Feline infectious peritonitis virus (FIPV) (ATCC-990), an enteric feline coronavirus, was propagated and assayed in the Crandell Reese feline kidney cell line (CRFK) (ATCC-94). Human coronavirus 229E (HCoV) (ATCC-740), a respiratory virus, was propagated and assayed in the fetal human lung fibroblast, MRC-5 cell line (ATCC-171). Poliovirus 1 LSc-2ab (PV-1), a human enteric virus known to be very stable in the environment, was propagated and assayed in the Buffalo green monkey kidney (BGM) cell line. Infected cells were frozen and thawed three times at −20°C to release virus after cytopathogenic effects (CPE) were observed in the monolayer. This was followed by centrifugation at 1000g for 10 min to remove cell debris, addition to the virus suspension of 9% polyethylene glycol (MW 8000) and 0.5 M sodium chloride, and stirring overnight at 4°C. After centrifugation at 10,000g for 30 min, the pellet was resuspended in 0.01 M phosphate buffered saline (PBS; pH 7.4) (Sigma, St. Louis, MO) to 5% of the original virus suspension volume. The coronaviruses were then titered and stored at −80°C. The poliovirus was further purified by extraction with equal volumes of Vertrel XF (Dupont, Wilmington, DE), emulsified and centrifuged at 7,500g for 15 min, and subsequently the top aqueous layer was collected, titered, and stored at −80°C.
Tap water samples were collected from a cold tap faucet in the laboratory. The source is groundwater with water quality parameters: pH 7.8, 297 mg/l dissolved solids, and 0.1 mg/l total organic carbon. The water was allowed to run for 3 min before collection of the sample. Virus survival was determined in both nonfiltered tap water and tap water passed through a 0.2-μm pore size filter to remove bacteria. Tap water (30 ml) was added to sterile 50 ml polypropylene centrifuge tubes, to reduce loss of virus by adhesion to the container. Sterile sodium thiosulfate was added to a final concentration of 33 μg/ml to dechlorinate the water. After vortexing, virus was added to each tube to a final concentration of 105 TCID50/ml. Again the tubes were vortexed and a sample was immediately taken (zero time point). The tubes of tap water were then stored at either 4°C or room temperature (23°C). The tubes stored at room temperature were covered in aluminum foil to prevent exposure to light. Tubes were sampled after 1, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until they were assayed on cells.
Samples of primary and activated sludge (secondary) effluent were collected in sterile polypropylene bottles from the Roger Road Wastewater Treatment Plant in Tucson, AZ, USA. Primary effluent was collected after settling and secondary effluent was collected prior to chlorination. Typical wastewater quality parameters for this facilities primary effluent are biological oxygen demand (BOD) and suspended solids of 110–220 mg/l. Secondary effluent at Roger Road typically reflects a 90–95% reduction in both BOD and suspended solids from the primary effluent. The effluent (30 ml) was added to sterile 50 ml polypropylene centrifuge tubes. Primary effluent was filtered through a 0.2-μm pore size filter before addition of the virus and was also tested unfiltered. Secondary effluent was only tested unfiltered. Virus was then added to each tube to a final concentration of 105 TCID50/ml. The tubes were vortexed and a sample was immediately taken (time zero). The tubes were then covered and held at room temperature (23°C). Samples were collected after 1, 2, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until assay.
Viruses were enumerated on cell cultures using either the plaque assay or TCID50 technique. PV-1 was titered in 6-well plastic cell culture plates by the plaque assay method (Payment and Trudel 1993 ). This is a direct quantitative method with a minimum detection limit of 10 pfu/ml. Each dilution was plated in duplicate wells. Coronaviruses, which do not form plaques in cell culture, were titered in 24-well plastic cell culture plates by the tissue culture infectious dose 50% technique (TCID50) (Payment and Trudel 1993 ). This technique determines the dilution at which 50% of the wells show CPE. Taking the inverse log of this dilution gives a titer of the virus per ml TCID50. The minimum detection for this method was 3.7 viruses per ml. Each dilution was plated in a minimum of 8 wells. Any samples that were not from test waters filtered prior to adding virus had to be filtered before assaying on cell culture to eliminate bacterial contamination. The 0.2 μm low protein binding Millex filters (Millipore, Billerica, MA) with polyethersulfone (PES) membrane were prepared by passing 3% beef extract (Becton Dickinson, Sparks, MD) at pH 7 through to block sites that might adsorb virus. All experiments were performed in triplicate.
Tap water samples were collected from a cold tap faucet in the laboratory. The source is groundwater with water quality parameters: pH 7.8, 297 mg/l dissolved solids, and 0.1 mg/l total organic carbon. The water was allowed to run for 3 min before collection of the sample. Virus survival was determined in both nonfiltered tap water and tap water passed through a 0.2-μm pore size filter to remove bacteria. Tap water (30 ml) was added to sterile 50 ml polypropylene centrifuge tubes, to reduce loss of virus by adhesion to the container. Sterile sodium thiosulfate was added to a final concentration of 33 μg/ml to dechlorinate the water. After vortexing, virus was added to each tube to a final concentration of 105 TCID50/ml. Again the tubes were vortexed and a sample was immediately taken (zero time point). The tubes of tap water were then stored at either 4°C or room temperature (23°C). The tubes stored at room temperature were covered in aluminum foil to prevent exposure to light. Tubes were sampled after 1, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until they were assayed on cells.
Samples of primary and activated sludge (secondary) effluent were collected in sterile polypropylene bottles from the Roger Road Wastewater Treatment Plant in Tucson, AZ, USA. Primary effluent was collected after settling and secondary effluent was collected prior to chlorination. Typical wastewater quality parameters for this facilities primary effluent are biological oxygen demand (BOD) and suspended solids of 110–220 mg/l. Secondary effluent at Roger Road typically reflects a 90–95% reduction in both BOD and suspended solids from the primary effluent. The effluent (30 ml) was added to sterile 50 ml polypropylene centrifuge tubes. Primary effluent was filtered through a 0.2-μm pore size filter before addition of the virus and was also tested unfiltered. Secondary effluent was only tested unfiltered. Virus was then added to each tube to a final concentration of 105 TCID50/ml. The tubes were vortexed and a sample was immediately taken (time zero). The tubes were then covered and held at room temperature (23°C). Samples were collected after 1, 2, 3, 6, 10, 15, and 21 days and the samples frozen at −80°C until assay.
Viruses were enumerated on cell cultures using either the plaque assay or TCID50 technique. PV-1 was titered in 6-well plastic cell culture plates by the plaque assay method (Payment and Trudel 1993 ). This is a direct quantitative method with a minimum detection limit of 10 pfu/ml. Each dilution was plated in duplicate wells. Coronaviruses, which do not form plaques in cell culture, were titered in 24-well plastic cell culture plates by the tissue culture infectious dose 50% technique (TCID50) (Payment and Trudel 1993 ). This technique determines the dilution at which 50% of the wells show CPE. Taking the inverse log of this dilution gives a titer of the virus per ml TCID50. The minimum detection for this method was 3.7 viruses per ml. Each dilution was plated in a minimum of 8 wells. Any samples that were not from test waters filtered prior to adding virus had to be filtered before assaying on cell culture to eliminate bacterial contamination. The 0.2 μm low protein binding Millex filters (Millipore, Billerica, MA) with polyethersulfone (PES) membrane were prepared by passing 3% beef extract (Becton Dickinson, Sparks, MD) at pH 7 through to block sites that might adsorb virus. All experiments were performed in triplicate.
