Disinfectant Investigation, March/April 2009
Feline herpesvirus (FHV), feline calicivirus (FCV) and canine parvovirus (CPV) were propagated in cell culture to achieve significant viral titer. The virus-infected cultures were harvested, subjected to a freeze-thaw cycle, and the homogenous mixture was divided into 5 ml portions and frozen at -80C. An aliquot of the frozen virus was thawed and titrated according to standard procedure.
Titrated virus stock was diluted 1:100 in PBS to reduce the concentration of FBS (from 5% to .05%) thereby avoiding disinfectant inhibition. Ten ml of the diluted stock was mixed with an equal volume of 2x disinfectant (final pH after dissolution in water was 9.4) in a polypropylene tube (this would achieve a 1:200 final dilution of the virus stock, and 1X dilution or working concentration of the disinfectant). Contact was done at room temperature for 10 minutes. The solution was then dialyzed using 10,000 MWCO dialysis tubing and Hank’s balanced salt solution, through five buffer changes over a 48 hour period at 4C, to remove the disinfectant. The remaining solution (virus free of disinfectant; approximately 10ml total volume) was removed from the tubing and a portion was used to make 10-fold serial dilutions in DMEM. For FHV and FCV, each dilution was applied to CRFK cells in 96-well plates in five replicates. Comparing to original viral stock concentration, viral dilutions of 2x10-2 through 10-8 were titrated post disinfectant treatment. For CPV, viral propagation using dilutions of dialyzed virus were done in six-well plates in two replicates given that splitting (1:2) of cell cultures was required 24 hrs post-inoculation to enhance viral growth. In addition, titers of 10-3 through 10-7 and one uninoculated well were made for each replicate. Cell cultures were monitored for 5 days total (with day 1 being the day of inoculation). Resultant titers were determined based on cytopathic effects (FHV and FCV) or antigen detection by immunofluorescence (CPV).
Negative control consisted of water (FCV, FHV) or PBS (CPV) only (no disinfectant), while positive control utilized a 3% sodium hypochlorite solution.