Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing.

To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were marked genetically by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex(®) technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex(®) technology and quantitative sequencing provided semi-quantitative or qualitative measurements, a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses.

Comparison of enzootic risk measures for predicting West Nile disease, Los Angeles, California, USA, 2004-2010.

In Los Angeles, California, USA, 2 epidemics of West Nile virus (WNV) disease have occurred since WNV was recognized in 2003. To assess which measure of risk was most predictive of human cases, we compared 3 measures: the California Mosquito-Borne Virus Surveillance and Response Plan Assessment, the vector index, and the Dynamic Continuous-Area Space-Time system. A case-crossover study was performed by using symptom onset dates from 384 persons with WNV infection to determine their relative environmental exposure to high-risk conditions as measured by each method. Receiver-operating characteristic plots determined thresholds for each model, and the area under the curve was used to compare methods. We found that the best risk assessment model for human WNV cases included surveillance data from avian, mosquito, and climate sources.

Early warning system for West Nile virus risk areas, California, USA.

The Dynamic Continuous-Area Space-Time (DYCAST) system is a biologically based spatiotemporal model that uses public reports of dead birds to identify areas at high risk for West Nile virus (WNV) transmission to humans. In 2005, during a statewide epidemic of WNV (880 cases), the California Department of Public Health prospectively implemented DYCAST over 32,517 km2 in California. Daily risk maps were made available online and used by local agencies to target public education campaigns, surveillance, and mosquito control. DYCAST had 80.8% sensitivity and 90.6% specificity for predicting human cases, and k analysis indicated moderate strength of chance-adjusted agreement for >4 weeks. High-risk grid cells (populations) were identified an average of 37.2 days before onset of human illness; relative risk for disease was >39× higher than for low-risk cells. Although prediction rates declined in subsequent years, results indicate DYCAST was a timely and effective early warning system during the severe 2005 epidemic.

Temporal connections between Culex tarsalis abundance and transmission of western equine encephalomyelitis virus in California.

Definition of targets for vector control requires an understanding of the relationship between vector abundance and the intensity of arbovirus transmission. Using an extensive surveillance dataset with observations from sentinel chicken flocks and mosquito traps paired in time and space, hierarchical autoregressive logistic regression models were developed to predict the probability of seroconversion in chickens for western equine encephalomyelitis virus (WEEV) based on the relative abundance of the principal vector, Culex tarsalis. After adjustments for confounders, the abundance of Cx. tarsalis 29-42 d before the date of chicken sampling was credibly associated with the risk of WEEV transmission in both the Central and Coachella Valleys, and a doubling of relative Cx. tarsalis abundance was associated with a 58% increase in the odds of seroconversion. The critical time windows identified in our study highlight the need for surveillance of vector populations and forecasting models to guide proactive vector control measures before the detection of transmission to sentinel chickens.