Observed data: (A): Calculated pooled Ct value vs. disease week from 2022 to 2024. Pooled Ct values presented here are calculated by using species Culex pipiens/restuans/salinarius and Culex tarsalis. (B): RT-qPCR test results vs. disease week from 2022 to 2024.

Overall structure of the agent-based model.

(A): Transmission cycle of WNV between birds (hosts) and mosquitoes (vectors). Female mosquitoes lay eggs, which hatch after an average of 8 days, before becoming female adult mosquitoes that contribute to the transmission cycle. (B): Transmission occurs with some probability when an infected female mosquito bites a susceptible bird, or a susceptible female mosquito bites an infected bird. (C): Within-host viral kinetics model. Infected birds follow an acute viral load trajectory with rising and clearance phases, initiating from the RT-qPCR limit of detection. Female mosquitoes may follow one of two different viral kinetics models: the mosquito’s initial viral load is proportional to the infector bird’s viral load at the time of bite and either leads to a non-replicating viral trajectory (static viral load) (i) or a replication-competent infection (dynamic viral load) (ii). Note the Ct value scale is inverted, with lower Ct values depicting higher on the scale. (D): Overall population-level dynamics of the agent-based model. If bitten by an infected mosquito, susceptible birds (S) can become infectious (I). Once infectious, they will eventually recover (R) with lifelong immunity following their infectious period. Susceptible mosquitoes (S), if they bite infectious birds, will become infectious (I) for the rest of their lifespan. Births and deaths are possible in all these states for both birds and mosquitoes. (E): Simulation of routine mosquito trapping and pooled testing accounting for blood meal-seeking behaviour. For each pool, a combined pooled Ct value is calculated as the mean viral load of captured WNV-positive mosquitoes, diluted by the contribution of WNV-negative mosquitoes. (F): Example population-level Ct dynamics from the simulation. Note the Ct value scale is inverted, with lower Ct values depicting higher on the scale.

Simulated population-level WNV infection dynamics in mosquitoes.

Each line represents one year of simulated data, and the bold black line highlights the simulated trajectory used for (C). (A): Weekly mosquito infection prevalence per capita. (B): Weekly mosquito infection incidence per capita. (C): Individual-level Ct values of the female mosquito population from one year of a single simulation. Each point represents a Ct value from a single randomly selected mosquito, representing perfect observations of the entire population at all times. (D): Calculated pooled Ct values from the mosquito population based on the individual-level Ct values in (C)

Distributions of the pooled Ct values within the limit of detection.

(A): simulated Ct values by assuming all mosquitoes undergo dynamic infection from Ct=40. (B): simulated Ct values assuming mosquitoes inherit 100% of viral load from birds, and 50% of mosquitoes undergo a static viral kinetics trajectory. The bottom left and right panels. (C) and (D): the density of pooled Ct values from Nebraska and Colorado states over three years, respectively.

(A): Weekly pooled Ct values from the captured mosquitoes (using the sampling procedure used in the agent-based model). Each dot represents the Ct value calculated per pool. (B): Binarised data based on the pooled Ct values in (A). Binarisation was based on whether or not the pooled Ct values were within the limit of detection. (C): Estimated proportion of productive infection using the pooled Ct values. The pink ribbon shows the 95% confidence interval. (E): The estimated weekly WNV prevalence among mosquitoes using the binarised data with the PooledInfRate R package (green line). The green ribbon shows the 95% confidence interval. These values are compared with the true prevalence from the simulation (blue dashed line). (F):: Estimated productive infection prevalence using pooled Ct values. The purple ribbon shows the 95% confidence interval.

Comparison of prevalence estimates based on the Pooled Ct values and the PooledInfRate R package with varying pool sizes.

The beige colour dots present maximum likelihood prevalence estimates calculated from pooled Ct values from a different simulation. The estimated prevalence calculated using the binarised data is in dark red. Each panel presents the estimates based on 100 pools containing up to 50 mosquitoes (pool size). The black dashed line represents perfect estimates. Labels along with the red dotted lines illustrate the maximum prevalence level at which the PooledInfRate R package returns accurate estimates.

Overall prevalence estimation per year for Nebraska and Colorado from 2022-2024 using pooled Ct values (green) and binarised data (red), and the estimated productive infection prevalence (beige).

The lines show mean estimates and the ribbons are 95% confidence intervals. Red dashed lines are the estimated prevalence based on binarised data.