4.1 Introduction
4.3.2 Phylogenetic analysis of detected human adenoviruses and enteroviruses The evolutionary relationships of enteroviruses detected in the treated effluent of NWWTP and
the receiving Umgeni River is depicted in Figure 4.2, while those of the NGTW and receiving Aller River is shown in Figure 4.3. Enteroviral isolates detected in samples collected before chlorination during spring clustered together with isolates detected at the discharge point during winter and summer for the NWWTP whilst isolates detected upstream of the Umgeni River during spring and downstream during autumn clustered closely together (Figure 4.2). Similarly, enteroviral isolates detected downstream of the Aller river during autumn clustered closely with isolates at the discharge point for the NGTW during autumn as well, whilst isolates detected at the before chlorination sampling point during autumn and winter clustered together for the NGTW (Figure 4.3).
Evolutionary relationships of Human adenoviruses detected in the treated effluent of the NWWTP and the receiving Aller River is depicted in Figure 4.4, whilst those of the NGTW and receiving Aller River is shown in Figure 4.5. Human adenoviral isolates detected upstream and downstream of the Umgeni River for the NWWTP during summer clustered closely with isolate detected at the NWWTP discharge point during autumn (Figure 4.4). Similarly isolates detected upstream and downstream of the Aller River during summer clustered closely together (Figure 4.5).
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Figure 4.2: Evolutionary relationships of enteroviral taxa sequenced from NWWTP and receiving Umgeni River across all seasons. The evolutionary history was inferred using the Neighbour- Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analysed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (Jukes and Cantor, 1969) and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated.
There were a total of 60 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
NW DS SPRING NW US SUMMER
NW BC SPRING NW DP WINTER NW DP SUMMER
NW US AUTUMN NW DS WINTER
NW BC SUMMER NW DS AUTUMN NW BC WINTER
NW US SPRING NW BC AUTUMN 37
37
35 22
23
16
40 7 13
0.002
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Figure 4.3: Evolutionary relationships of enteroviral taxa sequenced from NGTW and receiving Aller River across all seasons. The evolutionary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analysed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site (Jukes and Cantor, 1969). The analysis involved 13 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
NG DS SPRING NG DP WINTER NG DS AUTUMN
NG DP AUTUMN NG BC AUTUMN NG BC WINTER
NG DP SUMMER NG US SPRING
NG US SUMMER
NG DS WINTER NG US WINTER
NG US AUTUMN
NG BC SPRING 52
47
42
42 32
21 11
8
29
0.02
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Figure 4.4: Evolutionary relationships of adenoviral taxa sequenced from NWWTP and receiving Umgeni River across all seasons. The evolutionary history was inferred using the Neighbour- Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analysed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site (Jukes and Cantor, 1969). The analysis involved 8 nucleotide sequences.
Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 79 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
Figure 4.5: Evolutionary relationships of adenoviral taxa sequenced from NGTW and receiving Aller River across all seasons. The evolutionary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.22952000 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site (Jukes and Cantor, 1969). The analysis involved 4 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 96 positions in the final dataset.
Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
NW US SUMMER NW DS SUMMER
NW DP AUTUMN
NW BC AUTUMN NW BC AUTUMN
NW BC SPRING
NW DS AUTUMN
NW US SPRING 45
41
96 54
39
0.05
NG DS SUMMER NG US SUMMER
NG DS AUTUMN NG DS SUMMER 53
0.02
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Surface water sources may be contaminated with a range of enteric viruses from a variety of sources such as raw sewage, wastewater discharges as well as domestic and animal run-off with the most commonly reported enteric viruses being adenovirus, enterovirus, norovirus, rotavirus and hepatitis A and E viruses (Wong et al., 2012). The main aim of this chapter was to recover and detect the presence of two important enteric viruses commonly implicated in waterborne diseases, namely enteroviruses and human adenoviruses, in treated wastewater effluent of two WWTPs and receiving river water sources in the Durban area using a glass wool adsorption- elution method and a PCR assay. Numerous methods have been developed in order to evaluate and monitor the microbiological quality of surface water sources as well as the efficacy of treatment plant processes with the majority of emphasis being placed on pathogenic bacterial contamination. The traditional method for human pathogenic viral detection is cell culture, however it has proven to be costly, time consuming and impractical for continuous monitoring.
Thus, numerous methods have been developed and whilst there has been an increase in the number of studies investigating the presence of human enteric viruses in wastewater within South Africa, the absence of sufficient expertise and knowledge of a viral concentration method that is cost effective and operates well with high recovery efficiency is one of the main reasons for the low detection and report rates of human enteric viral contamination (Chigor and Okoh, 2012). Thus, there is a growing demand for more rapid, cost effective and more robust method that will aid in the detection of viruses from environmental samples. Previous studies have highlighted the efficiency of glass wool as a cost effective and simple means to recover and concentrate viruses, however; the effectiveness depends on the type of virus, water pH and water constituents present (Gantzer et al., 1997). Lambertini et al. (2008) reported glass wool recovery
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efficiencies to be significantly affected by water pH with recovery efficiencies decreasing at pH’s greater than 7.5. Thus, the method should be optimised based on the isoelectric points and adsorptive behaviours of individual viral types being detected (Lambertini et al., 2008).
Although most molecular-based methods fail to distinguish between infectious and non- infectious viral particles, the presence of viral nucleic acid in contaminated water sources suggests the presence of infective viral particles as the survival of naked nucleic acids is usually low within the water environment (Lambertini et al., 2008). In addition, the glass wool adsorption-elution method has been found to be more effective in retaining infectious intact viral particles rather than naked viral nucleic acid, thus only viral capsids adsorb to the glass wool whilst naked DNA passes through the columns during filtration. Hence, the enteric viruses detected in this study were probably intact and infectious, thus indicating their potential threat to those utilising these water sources for direct domestic and recreation use. The detection of both viral types at the discharge point (Table 4.2) indicates both, the resistance of these viruses to currently used chlorination treatment as well as inefficient chlorination procedures noted within the treatment plant itself. This corroborates previous studies that have highlighted the resistance of these viruses to common conventional tertiary treatments with no correlation between viral loads and the reported high removal efficiency of commonly used bacterial indicators (Le Cann et al., 2004; Meleg et al., 2006). In addition, numerous studies have highlighted the stability of human adenoviruses compared to other commonly detected enteric viruses when subjected to UV irradiation (Bofill-Mas et al. 2006; Eischeid et al. 2009; Lee and Shin 2011; Nwachuku et al.
2005). In this study however, human adenoviruses were detected in 62.5% of all samples collected for both plants whilst enteroviruses were detected in 100% and 87.5% of samples collected at the NGTW and NWWTP respectively (Table 4.2).
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Analysis of adenoviral sequences revealed a 91 – 99% similarity to previously confirmed human adenoviral isolates in the GenBank database with phylogenetic analysis indicating isolates detected upstream and downstream of the receiving Umgeni river during summer as well as those detected at the discharge point during autumn being closely related and clustering together (Figure 4.4). Similarly, human adenoviral isolates confirmed in the upstream and downstream samples of the Aller river during summer exhibited the closest similarity (Figure 4.5). Previous studies have confirmed the specificity of human adenoviral strains, with only human waste and infected individuals serving as a source of these strains (Motes et al., 2004). Also, human adenoviral infections are known to occur throughout the year with the infection rate being more common in late winter, spring and early summer which may be due to increased rainfall and hence increased run-off from surrounding areas (Fong et al., 2010). In addition, these viral agents are generally associated with nosocomial infections, with type 40 and 41 being considered the most important cause of childhood gastrointestinal illnesses (Magwalivha, 2009). Thus the lack of seasonal variability associated with human adenoviral detection in this study indicates that the occurrence of human adenoviruses within the aquatic environment is most likely due to contamination with untreated or improperly treated human sewage (Hang, 2006).
Viral distribution within water systems depends on a range of factors such as temperature, pH, humidity and season with previous studies indicating enteroviral loads of approximately 2 - 500 PFU/l with loads as high as 5600 PFU/l recorded during disease outbreaks and epidemics (Kocwa-Haluch, 2001). Analysis of enteroviral sequences showed isolate similarity (99%, 98%
and 97%) to that of Human Enterovirus 90 isolate 01421 (Accession Number: KC570453.1), Human Enterovirus 71 strain (Accession Number: JX390655.1) and the Human coxsackievirus
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A20 strain CVA20a (Accession Number: EF015021.1) amongst others (Figure 4.2 and 4.3).
Detection of these viruses throughout the sampling period and within the receiving watershed indicates that these viruses are frequently excreted and relatively resistant not only to tertiary treatments but also to natural inactivation processes within surface water resources (Hundesa et al., 2006). Phylogenetic analysis was used to determine similarities between viral isolates detected at the different sampling points with the results obtained for enteroviral isolates from the NWWTP and receiving Umgeni River (Figure 4.2) indicating that those isolates detected upstream and at the discharge point during summer, those detected before chlorination and downstream sampling point during spring as well as those at the discharge point during winter clustering together and are thus closely related. Generally, filtration of large volumes of water may result in the concentration of high levels of PCR inhibitors such as humic acids which may inhibit downstream detection assays (Kocwa-Haluch, 2000). Thus, failure to detect human adenoviruses at the NGTW in samples collected before chlorination whilst discharge point samples tested positive could be attributed to high concentrations of inhibitors which may have affected the PCR assay.
Overall, the data obtained in this study confirm the effectiveness of a more economical method for viral recovery and concentration, namely the glass wool adsorption-elution procedure. In addition, the results have also indicated that tertiary treatment processes within the two treatment plants monitored, whilst possibly effective for currently monitored bacterial indicators, it is not effective for the removal of enteroviruses and human adenoviruses. This further emphasises the need to incorporate viral indictors into the current water quality guideline as the treated effluent discharge from these WWTPs could further contaminate the receiving surface water and pose a serious threat to the end users of these surface water resources.
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CHAPTER FIVE
GENERAL DISCUSSION AND CONCLUSION