TEST CONDITIONS Challenge Virus: TGEV Product: Stalosan F powder. Exposure temperature: Ambient room temperature (approx. 25ºC). Virus growth medium: Minimum Essential Medium (MEM) with Earle's Salts supplemented with L-glutamine, antibiotics, 10% TPB (tryptone phosphate broth) and 3.0 µg/mL trypsin Virus titration on: ST (swine testicular) cells. Specific Aim: To determine the virucidal efficacy of 2 doses of powder Stalosan F against TGEV in a surface test for contact times of 5 min, 10 min, 60 min, and 6 hrs. Test procedure: 1. A suspension of TGEV was applied to wells of 24-well microtiter plates @40 µ L of TGEV/well. 2. After the virus inoculum is dry, a dose of powder Stalosan F was sprinkled on four virus- containing wells making sure to cover all of the virus inoculum with Stalosan F. Two other doses were applied to eight other virus-containing wells. 3. Beef extract powder was applied to 12 wells to serve as negative control; four wells each were used to mimic the three doses of Stalosan F. 4. Surviving virus from pairs of wells (one experimental and one control) were eluted in 360 µ L of eluent (3% beef extract solution in 0.05M glycine, pH 7.2) after 5 min, 10 min, 60 min, and 6 hrs of contact time. 5. The eluted samples were tested for any surviving virus by preparing serial 10-fold dilutions of all eluates and inoculating them in MDCK cells contained in 96-well microtiter plates after washing two times with HANKS solution. Three wells were used per dilution. 6. The inoculated cells were incubated at 37 ⁰ C and 5% CO 2 for up to two days. The plates were examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (CPE). 7. The highest dilution of the sample showing positive CPE was considered as the end point. 8. Virus titers in various samples were calculated by the Karber method (1931). 9. Comparison of virus titers in control and experimental wells indicated the amount and per cent of virus inactivated after a given contact time. Results: 1. Surface test was tried using 10 mg and 20 mg of Stalosan powder with four different contact times (5, 10, and 60 minutes and 6 hours). For each experiment the inoculated cells were washed twice with HANKS solution after 90 minutes of incubation with different dilutions of the samples, and then followed by incubation at 37°C. This approach was successful to avoid the cytotoxic effect of the sanitizer itself on MDCK cells . The 90 min was chosen because it enough for the survived virus to attach with the hosting cells if it presents. 2. This experiment was done in triplicate and the results are shown in Tables 1-3. 3. As shown in the tables, there is a reduction in virus titer in the control wells after 6 hrs. This is attributed to the low survivability of the virus under dry conditions. This noticed reduction showed minor decrease in the calculated percentage of the virus titer reduction of the sanitizer. 2
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