An automated colorimetric micro-neutralization assay (CmNt) was developed for confirmation and differentiation of West Nile Virus (WNV)-positive human sera as a higher throughput alternative to the standard six-well plaque-reduction neutralization test (PRNT). CmNt was performed in high-capacity 96-well micro-titer plates and required 4-6 days to complete. Inhibition of infection was determined by reduced neutral red-dye retention and conveniently recorded by a colorimetric plate reader. Human sera previously confirmed by PRNT as either negative (N = 52), WNV positive (N = 81), or St. Louis encephalitis virus positive (N = 12) were tested by CmNt; interpreted results were virtually identical to PRNT with a reduced turnaround time and higher throughput. Additionally, a handful of dengue virus positive and negative specimens (four each) were tested by CmNt; interpreted results were identical to PRNT.

Jamestown Canyon (JC) and Saint Louis encephalitis (SLE) viruses are mosquito-transmitted viruses that have long been present in California. The objective of this study was to determine the seroprevalence of these two viruses in horses prior to the introduction of West Nile (WN) virus. Approximately 15% of serum samples collected in 1998 from 425 horses on 44 equine operations horses throughout California had serum antibodies to JC virus, whereas antibodies were not detected to SLE virus. The results indicate that horses in California were commonly infected prior to 1998 with mosquito-transmitted Bunyaviruses that are identical or closely related to JC virus, but not with SLE virus. The different seroprevalence of SLE and JC viruses in horses likely reflects the unique ecology of each virus, and it is predicted that WN virus will have a wider distribution in California than closely related SLE virus.

West Nile virus (WNV) was first isolated in California during July 2003 from a pool of Culex tarsalis collected near El Centro, Imperial County. WNV transmission then increased and spread in Imperial and Coachella Valleys, where it was tracked by isolation from pools of Cx. tarsalis, seroconversions in sentinel chickens, and seroprevalence in free-ranging birds. WNV then dispersed to the city of Riverside, Riverside County, and to the Whittier Dam area of Los Angeles County, where it was detected in dead birds and pools of Cx. pipiens quinquefasciatus. By October, WNV was detected in dead birds collected from riparian corridors in Los Angeles, west to Long Beach, and through inland valleys south from Riverside to San Diego County. WNV was reported concurrently from Arizona in mid-August and from Baja, Mexico, in mid-November. Possible mechanisms for virus introduction, amplification, and dispersal are discussed.

House finches, Carpodacus mexicanus, were experimentally infected with high and standard doses of western equine encephalomyelitis virus (WEEV) or St. Louis encephalitis virus (SLEV) to determine whether high doses would produce an elevated viremia response and a high frequency of chronic infections. Finches inoculated with approximate to100,000 plaque forming units (PFU) of WEEV or SLEV produced viremia and antibody responses similar to those in finches inoculated with approximate to100 PFU of WEEV or 1000 PFU of SLEV, the approximate quantities of virus expectorated by blood-feeding Culex tarsalis Coquillett. infected finches were held through winter and then necropsied. Only one finch inoculated with the high dose of SLEV developed a chronic infection. Our data indicated that elevated infectious doses of virus may not increase the viremia level or the frequency of chronic infection in house finches.

Female Culex tarsalis Coquillett in reproductive diapause were infected per os or by intrathoracic inoculation with western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during "fall," maintained over a simulated "winter," and then tested for virus infection and transmission in vitro and in vivo after "vernal" termination. Exposure of F1 progeny of field-collected females to cool temperatures and short daylength produced females in reproductive diapause that were reluctant to imbibe infectious virus from pledgets soaked with suspensions of virus, blood and sucrose (2.5% by volume). Those infected per os maintained virus at very low or undetectable titers. Some females that originally tested negative for WEE by plaque assay on Vero cell culture tested positive by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Vero cell culture after passage in mosquito cells. Few females became infected orally with SLE, but these infected females developed elevated titers. Females inoculated with SLE retained their infection through winter and then transmitted readily in vitro and in vivo. Feeding on a vertebrate host after diapause termination significantly increased the titer of SLE in previously infected females. These experiments simulated how infections acquired either horizontally or vertically may provide mechanisms for WEE and SLE overwintering. Attempts to detect infected females during winter following a summer with enzootic WEE activity were negative by both RT-PCR and plaque assay.