TEST CONDITIONS Challenge Virus: AIV (H9N9 subtype) 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: MDCK (Madin-Darby canine kidney) cells. Specific Aim: To determine the virucidal efficacy of 2 doses of powder Stalosan F against AIV in a surface test for contact times of 5 min, 10 min, 60 min, and 6 hrs. Test procedure: A suspension of AIV was applied to wells of 24-well microtiter plates @40 L of virus/well. After the virus inoculum was dry, a dose of powder Stalosan F (10 mg) was sprinkled on 12 virus-containing wells making sure to cover all of the virus inoculum with Stalosan F. The other dose (20 mg) was applied to the other 12 virus-containing wells. Tris buffer saline (in powder form) was applied to 24 wells to serve as negative control; twelve wells each were used to mimic the two doses of Stalosan F. Surviving virus from triple wells (three experimental and three control) was eluted in 400 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. 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. Three wells were used per dilution. The inoculated cells were incubated at 37°C and 5% CO 2 for up to 3 days. The plates were examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (CPE). The highest dilution of the sample showing positive CPE was considered as the end point. Virus titers in various samples were calculated by the Karber method (1931). Comparison of virus titers in control and experimental wells indicated the amount of virus inactivated after a given contact time. The results are shown in Table 1. 2
Download PDF file