Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces
- 198 Downloads
A method for studying the behavior of viruses on surfaces has been developed and is illustrated by determining the temperatures that inactivate adsorbed viral hemorrhagic septicemia virus (VHSV) and the concentration of 1-propanol that disinfected surfaces with adsorbed VHSV and chum salmon virus (CSV). VHSV is a rhabdovirus; CSV, a reovirus, and they were detected with two fish cell lines, EPC and CHSE-214, respectively. When polystyrene tissue culture surfaces were incubated with virus, rinsed, and left to dry, they still supported the attachment and spreading of cell lines and after 7 days these cells showed the characteristic CPE of the viruses. Thus cells appeared to be infected directly from surfaces on which viruses had been adsorbed. Applying this property to 96-well plates allowed duplicate surfaces to be examined for their infectiousness or support of CPE. For each treatment 80 replicate surfaces in a 96-well plate were tested at one time and the results expressed as the number of wells showing CPE. VHSV adsorbed to polystyrene was inactivated by drying in the dark at temperatures above 14 °C, but remained infectious for at least 15 days of drying at 4 °C. For chemical sterilization of polystyrene surfaces with adsorbed virus, disinfection was achieved with 1-propanol at 40% for VHSV and at 60% for CSV. As CPE can be conveniently monitored in 96-well plates with a fluorescence plate reader, this method can be used to rapidly evaluate a variety of treatments for their ability to inactivate surface-bound viruses.
KeywordsViral adsorption Surface disinfection Viral inactivation Fomites Fish VHSV CSV
The research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to Niels Bols. The authors thank Dr. John Lumsden U of Guelph for VHSV.
- Abad FX, Pinto RM, Diez JM, Bosch A (1994) Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine. Appl Environ Microbiol 60:2377–2383Google Scholar
- Berman D, Hoff JC (1984) Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine. Appl Environ Microbiol 48:317–323Google Scholar
- Bitton G (1980) Adsorption of viruses to surfaces: technological and ecological implications. In: Bitton G, Marshall KC (eds) Adsorption of microorganisms to surfaces. Wiley, New York, pp 332–374Google Scholar
- Inouye S (1973) Nonspecific adsorption of proteins to microplates. Appl Microbiol 25:279–283Google Scholar
- Lumsden JS, Morrison B, Yason C, Russell S, Young K, Yazdanpanah A, Huber P, Al-Hussinee L, Stone D, Way K (2007) Mortality event in freshwater drum Aplodinotus grunniens from Lake Ontario, Canada, associated with viral haemorrhagic septicemia virus, Type IV. Dis Aquat Org 76:99–111CrossRefGoogle Scholar
- Mbithi JN, Springthorpe VS, Sattar SA (1990) Chemical disinfection of hepatitis A virus on environmental surfaces. Appl Environ Microbiol 56:3601–3604Google Scholar
- Mbithi JN, Springthorpe VS, Sattar SA (1991) Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces. Appl Environ Microbiol 57:1394–1399Google Scholar
- Schaub SA, Sagik BP (1975) Association of enteroviruses with natural and artificially introduced colloidal solids in water and infectivity of solids-associated virions. Appl Microbiol 30:212–222Google Scholar
- Thompson SS, Yates MV (1999) Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems. Appl Environ Microbiol 65:1186–1190Google Scholar
- Ward RL, Ashley CS (1977) Inactivation of enteric viruses in wastewater-sludge through dewatering by evaporation. Appl Environ Microbiol 34:564–570Google Scholar