potentially infectious for a long time in water, including diverse environmental conditions and water and sewage treatment processes that are normally intended for bacterial control. Moreover, they can adsorb solid particles of the aquatic environment, promoting greater stability. Due to all these characteristics, AdVs may be used as viral markers for detection of fecal contamination in water (García 2006; Yates et al. 2006; Ogorzaly et al. 2015).

Belo Stream is one of the tributaries of the Caí River Basin, located in the mountainous region of the state of Rio Grande do Sul (RS), in southern Brazil. Its springs are located in the city of Caxias do Sul, in both urbanized and industrialized areas, which add to domestic and industrial effluents. In the countryside, the impacts are the result of agricultural activities and animal husbandry when the area near to its mouth is used for recreation. Until now, no investigation has been carried out on the presence of enteric viruses in this region.

In this context, the aim of the present work was to compare results obtained from concentrated and unconcentrated samples for the presence and diversity of the mammalian polymerase DNA gene of AdV along a river known to be polluted by human and animal waste.

Sampling was performed from March 2015 to April 2016, monthly, at four points along Belo Stream in Caxias do Sul (RS, Brazil) as shown in Figure 1, in accordance with the Brazilian Association of Technical Standards (ABNT) 9897 (Planning of sampling of liquid effluents and receiving bodies) and ABNT 9898 (Preservation and sampling techniques of liquid effluents and receiving bodies) (ABNT 1987a, 1987b). Belo Stream drains 21% of the city urban perimeter; it has an area of 75.10 km² and a perimeter of 63.11 km.

A total of 55 samples were collected from four different points: 13 samples from P1, and 14 from P2, P3, and P4. A volume of 500 mL of surface water was collected in sterile flasks. These samples were stored at 4 °C until the concentration process took place. A short description of the points is given in Table 1.

A total of 55 samples containing 500 mL were collected. From these 500 mL, 36 mL were concentrated by ultracentrifugation method, following the protocol: An aliquot was centrifuged at the rate of 41,000 × g at 8 °C for 3 h. Thereafter, the precipitate was resuspended in 2 mL of Tris-EDTA buffer (pH 8.0), and vigorously homogenized in vortex for 1 min. The resuspended samples were aliquoted and stored in microtubes at −80 °C until the DNA extraction process. Unconcentrated samples (55) were also evaluated and aliquoted into microtubes and stored at −80 °C for further DNA extraction.

Genetic material from the concentrated and unconcentrated samples was extracted with a BioPur^® Kit from an initial volume of 200 μL according to the instructions described by the manufacturer. The final elution was performed in microtubes free of DNAse/RNAse, in which they were stored and maintained at −80 °C until further processing.

To evaluate the presence of different AdV species, a partial sequence of the DNA polymerase (pol) gene was amplified by nested polymerase chain reaction (nested-PCR). Measurements of the reaction were carried out for a final volume of 50 μL, as follows: 1 μL of Pol-F primer (5′-CAGCCKCKGTTRTGYAGGGT-3′), 1 μL of the primer Pol-R (5′-GCHACCATYAGCTCCAACTC-3′), both primers at 20 pmoles, 18 μL of DNAse/RNAse free water, 25 μL of GoTaq^® Green Master Mix (Promega, USA) and 5 μL of nucleic acid extracted from each sample. After initial incubation at 94 °C for 5 min, 30 cycles of amplification were performed. These consisted of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s, extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. Amplification was performed in a Bio-Rad^® thermal cycler. The second PCR reaction was performed using the same reagents and quantities from the first one, as well as the same amplification cycles carried out with the products of the first PCR; however, both primers were replaced with Pol-nF (5′-GGGCTCRTTRGTCCAGCA-3′) and Pol-nR (5′-TAYGACATCTGYGGCATGTA-3′) (Li et al. 2010).

PCR products were analyzed on 2% agarose gel, 0.5 mg of ethidium bromide/mL was added and the running time was 60 min at 70 V. Molecular sizes of the products were compared with a 100 bp DNA standard (Ludwig brand). The bands stained with ethidium bromide were visualized in UV light; subsequently images were photographed with an Easy Doc 200 UV transilluminator equipment.

Samples testing positive for the DNA polymerase gene by nested-PCR were submitted to DNA sequencing for species identification. Sequencing was carried out by Ludwig Biotec (Sequencing Service) using an automated sequencer (ABI-PRISM 3500 Genetic Analyzer Applied Biosystems). Phylogenetic analysis was performed by comparing the genomic sequences obtained by direct DNA sequencing with other nucleotide fragments available from GenBank, in accordance with the Neighbor-Joining methodology (Saitou & Nei 1987). A phylogenetic tree was elaborated from the calculation of evolutionary distances, using the Kimura-parameter 2 method (Kimura 1980) and operating with Molecular Evolutionary Genetics Analysis software version 5 (MEGA5) (Tamura et al. 2011).

The AdV DNA polymerase gene was detected in 43.6% (24/55) of the concentrated samples. On the other hand, 25.4% (14/55) of the unconcentrated samples were positive for AdV. Thus, more positive samples were obtained when submitted to the concentration step in contrast to the unconcentrated ones.

The greatest number of positive samples for both concentrated and unconcentrated samples was from the P3 site (concentrated samples 57.1% (8/14); unconcentrated: 50.0% (7/14)) as seen in Tables 2 and 3. The sites with the lowest positivity were P2 (21.4% (3/14)) and P4 (7.1% (1/14)) (Tables 2 and 3) for concentrated and unconcentrated samples, respectively.

Among the concentrated samples (Figure 2), AdV was found in at least one collection point every month. Furthermore, October and November of 2015 and February of 2016 were the months which presented the highest number of positive results for AdV (three samples tested positive in each of these months). Based on unconcentrated samples (Figure 3), July, October, and November of 2015 and March of 2016 presented greater positivity, as two positive samples were detected in each of these months.

Based on the results obtained by nucletotide sequencing and phylogenetic analysis, it was found that the most prevalent species of AdV was F in both unconcentrated and concentrated samples, being found at a rate of 16.4 and 12.7%, respectively. On the other hand, the BAdV species was the one with less predominance in concentrated samples, being present in only one sample (1.8%) from P1. In the unconcentrated samples group D was the one less found, being in only 1.8% of the samples. It is important to point out that the HAdV-E, BAdV and MadV species were only identified among the water samples that were submitted to the concentration method (Tables 2 and 3, and Figure 4). Therefore, it may be claimed that the concentration method allows superior results to be obtained, since a great diversity of AdV species was identified, including from other hosts in addition to the human ones.

Water resources suffering anthropogenic influence can harbor immense microbiological diversity, as they can be contaminated by different effluent sources. AdV is often found in aquatic ecosystems, since current sewage treatment methods are not fully effective in the removal of viral particles. In the present study, the presence of AdV was detected in 43.6% of the concentrated samples. Taking into consideration this study and the sites investigated, the occurrence of AdV was higher than in other studies. Aw & Gin (2011) evaluated AdV in surface waters in Singapore and reported positivity in 38% of the samples. Kundu et al. (2013) found the presence of AdV in 11% of recreational water samples from the USA. However, Maurer et al. (2015) found superior results compared with this study. They described the presence of the AdV genome in 77.8% of samples collected in recreational waters in southern Brazil.

Usually, when comparing concentrated with unconcentrated samples, the number of samples found positive is higher after the concentration stage, which allows the finding of a greater diversity of AdV species. This is an important fact to consider when comparing both protocols, since it is not always possible to find the same diversity if samples are not concentrated. Therefore, the concentration method is fundamental to surface water samples, because it allows an increase in the frequency of positive samples. Furthermore, unconcentrated samples contain a number of substances that inhibit or interfere with the viral detection and quantification, due to the receipt of different effluents, such as domestic, industrial, and agricultural (Fumian et al. 2010; Silva et al. 2011).

HAdV found in recreational waters has been considered to cause gastroenteritis outbreaks and other diseases of the respiratory system due to one of its main characteristics: the resistance to variations in environmental conditions (Sinclair et al. 2009). Considering concentrated samples, the total number of positive samples found at P4 (place used for leisure and aquatic recreation) was six (6/14 (42.8%)) (Table 1). It is worth highlighting that among these six positive samples, three belong to group F, a major virus that causes gastroenteritis, and is also considered one of the major etiological agents responsible for infantile gastroenteric infections. Thus, this constitutes a health risk for the population that bathe in these waters, since viral analysis in recreational waters in Brazil is not mandatory (Filho et al. 2007; Kundu et al. 2013; La Rosa et al. 2015). Caxias do Sul (RS, Brazil) is one of the cities that face gastroenteritis problems. Paesi & Magrini (2015) conducted a study on the number of cases of acute diarrheal disease in this municipality from 2004 to 2013. The authors reported that during this period 61,246 cases were recorded, the highest numbers being in low-income neighborhoods. Furthermore, the authors also reported that the area with the second highest register was Desvio Rizzo. In this same area is located P1, which, as previously mentioned (Table 2), was the point with the second highest number of positive samples (7/13 (53.8%)).

In contrast to what is found in the literature, HAdV belonging to the F group was detected more frequently in this study. In the literature, water sample analyses have shown that AdV belonging to group C are in a greater quantity (Bibby & Peccia 2013; Barrios et al. 2016; Staggemeier et al. 2017). On the other hand, there are studies that have found similar results. Kuo et al. (2015), and Wieczorek et al. (2015), when analyzing the presence of AdV genome in sewage samples from Taiwan and Poland, respectively, described a higher number of AdV from the F group. Other species of HAdV have also been identified, such as those of groups D and E, viruses that affect the gastrointestinal and respiratory tract, respectively. It is described in literature that HAdV-A, C, and F are more frequently found in water bodies when compared with groups B, D, and E (Kuo et al. 2015; Ogorzaly et al. 2015; Wieczorek et al. 2015). Serotypes 40 and 41 belonging to the F group have been considered one of the most prevalent viruses in acute gastroenteritis in children, and may also cause mortality in immunocompromised individuals. These viruses are released for long periods in feces, urine and respiratory secretions of infected persons. Thus, the high number of positivity found in this species, when compared with others, suggests a high incidence of HAdV-F infections in the population of the region of the present study (Jiang 2006; Filho et al. 2007; La Rosa et al. 2015).

AdV from other mammalian hosts were also detected, such as MAdV and BAdV. Recently, three types of MAdVs were described, and in this study, type 2 was identified. MAdVs have potential for vector screening in genes and cancer therapy, and may complement vector studies derived from HAdV. Furthermore, MAdVs provide insight into the pathogenesis process of the host, which includes acute and persistent infections (Nguyen et al. 1999; Weinberg et al. 2005; Klempa et al. 2009; Robinson et al. 2009; Hemmi et al. 2011). In spite of the low detection of BAdV (1.8% (1/55)) in the present study, it is worth noting that this virus can be used as a marker of fecal animal contamination in groundwater and surface waters in rural environment. Due to its structural characteristics, this type of the virus is more stable in the environment and may cause disease in animals. Bovine excrements are often applied to the soil as untreated fertilizer. Because of this, both groundwater and surface water can be contaminated through the dragging of fecal material to water due to the runoff. As a result of this, environmental degradation and the transmission of diseases caused by these pathogens are concerning, since the animals have access to contaminated rivers and streams (Spilki et al. 2009; Wong & Xagoraraki 2010).

In the face of the presented results, it is worth mentioning that the great diversity of AdV identified by the nested-PCR technique followed by sequencing represents a result that has not been reported in the literature. Most of the studies on diversity use techniques that are restricted to PCR or quantitative PCR (qPCR) using specific primers for a given species, thus requiring more detailed work (Filho et al. 2007; La Rosa et al. 2015; Wieczorek et al. 2015; Adefisoye et al. 2016; Barrios et al. 2016).

In general, AdV detection occurred during all the months of the study, and it can be stated that they did not present seasonally, a fact also observed by other studies (Wieczorek et al. 2015; Adefisoye et al. 2016). Among the months that presented the highest number of positive samples, the month of October 2015 stands out the most. According to INMET (National Meteorological Institute), the months with the highest rainfall in 2015 were September and October. Therefore, a possible reason for the increase in the number of positive samples in October is the increase in precipitation index during this period. Rainfall events can introduce a large quantity of microbial contaminants, which include human enteric viruses, through flow of contaminants and flow of fecal material to the sampling sites (Hata et al. 2014; Rodrigues et al. 2015).

In this study, the identification of AdV was performed by nested-PCR, which does not necessarily mean the presence of infectious viral particles, since PCR techniques detect both infective and non-infective genomes. However, literature confirms the risk of individuals getting sick when they bathe in water contaminated with AdV (Ogorzaly et al. 2010). In order to identify the true presence of infectious particles in water, PCR-integrated cell culture (ICC-PCR) studies should be performed (Rigotto et al. 2010).

The results obtained in the present study are rare in literature regarding diversity, since it is not always possible to find the same amount of species by using the nested-PCR technique. AdV belonging to the F group, an important virus that causes gastroenteritis, was present in most samples (in both unconcentrated and concentrated samples). The concentration step is necessary, for it allows the identification of a greater diversity and quantity of AdV in analyzed samples when compared with unconcentrated ones. However, results obtained from unconcentrated samples should be considered, since three different species of HAdV were found. Because they were detected in all analyzed months, the AdV detected did not present seasonally. The analysis of AdV in these waters reveals mainly human fecal contamination along Belo Stream, which demonstrates the inefficiency or the absence of adequate sewage treatment processes to remove AdV. Considering that part of this stream is used for leisure and recreation, such contamination can put the exposed population at risk.

The authors are grateful for the financial and structural support from University Feevale, the Brazilian Coordination for the Improvement of Higher Level Personnel (CAPES), the Foundation for Research Support of the State of Rio Grande do Sul (FAPERGS), the Brazilian National Council for Scientific Development (CNPq), and the Institute of Environmental Sanitation/University of Caxias do Sul.