RESEARCH by plaque assays on pig kidney epithelial cells as described previously (19), by using a 3% carboxymethylcellulose overlay. Tick Infection and Co-feeding Transmission Experiments Seven groups of six BALB/c mice (female, 4–6 weeks old) were injected subcutaneously with 104 PFU of WNV. Three of the mice were bled daily from the tail to follow the course of viremia by plaque assay. Two groups of mice were infested with I. ricinus nymphs (20 per mouse); one group was infested 3 days before infection, the other 4 days after infection. The other five groups of mice were infested with second instar O. moubata ticks (10 per mouse) on either the same day (day 0) or 1, 2, 3, or 4 days after infection. After the initial experiment, and to increase the number of positive ticks available for experimentation, 12 additional mice were infested with O. moubata 2 days after infection with WNV. Ticks housed in gauze-covered neoprene feeding cham- bers on mice (18) were removed when fully engorged, 24 hours after infestation in the case of O. moubata ticks and 6 days after infestation in the case of I. ricinus nymphs. The engorged ticks were stored at 20°C in KCl-saturated desiccators until testing for WNV or until ready for a fur- ther bloodmeal, as indicated in Table 1. After storage, the ticks (pools and individual ticks) were homogenized in 500 µ L of PBS by using plastic homogenizers under sterile conditions. The homogenates were frozen and stored at –70°C until analyzed. Tick homogenates were assayed for infectious virus antigen (by immunofluorescence assay) and viral RNA (by RT-PCR) as shown in Table 1. Co-feeding transmission experiments were carried out by infesting clean BALB/c mice (n = 7, Harlan, UK) with 10 third instar O. moubata ticks 57 days after they had taken an infectious bloodmeal, and 10 uninfected ticks (second instar) in separate feeding chambers. The two feeding chambers were separated by at least 1 cm. To investigate tick-to-host transmission, 10 BALB/c mice were infested with cohorts of 5, 10, 15, or 20 third instar O. moubata ticks 57 days after an infectious blood- meal. Fifteen days after infestation, the mice (including those used for co-feeding) were euthanized; brains were removed, homogenized in 1 mL of sterile PBS, and stored at –70°C until they were tested for WNV. Immunofluorescence Assay Samples of tick (or mouse brain) homogenate (100 µ L) were used to infect 2 x 106 C6/36 mosquito cells in a total of 3 mL L-15 medium containing 7% fetal calf serum (Gibco-BRL, Paisley, UK) in 30 mm Petri dishes that con- tained glass coverslips. Infected cells were incubated at 28°C for 3 days. Cells were fixed in cold acetone and treat- ed according to standard methods (19). Infected cells were fluorescein isothiocyanate–labelled with a broadly reactive flavivirus E-protein monoclonal antibody (MAb 813) (20) Table 1. Results of immunofluorescence assay (IFA) or nested reverse transcriptase–polymerase chain reaction (RT-PCR) from Ornithodoros moubata and Ixodes ricinus ticks fed on West Nile virus–inoculated BALB/c mice or noninfected mice (co-fed ticks) Days from infection RT-PCRc Species Developmental stage to infestation Days after engorgementa IFAb +/– (no. positive/no. tested) O. moubata 0 1, 2, 7 – (8) ND First bloodmeal Second instar 1 1–7 – (5) ND (infected mice) 2 1–7, 14 + (5) + (5) 3 1–7, 14 + (5) + (5) 4 1, 3, 7 – (5) ND Third instar 2 22 + (5) ND 3 22 + (5) ND 2 132 + (5) 7/14 Second bloodmeal Third instar 2 60 (3) + (5) + (5) (uninfected mice) 2 64 (7) + (5) + (5) Fourth instar 2 75 (25) + (5) + (5) Uninfected co-fed Second instar 5 ND 15/66 O. moubata ticks Third instar 45 ND 4/15 I. ricinus Nymph 4 2 ND 0/12 First bloodmeal –3 2 ND 2/12 (infected mice) –3 30 ND 0/25 BALB/c miced – (1) 1/17 aNumber of days after the ticks had completed feeding on inoculated mice when ticks were tested for virus infection. Where given, parentheses depict ticks that had fed a second time and the number of days after which the ticks were tested. bTick homogenate samples were scored positive if >10% of inoculated C6/36 cells showed specific fluorescence with both 813 and 546 monoclonal antibodies. Numbers of ticks in each pool are shown in parentheses. cWhere indicated by +, pools of ticks were tested; numbers of ticks in each pool are shown in parentheses. ND, not done. dMice were infested with infected O. moubata ticks and after 14 days were sacrificed and the brain homogenates tested by IFA and RT-PCR. N/A, not applicable. 654 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 4, April 2004