Title: Canonical Correspondence Analysis (CCA) for microbiological, physicochemical and heavy metal assessment of the Umgeni River-South Africa. Title: Virus Population Assessment in the Umgeni River-South Africa Authors: Atheesha Ganesh and Johnson Lin.
This work is dedicated to my beloved parents, father Diplall Ganesh and mother Reetha Devi Ganesh, for their love, endless support and prayers
Thank you for always believing in me and giving me the opportunity to dream big…
4 CCA ordination plot for bacteriophage and virus-like particle populations and water quality variables at the five study sites during fall, winter, spring, and. 84 Figure 3.10 TEM images of putative naked Picornaviridae (Enterovirus)-like particles (a-e). and f) Coxsackievirus (Schramlová et al., 2010), present in the Umgeni River at sampling sites U1, U2 and U3 during the tested spring and summer seasons.
CHAPTER ONE
LITERATURE REVIEW
Current Environmental Water Situation in South Africa
These pressures are further exacerbated by climate change, particularly its impacts on water resources (Turpie et al., 2003). An accurate and comprehensive assessment of microbial water quality is of paramount importance if both existing and new water sources are to be used safely (Payment et al., 1991b).
Surface Water Pollution: Sources, Indicator Organisms and Detection Methods
- Faecal Streptococci and Enterococci
- Bacteriophages
- Somatic Coliphages
- Male-Specific F-RNA Coliphages
To increase the assurance of water quality results, especially when monitoring for faecal contamination, analysis for enterococci has been used (Stevens et al., 2003). 9 the toxin-regulated pilus (TCP) is also essential for pathogenicity (Faruque and Mekalanos, 2003; . Olaniran et al., 2011).
Viral Studies in Freshwater Environments
14 have been found in many groundwater supplies, including private domestic wells, municipal wells, and unconfined aquifers (Borchardt et al., 2003). The development of tools for analyzing viruses in aquatic ecosystems is thus essential for obtaining accurate measurements of their activity and for predicting the consequences of these activities (Miki et al., 2008).
Human Pathogenic Viruses Present in Environmental Waters
Enteric viruses can pose a significant risk of infection to vulnerable individuals even at low levels of viral contamination (Fong and Lipp, 2005; Teunis et al., 2008). The high prevalence of enteric viruses in surface water highlights the importance of evaluating water sources used for domestic purposes for viral contamination (Kiulial et al., 2010).
Waterborne Human Pathogenic Viruses of Public Health Concern and their Associated Illnesses
- Adenovirus (AdV)
- Enterovirus (EV)
- Hepatitis A Virus (HAV)
- Norovirus (NoV)
- Rotavirus (RV)
- Astrovirus (AstV)
Many studied human HAV strains belong to genotype I or III (Lu et al., 2004). Reverse transcription-PCR (RT-PCR) is currently the most widely used test for the detection of NoV in environmental water (Greening and Hewitt, 2008; Karim and LeChevallier, 2004), in the Netherlands (Lodder and de Roda Husman, 2005) and in Germany (Pusch et al. 2005).
Hepatitis B Virus
Astroviruses have been detected in environmental samples by RT-PCR, which has been shown to be more sensitive than EM and EIA (Chapron et al., 2000; Le Cann et al., 2004).
Methods for Isolating Viruses in Environmental Waters
- Glass-Wool Adsorption-Elution Method
- Ultrafiltration and Tangential Flow Filtration
- Organic Flocculation
The main reason is that viruses can differ in biophysical characteristics, so not all viruses are concentrated equally efficiently by adsorptive elution (Williamson et al., 2008). 29 simultaneous recovery of different microbes, including bacteria and viruses, from different water samples (Grabow et al., 2001).
Methods for Detecting the Presence of Viruses in Environmental Waters
- Cell-Culture Techniques
- Electron Microscopy
- Epifluorescence Microscopy (EFM)
- Transmission Electron Microscopy (TEM)
- Flow Cytometry (FCM)
- Polymerase Chain Reaction (PCR)
- Real-Time (RT) PCR
- Pulsed-Field Gel Electrophoresis (PFGE)
- Metagenomic Sequencing
- Microfluidic Digital PCR
Transmission electron microscopy that can distinguish viruses by morphotypes (Middelboe et al., 2003) offers very limited resolution. Thus, PFGE reveals only a minimal estimate of the dominant genotypes present within a sample (Danovaro et al., 2007).
Scope of Present Study
This has shown that viral communities are extremely diverse on both local and global scales (Angly et al., 2006; Breitbart et al.). Water supply monitoring and research into water-borne viruses (mainly in Gauteng) in South Africa have been inadequate (Grabow et al., 2004).
Hypothesis
This is despite the fact that viruses are generally more stable than common bacterial indicators in the environment (Okoh, 2010). The main objective of this study was to establish a virus concentration system and estimate the abundance of viruses found in water samples of the Umgeni River from Durban, South Africa.
Research Objectives
This virus study thus focused on the Umgeni River catchment in (KwaZulu-Natal) South Africa, as this water source is widely used for recreational, agricultural and domestic activities (DWAF, 1996a; b). The river supplies water to more than 3.5 million people and supports an area responsible for approximately 65% of the province's total economic output (WRC, 2002).
Experimental Design
The second chapter presents the first part of the study; here, the water quality of the Umgeni River is studied in terms of its microbial load and physicochemical aspects. In addition, the effects of seasonal variability on the microbial load and physicochemical properties of river water were determined by canonical correspondence analysis (CCA).
CHAPTER TWO
Description of Study Area and Sampling Procedure
The pots were sterilized with 70% (v/v) alcohol and rinsed with river source water before collection. Water samples were collected by holding the container by the handle and submerging it knee-deep (± 0.5 m) below the water surface facing away from the water current.
Determination of Physico-Chemical Parameters
- Chemical oxygen demand
If there was no water flow, this was artificially simulated by pushing the container forward. The container was filled, leaving approximately 50 mm of free space to allow mixing during laboratory analysis. All samples were protected from direct sunlight and transported to the Discipline of Microbiology laboratory, University of KwaZulu-Natal (Westville campus) within one hour of sampling and stored at 4°C until further analysis (Buckalew et al., 2006 ). The chemical oxygen demand (COD) of each sample was determined photometrically using the SpectroQuant Nova 60 COD cell test (Merck), which measures in the range 0-15,000 mg/ℓ COD or O2.
Each COD test vial containing all required reagents was vortexed to resuspend the bottom sediment before adding 3 mℓ of each water sample and mixing vigorously.
Enumeration of Bacterial Indicator Microorganisms
The biological oxygen demand (BOD5) of each water sample was measured using the OxiDirect BOD (HACH) system over a 5-day period. The selected range of BOD5 was 0-40 mg/ℓ and the corresponding sample volume (3 mℓ) according to the manufacturer's instructions was used for analysis. The analysis was performed according to the manufacturer's instructions and the measured BOD was expressed in mg/ℓ.
47 Table 2.2 Selective media and incubation conditions used for isolation and enumeration of bacterial indicator organisms.
Statistical Analysis
- Physico-chemical Characteristics of Water Samples
- Canonical correspondence analysis (CCA)
Low sulfate concentrations were detected for the rest of the sampling points during all seasons. 3 Physico-chemical quality of the Umgeni River water samples during autumn, winter, spring and summer seasons. The Total Heterotrophic Bacterial (THB) population of the Umgeni River water samples over the four seasons is presented in Figure 2.2.
59 Table 2.8 Properties of the directional binomial plot of canonical correlation analysis for the quality variables of heavy metals and total bacterial growth at the five study sites during the autumn, winter, spring and summer seasons.
Materials and Methods .1 Sample Collection
- Bacteriophage Determinations
- Preparation of Bacterial Hosts for Bacteriophage Detection
- Presence –Absence Spot Test
- Double Agar Overlay Plate Assay
- Tangential Flow Filtration (TFF) for Viral Recovery
- Enumeration and Visualisation of Virus-Like Particles (VLP) .1 Epifluorescent Microscopy
- Viral Infectivity Assay Using Cell-Culture
Samples of the viral community (virioplankton) were concentrated using a two-step tangential flow filtration process as shown in Figure 3.1, according to the method of Wommack et al. SYBR gold staining combined with epifluorescence microscopy (Chen et al., 2001; Patel et al., 2007; Shibata et al., 2006) was used to count virus-like particles (VLPs) from viral filtrate of the Umgeni River. These TEM images were then compared with known viral images of human origin where possible (Rosario et al., 2009).
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) assay was performed using the aforementioned cell lines (Heldt et al., 2006).
Statistical Analysis
Results
- Enumeration of Bacteriophages and Virus-Like Particles (VLPs)
- Canonical correspondence analysis (CCA)
CCA axis 1 (Figure 3.3) accounted for 77.6% of the total variance of the species dataset, and in total the species-environment relationship accounted for 94.1% of the cumulative variance, suggesting that there may be a relationship between VLP and phage populations at different sites and water quality variables in all seasons. 2 Properties of the canonical correlation analysis ordination bi-plot for all water quality variables and the total viral and bacteriophage growth at the five study sites in autumn, winter, spring and summer seasons. CCA axis 1 (Figure 3.4) accounted for 60.5% of the total variance of the species dataset, and in total the species-environment relationship accounted for 83.7% of the cumulative variance.
3 Bi-plot ordination properties of Canonical Correlation Analysis for bacteriophage and virus particle populations and water quality variables at the five study sites during fall, winter, spring, and summer seasons.
Visualisation of Virus-Like Particles (VLP) by Transmission Electron Microscopy (TEM) Table classifies the different type of bacteriophage that could be detected in the Umgeni River
83 Figure 3.7 TEM images of long-tailed phage particles of different morphotypes present in the Umgeni River at sampling sites U1, U2, U3, U4 and U5 during the winter, spring and summer seasons tested. 85 Figure 3.10 TEM images of presumed naked Picornaviridae (Enterovirus)-like particles (a-e) and f) Coxsackievirus (Schramlová et al., 2010), present in the Umgeni River at sampling sites U1, U2 and U3 in the spring and summer seasons tested. 86 Figure 3.12 TEM images of putative (a, b, c) Reoviridae virus-like particles, d) known Rotavirus (Marshall, 2005), (e, f, g) putative Caliciviridae virus-like particles, h) Norovirus (Humphrey, 2008 ) , present in the Umgeni River at the different sampling sites during all seasons tested.
15 Cytopathic effect (CPE) of the concentrated virus-like particles on six cell lines for the Umgeni River at the different sampling sites during all seasons tested.
CHAPTER FOUR
Materials and Methods .1 Sample Collection
- Tangential Flow Filtration (TFF) for Viral Recovery
- Extraction of Viral Nucleic Acids from Water Samples and cDNA Synthesis
- Adenoviruses
- Enteroviruses
- Rotaviruses
- Hepatitis B Viruses
- Detection and Sequencing of PCR/RT-PCR Positives
- Integrated Cell Culture PCR (ICC PCR) .1 Extraction of Viral Nucleic Acids
- cDNA Synthesis
- Real-time PCR amplification of Human Viruses from the Umgeni River Water Custom TaqMan ® Gene Expression Assays (containing a primer and probe mix) were developed by
- Analysing Real-Time PCR Data
- Quality Control
Nested PCR was used to amplify the hexon gene which is conserved in approximately 47 different adenovirus serotypes (Allard et al., 1992). Nested PCR was used to amplify the 5' untranslated region conserved in approximately 25 different Enterovirus genomes (Fong et al., 2005). Nested PCR was used to amplify the VP7 gene of group A rotaviruses (Gilgen et al., 1997).
Nested PCR was used to amplify the S gene of hepatitis B viruses (HBV) (Koike et al., 1998).
Results
- Detection of Pathogenic Human Viruses using PCR / RT-PCR
- Detection of Human Enteric Viruses by Integrated Cell Culture PCR (ICC-PCR)
- Phylogenetic Analysis
111 Figure 4.7 Neighbor-joining tree representing the phylogenetic relationship between nucleotide sequences of amplicons (154 bp) of the 5'-untranslated region of the Enterovirus genome from different river water samples (U1-U5, Autumn, Winter, Spring and Summer). However, the conventional nested PCR/RT-PCR step detected viral particles in 100% of the concentrated river water samples (Figures 4.1 to 4.4). The hexon gene region of Adenovirus was successfully amplified from 100 % of the VLPs isolated from the water samples (Figure 4.1).
This study discovered the presence of Rotavirus in water samples during all seasons of the year.
CHAPTER FIVE
Future Recommendations
Where virological facilities can be provided, it is necessary to monitor sewage effluents, raw water sources and drinking water for the presence of viruses. These studies should include the development and evaluation of virus detection methods and alternative indicators of virus contamination (eg, phages) and the improvement of treatment methods for the inactivation and removal of viruses from water and wastewater. A standard method for the concentration and detection of viruses in large volumes of drinking water (eg, 20-100 ℓ) should be established based on a thorough evaluation in the various laboratories present.
Future studies are needed to provide effective and reproducible methods for the detection of waterborne viral pathogens to control the extent of contamination of aquatic environments, the types of pathogens involved, and the association between viral contamination and environmental factors.
Media and Buffers
Media
Buffers
206 Table 23: Size range of tail phages observed by electron microscopy for water samples collected along the Umgeni River during summer.
