Vero 76 cells were used for virus propagation, plaque assays, and virus isolation. Growth medium (GM) was Dulbecco modified Eagle medium (GIBCO, Invitrogen Corp., Carlsbad, CA, USA) that contained 10% heat-inactivated fetal bovine serum (FBS) and 2.0 mmol/L L-glutamine. Maintenance medium (MM) was GM with 1% FBS. Overlay medium (OM) for virus assay was 1 part MM and 1 part 2% agarose (Difco, Becton, Dickinson and Co., Sparks, MD, USA), prepared in Hanks’ balanced salt solution (GIBCO, Invitrogen Corp.). Virus diluent was 1 part MM and 1 part CO₂-independent medium (GIBCO, Invitrogen Corp.) containing 1% FBS. Mosquito grinding diluent (GD) was GM supplemented with 20% FBS. Twenty milligrams of gentamicin sulfate (GentaMax100, Phoenix Pharmaceuticals, Inc., Belmont, CA, USA) was added per 100 mL of all media and diluents. Feeding solution for capillary tubes used for collecting saliva in artificial transmission assays consisted of 5% (w/v) sucrose and 0.5% FBS in 0.15 mmol/L phosphate-buffered saline, pH 7.0.

WNV strain IA02-crow was used in all experiments. The virus was isolated from a crow in Iowa in 2002 and passed 4× in Vero 76 cell cultures (15). Virus was assayed by the plaque method. Serial 10-fold dilutions of serum were made in cold (≈4°C) virus diluent. Individual 6-well cell culture plates containing confluent Vero 76 cell monolayers were inoculated in duplicate with 0.75 mL of each virus dilution and incubated for 75 min at 37°C in a 5% CO₂ atmosphere. The inoculums were replaced with 3 mL of OM, and plates were maintained at 37°C. Three days later, an additional 3 mL of OM containing 2% stock neutral red solution (Sigma Aldrich, Saint Louis, MO, USA) was added to the original overlays. Plates were maintained at 37°C overnight; plaques were then counted and titers were expressed as PFU/mL.

Mature eastern chipmunks were captured with live traps (H.B. Sherman Traps, Tallahassee, FL, USA) in Story County, Iowa, in the fall of 2005 under a state of Iowa permit. Chipmunks were verified to be free of antibodies to WNV by an epitope-blocking ELISA (16). Chipmunks were housed in individual cages in the biosafety level 3 animal facility at Iowa State University (ISU) and treated and handled in accordance with guidelines established by the ISU Institutional Animal Care and Use Committee. Chipmunks were anesthetized with ketamine/aceopromazine at 10:1.0 mg/kg for mosquito feeding and blood collection. Chipmunks were bled once from the retroorbital plexus of each eye and once from the heart. Blood was not collected again from the retroorbital plexus of either eye for at least 7 days, or from the heart unless the bleeding was terminal. Two-day-old WNV antibody–free broiler chickens (Hoover’s Hatchery, Inc., Rudd, IA, USA) were used to detect virus transmission by mosquitoes that previously fed on infected chipmunks.

Ae. triseriatus and Cx. pipiens were collected in Iowa and colonized in 2002. Ae. vexans were first-generation mosquitoes collected in central Iowa during the summer of 2004. Mosquitoes were maintained on 10% sucrose (w/v) in controlled conditions (27°C ± 1°C and 80% ± 5% relative humidity) with a 16:8-hour photoperiod. Mosquitoes were deprived of sucrose for 48 hours and water for 24 hours before feeding on chipmunks and chickens or before artificial transmission experiments.

Mosquitoes were killed by freezing. Whole insects, bodies without legs and wings, and legs alone were triturated individually in 1.5-mL microcentrifuge tubes containing 300 μL of cold (≈4°C) GD. For artificial transmission assays, the contents of individual capillary tubes containing saliva of mosquitoes were deposited into 300 μL of GD and stored at –70°C until assayed. Samples were thawed at 37°C and brought to a final volume of 2 mL with cold MM. Approximately 1.6 mL of each sample was passed through a 0.45-μm filter onto a confluent cell monolayer in a 25-cm² cell culture flask. The remaining volume was stored at –70°C. Inoculated flasks were incubated for 1 h at 37°C, and then 5 mL of MM was added. Inoculated cell cultures and controls were observed daily for cytopathic effects (CPE) for up to 7 days postinoculation (p.i.). WNV specificity of CPE was confirmed by the VecTest WNV/SLE antigen assay (Microgenics Corp., Freemont, CA, USA).

Serum samples were tested for antibodies to flaviviruses by a blocking ELISA as previously described (16). The ELISAs were performed by using the WNV-specific monoclonal antibody (MAb) 3.1112G (Chemicon International, Temecula, CA, USA) or the flavivirus-specific MAb 6B6C-1, obtained from the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA. The ability of test sera to block binding of the MAbs to WNV antigen was compared to the blocking ability of control sera from chipmunks without antibodies to flaviviruses. Data were expressed as relative percentages and inhibition values; a threshold >30% was considered to indicate viral antibodies.

Tissue samples were fixed in 10% formalin and processed for histologic examination by the Veterinary Diagnostic Laboratory at ISU following standardized protocols. WNV-specific mouse ascites fluid (ATCC Catalog #VR01267CAF, Manassas, VA, USA) was used as the primary antibody at a dilution of 1:2,000 to detect WNV antigen. A 30-minute staining period was used. Processed tissues, including negative controls, were evaluated blindly by a veterinary pathologist for evidence of microscopic lesions and scoring of the amount of WNV antigen in the tissues. The amount of WNV antigen in the tissues was given a score of 0 (no staining), + (mild multifocal staining of individual cells), ++ (moderate multifocal staining of cells), or +++ (large amounts of antigen and large numbers of cells staining).

Initially, 1 chipmunk was inoculated intramuscularly (i.m.) with 10^5.7 PFU of WNV. Blood was collected 2 days later and found viremic for WNV. Subsequently, 6 chipmunks were divided into 2 equal groups. One group was inoculated i.m. with 10^3.5 PFU of WNV and the other with 10^1.5 PFU. An eighth chipmunk was subsequently inoculated with 10^2.6 PFU. Serum specimens for WNV assay were collected on days 1–4 p.i. and then not again until day 8 p.i. If we encountered difficulty in obtaining blood, we aborted the procedure. Swabs of oral and rectal cavities of chipmunks that were inoculated with WNV were taken on days 1–3 p.i. and assayed for WNV. Swabs of urine also were collected from chipmunks when possible.

Tissues—including the brain, spinal cord, lung, heart, skeletal muscle, liver, kidney, spleen, pancreas, small and large intestine, adrenal glands, and salivary glands—were collected from all chipmunks. These tissues were examined for gross and microscopic lesions and assayed by immunohistochemical tests for WNV antigen. Tissues from 1 chipmunk that had not been inoculated served as negative controls.

Groups of up to 15 Ae. triseriatus, Ae. vexans, and Cx. pipiens in the same container were fed on chipmunks between days 1 and 4 p.i. The blood-fed mosquitoes were separated by species, maintained for 14 to 15 days as described previously, and assayed for WNV infection. Ae. triseriatus also were assayed for disseminated infections.

Ae. triseriatus transmission of WNV was determined by feeding mosquitoes on 2-day-old chickens or by permitting mosquitoes, from which wings and legs were removed, to feed for 20 minutes from capillary tubes containing feeding solution. Transmission was confirmed by detecting WNV in blood that was collected from chickens ≈48 hours after mosquitoes fed, or in the contents of capillary tubes from which mosquitoes fed.

WNV viremia developed in all 8 chipmunks (Figure 1) after they were infected with the virus by needle inoculation. Viremia titers peaked on day 2 p.i. and were generally higher in those chipmunks that were inoculated with higher doses of virus. Peak WNV titers of 10^7.2 and 10^7.8 PFU/mL of serum occurred in 2 chipmunks that were inoculated with 10^2.6 or 10^3.5 PFU of WNV, respectively. The highest titer observed in chipmunks inoculated with 10^1.5 PFU of WNV was 10^6.5 PFU/mL. The mean estimated number of days and 95% confidence intervals that WNV serum titers remained >10^4.8 PFU/mL and >10^5.6 PFU/mL were 1.7 (1.1–2.3) and 1.4 (1.0–1.6), respectively. The longest period of time that WNV titers were >10^4.8 PFU/mL was at least 3 days and occurred in a chipmunk that was inoculated with 10^3.5 PFU of WNV. No WNV was detected in serum specimens from 3 chipmunks that were bled on day 8 p.i.

No WNV was isolated from oral or rectal cavities of infected chipmunks on day 1 p.i., but WNV was isolated on day 2 p.i. from the oral cavities of 4 chipmunks. On day 3 p.i., WNV was isolated from the oral cavities of 4, the rectal cavities of 3, and the urine of 2 chipmunks.

No signs of illness were observed in any of the WNV-infected chipmunks during the first 8 days p.i. Two chipmunks died on days 1 and 2 p.i. during sampling, and a third chipmunk was killed on day 4 p.i. to obtain blood for WNV assay. The first potential signs of WNV infection were observed in the remaining 5 chipmunks between days 9 and 11 p.i. In 3 chipmunks, neurologic symptoms developed, characterized by head tilt and incoordination; the chipmunks were humanely killed. A fourth chipmunk became lethargic and was reluctant to move; it also was humanely killed. A fifth chipmunk had no signs of illness but died unexpectedly on day 27 p.i. WNV-specific antibody was detected in 3 chipmunks that were bled on day 8, 11, or 14 p.i.

No gross lesions were observed in any chipmunk; however, microscopic lesions and/or WNV antigen were observed in tissues of 4 of 8 infected chipmunks (Table 1). Mild to moderate multifocal lymphoplasmacytic meningoencephalitis with gliosis and neuronal necrosis was observed in 2 chipmunks on days 9 and 11 p.i. Mild lymphohistiocytic perivascular cuffing of vessels with moderate to severe multifocal hemorrhage throughout the brain was present in 1 of these 2 chipmunks. WNV antigen also was detected in neurons of both chipmunks. Mild lymphoplasmacytic and histiocytic hepatitis were observed in 3 chipmunks. Viral antigen was also detected in Kupffer cells and macrophages of 2 of these 3 chipmunks. Mild multifocal lymphoplasmacytic interstitial nephritis with mild focal renal tubular necrosis was observed in 2 chipmunks on days 9 and 11 p.i. WNV antigen was present in renal tubular epithelial cells and in the renal arterial walls (Figure 2) of these 2 chipmunks and in 2 others in which no kidney lesions were found. WNV antigen in the absence of lesions was demonstrated in the muscularis mucosa, scattered mucosal epithelial cells, and mononuclear cells in the lamina propria of the small intestine of 2 chipmunks.

The feeding success rates of Ae. triseriatus, Ae. vexan, and Cx. pipiens on viremic chipmunks were 68%± 5%, 27% ± 6%, and 8% ± 4%, respectively. The WNV infection rates of the 3 mosquito species that fed on these chipmunks are summarized in Table 2. Disseminated infections developed in 5 of the 6 WNV-infected Ae. triseriatus that fed on chipmunks with WNV serum titers >10^5.6 PFU/mL. Two (67%) of 3 Ae. triseriatus with disseminated infections transmitted WNV in an artificial transmission assay. One Ae. triseriatus with a disseminated infection that fed on a 2-day-old chicken did not transmit WNV.