Ixodid and Argasid Ticks and West Nile Virus Table 2. Nucleotide sequences of primers used in first round (5′[1] and 3′[2]) and second round (5′[2] and 3′[2]) of nested RT-PCRa Primer Sequence Position in WNV (NY99)b 5′(1) CCATATGAATTCCATGAGTGCTATCAATCGGCGGAG 31 aa upstream (C gene) from start of PrM gene (373) 3′(1) CATATGCGGCCGCTTACTAGGTGATTGATCTGTTGTTCTCC 31 aa downstream (NS1) from end of E gene (2,562) 5′(2) CATATGCGGCCGCTTACTACCGGTCCATCCAAGCCTC Start of E gene (967) 3′(2) CCATATAGATCTCGGAGGTCATTCAACTGCCTTGGAATGAGC 395 aa into E gene (2,152) aRT-PCR, reverse transcriptase–polymerase chain reaction; aa, amino acids. bNucleotide position in relation to complete genome sequence of WNV (NY99) shown in parentheses (accession no. AF19685). or a WNV-specific monoclonal antibody (MAb 546) (21). Labeled cells were visualized by using an Olympus epiflu- orescence microscope. Uninfected cells were used as neg- ative controls and cells infected with the original viral stock as positive controls. Tick samples were deemed pos- itive when more than 10% of the cells showed specific flu- orescence with both monoclonal antibodies. Nested RT-PCR Assay RNA was extracted from homogenized samples (100 µ L) by using RNAgents total RNA extraction kit in accor- dance with the manufacturer’s instructions (Promega, Madison, WI). cDNA synthesis was carried out with Superscript II reverse transcriptase (Invitrogen, San Diego, CA) and 3′(1) primer (Table 2) for 50 min at 42°C, in a total volume of 20 µ L. PCR was carried out on the cDNA (1 µ L) by using 5′(1) and 3′(1) primers. Nested PCR was carried out on 1 µ L of the first-round PCR product using the nested primers 5′(2) and 3′(2). All PCR reactions were carried out in a 50-µ L volume with REDTaq DNA poly- merase (Sigma Chemical Co., St. Louis, MO). A Hybaid Touchdown thermal cycler was used with the following program: 94.5°C for 1 min, 30 cycles of 94°C for 40 s, 56°C for 1 min, and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. Viral stock, RNA extracted from uninfected ticks, and PBS-only samples were used as control reactions. Positive samples gave a PCR product of approximately 1.2 kbp. This method could detect RNA from a viral stock equivalent of 9 PFU (data not shown). To confirm the identity of RT-PCR products, PCR prod- ucts were gel purified with QIAquick (Qiagen, Crawley, UK) columns in accordance with manufacturer’s instruc- tions. The purified DNA was sequenced with an ABI auto- matic sequencer and the nested primers 5′(2) and 3′(1) and a primer based on the internal sequence of the E gene of WNV (not shown). Results neurologic disease developed in the mice after 5 or 6 days, and they were euthanized. O. moubata ticks that had fed on mice on days corresponding to the viremic period (i.e., days 2 and 3 after infection), but not those fed outside this period, contained viral antigen as measured by immunoflu- orescence assay (IFA) (Table 1). Two days after engorge- ment, 17% (n = 12) I. ricinus ticks that started to feed on hosts 3 days before WNV injection, but not those that had started to feed 4 days after injection, were positive for WNV RNA. When the former group of ticks was tested 28 days later, no evidence of infection was found. Infected O. moubata ticks, in contrast, maintained the virus after molt- ing into the next instar (i.e., third instar); following a sec- ond, noninfectious bloodmeal; and after molting a second time into fourth instars. Fifty percent of the individual ticks (n = 14) tested by RT-PCR were positive for WNV RNA when examined 132 days after the initial infectious bloodmeal. Co-feeding Transmission Five days after engorgement, 23% (n = 66) of uninfect- ed second instar O. moubata ticks that had co-fed with infected cohorts of third instar ticks on noninfected mice were positive for WNV RNA (Table 1). The remaining unfed ticks (n = 15) were tested after they had molted into third instars, 45 days after co-feeding. Four of these ticks (26%) were positive for WNV RNA. The identities of the PCR products obtained from three positive samples were confirmed by sequence analysis. Tick-to-Host Transmission Infected cohorts of O. moubata ticks (third instar) were fed on uninfected mice to investigate tick-to-host transmis- sion. Of the 17 uninfected mice used (including mice used in co-feeding experiments), none showed clinical signs of infection. One of the brains tested, from a mouse infested with an infected cohort of 20 ticks, was positive by RT- PCR but negative when tested by IFA (Table 1). The PCR product was sequenced to confirm the identity of WNV. Host-to-Tick Transmission BALB/c mice infected with WNV were weakly viremic 2 and 3 days after injection, with mean titers of 6 x 103 and 3 x 103 PFU/mL-1 blood respectively. After 4 days, viremia was no longer detectable by plaque assay, although severe Discussion Laboratory studies from the 1950s suggested that some tick species might serve as competent vectors for WNV. Hurlbut and Taylor (1956) showed that O. savignyi and O. erraticus ticks were infected after feeding on mice inocu- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 4, April 2004 655