Comparison of Multiple Passage Integrated Cell Culture-PCR and Cytopathogenic Effects in Cell Culture for the Assessment of Poliovirus Survival in Water

The goal of this study was to determine if a cytopathogenic effects (CPE) cell culture assay and an integrated cell culture PCR (ICC-PCR) assay would yield similar or different results when used to assess virus survival in water. Poliovirus type 1 was added to dechlorinated tapwater and stored at room temperature (22.5–24°C) for a total of 50 days. Samples were assayed at defined time intervals by the most probable number (MPN) method on Buffalo green monkey kidney cells (BGM) by CPE and additionally by ICC-PCR. Monolayers that were CPE negative on first passage were passed onto fresh monolayers of cells for a second and third time if still negative. By CPE assay, second passage was observed to yield a greater titer (2,300 vs. 24,000 MPN/ml) and third passage also resulted in an increased titer. ICC-PCR proved to be a more rapid and sensitive method than conventional cell culture for determining virus inactivation rates in water. Poliovirus survived in tapwater for up to 32 days, as assessed by both third passage ICC-PCR and CPE. There was no statistical difference in the inactivation rates between the two methods. To determine the total number of infectious viruses, these findings indicate the need for performing three cell culture passages or, alternatively, ICC-PCR on first passage.     

Assessment of the Antiviral Properties of Zeolites Containing Metal Ions

The antiviral properties of zeolite (sodium aluminosilicate) powders amended with metal ions were assessed using human coronavirus 229E, feline infectious peritonitis virus (FIPV), and feline calicivirus F-9. Zeolites containing silver and silver/copper caused significant reductions of coronavirus 229E after 1 h in suspension. The silver/copper combination yielded a >5.13-log₁₀ reduction within 24 h. It was also the most effective (>3.18-log₁₀) against FIPV after 4 h. Other formulations were ineffective against FIPV. On plastic coupons with incorporated silver/copper-zeolites, >1.7-log₁₀ and >3.8-log₁₀ reductions were achieved for coronavirus 229E and feline calicivirus within 24 h, respectively. Silver/copper zeolite reduced titers of all viruses tested, suggesting that it may be effective against related pathogens of interest [i.e., SARS coronavirus, other coronaviruses, human norovirus (calicivirus)]. Of note, it was effective against both enveloped and nonenveloped viruses. Metal-zeolites could therefore possibly be used in applications to reduce virus contamination of fomites and thus the spread of viral diseases.     

Simultaneous Concentration of Bovine Viruses and Agricultural Zoonotic Bacteria from Water Using Sodocalcic Glass Wool Filters

Infiltration and runoff from manured agricultural fields can result in livestock pathogens reaching groundwater and surface waters. Here, we measured the effectiveness of glass wool filters to simultaneously concentrate enteric viruses and bacteria of bovine origin from water. The recovery efficiencies were determined for bovine viral diarrhea virus types 1 and 2, bovine rotavirus group A, bovine coronavirus, poliovirus Sabin III, toxigenic Escherichia coli ,and Campylobacter jejuni seeded into water with three different turbidity levels (0.5, 215, and 447 NTU). Twenty liters of dechlorinated tap water (pH 7) were seeded with the test organisms, and then passed through a glass wool filter using a peristaltic pump (flow rate = 1 liter min^?1). Retained organisms were eluted from the filters by passing beef extract-glycine buffer (pH 9.5) in the direction opposite of sample flow. Recovered organisms were enumerated by qPCR except for C. jejuni, which was quantified by culture. Mean recovery efficiencies ranged from 55 to 33 % for the bacteria and 58 to 16 % for the viruses. Using bootstrapping techniques combined with Analysis of Variance, recovery efficiencies were found to differ among the pathogen types tested at the two lowest turbidity levels; however, for a given pathogen type turbidity did not affect recovery except for C. jejuni. Glass wool filtration is a cost-effective method for concentrating several waterborne pathogens of bovine origin simultaneously, although recovery may be low for some specific taxa such as bovine viral diarrhea virus 1.     

Assessment of a Portable Handheld UV Light Device for the Disinfection of Viruses and Bacteria in Water

Effective individual microbiological water purifiers are needed for consumption of untreated water sources by campers, emergency use, military, and in developing counties. A handheld UV light device was tested to assess if it could meet the virus reduction requirements established by the United State Environmental Protection Agency, National Science Foundation and the World Health Organization. The device was found capable of inactivating at least 4 log₁₀ of poliovirus type 1, rotavirus SA-11 and MS-2 virus in 500 mL volumes of general case test water. But in the presence of high turbidity and organic matter, filtration was necessary to achieve a 4 log₁₀ reduction of the test viruses.     

Occurrence of <Emphasis Type="Italic">Pepper Mild Mottle Virus</Emphasis> (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula,Mexico

The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels <40 MPN/100 ml. The highest average concentrations of somatic and male F+ specific coliphages were 920 and 330 plaque forming units per 100 ml, respectively, detected in freshwater during the rainy season. PMMoV RNA was detected in 85% of the samples with gene sequences sharing 99–100% of nucleotide identity with PMMoV sequences available in GenBank. Quantification of PMMoV genome copies (GC) by quantitative real-time PCR indicated concentrations ranging from 1.7 × 10¹ to 1.0 × 10⁴ GC/L, with the highest number of GC detected during the rainy season. No significant correlation was observed between PMMoV occurrence by season or water type (p > 0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.     

Viruses in surface and drinking waters

Over 114 different human enteric viruses are known. They are all excreted into sewage, and opportunities exist for them to find their way into water distribution systems. The characteristics of the illnesses caused by enteric viruses make their transmission by water difficult to recognize. Methods are not available for isolating all of the enteric viruses from water, so that finding any type of human virus in drinking water is an indication that other types may also be present. With improvements in technology, our laboratory has recently been able to detect group B coxsackieviruses, rotaviruses, and hepatitis A virus in chlorinated drinking water. At present, there are no uniform methods for concentrating, isolating, and identifying viruses in water. Emphasis should be placed on seeking uniformity in methods so that standards can be set for viruses in water.     

Environmental Surveillance of SARS-CoV-2 RNA in Wastewater and Groundwater in Quintana Roo,Mexico

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 10³ to 7.5 × 10³ genome copies per liter (GC l⁻¹), using the N1 RT-qPCR assay, and from 2.4 × 10² to 5.9 × 10³ GC l⁻¹ using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area.

     

Sources of pathogenic microorganisms and their fate during land application of wastes

The hazards associated with pathogens in land-applied animal and human wastes have long been recognized. Management of these risks requires an understanding of sources, concentrations, and removal by processes that may be used to treat the wastes; survival in the environment; and exposure to sensitive populations. The major sources are animal feeding operations, municipal wastewater treatment plant effluents, biosolids, and on-site treatment systems. More than 150 known enteric pathogens may be present in the untreated wastes, and one new enteric pathogen has been discovered every year over the past decade. There has been increasing demand that risks associated with the land treatment and application be better defined. For risks to be quantified, more data are needed on the concentrations of pathogens in wastes, the effectiveness of treatment processes, standardization of detection methodology, and better quantification of exposure.