Fig 1.
Overview of the Experimental Workflow.
Oropouche virus infection, dissemination, and transmission were evaluated across various genotypes, incubation periods in cell culture, and extrinsic incubation periods. Illustration figures were created with biorender.com.
Fig 2.
(A) Perforated bags containing Oropouche virus (OROV) suspension and defibrinated bovine blood placed on hand warmers.
(B) Perforated bag positioned atop a cylindrical cardboard mosquito cage for blood feeding. (C) Blood-engorged Culex quinquefasciatus females. (D) Immobilized female mosquitoes prepared for saliva collection using capillary tubes filled with immersion oil. Photo credit: Nathan Burkett-Cadena. Used with permission and published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
Table 1.
Mean infection, dissemination, and transmission rates (conditional rates) for Culex quinquefasciatus (Vero Beach strain, 2015) and Aedes aegypti (Orlando strain, 1952; Lower Keys strain, 2024) after exposure to two Oropouche virus (OROV) genotypes (TRVL9760 and 240023). Virus suspensions were harvested after 5 or 7 days of incubation in Vero cell culture, corresponding to higher or lower bloodmeal titers, respectively (incubation period in cell culture, IP; see footnote for titers). The Lower Keys strain represents F1 progeny from field-collected Ae. aegypti in 2024. Rates were evaluated at 7-, 14-, and 21-day extrinsic incubation periods (EIP). Infection rates were calculated as the number of mosquitoes with RT-qPCR–positive bodies divided by the total number blood-fed. Dissemination rates were calculated as the number with RT-qPCR–positive legs/wings divided by the number of body-positive mosquitoes. Transmission rates were calculated as the number with RT-qPCR–positive saliva divided by the number of dissemination-positive mosquitoes. All samples were tested for OROV RNA using RT-qPCR, with samples considered positive if Cq ≤ 38. Detection of viral RNA does not confirm the presence of infectious virus. A dash (–) indicates that samples were not collected or not tested under that condition.
Fig 3.
Titers of (A) stock virus suspensions and (B) blood-virus suspensions used for mosquito oral infection, containing two Oropouche virus (OROV) genotypes (TRVL9760 and 240023) incubated for 5 or 7 days in Vero cell culture.
Titers were quantified by plaque assay. Data points represent technical replicates; horizontal lines indicate the median and 95% confidence intervals. Statistical significance was assessed using one-way ANOVA followed by Tukey’s HSD test. Exact p-values for significant pairwise comparisons are shown (p < 0.05).
Fig 4.
Mean (A) infection (head, thorax, and abdomen), (B) dissemination (legs and wings), and (C) transmission (saliva) rates of Culex quinquefasciatus (CXQU, Vero Beach strain) and Aedes aegypti (AEAE, Orlando and Lower Keys strains) infected with Oropouche virus (OROV) genotypes TRVL9760 and 240023.
Positivity rates were calculated as follows: infection = positive bodies/ total mosquitoes tested; dissemination = positive legs/ infected bodies; transmission = positive saliva/ mosquitoes with disseminated virus. Data points represent sample-size–weighted mean positivity (%) calculated from individual mosquito-level binomial outcomes. Wilson score 95% confidence intervals for all infection, dissemination, and transmission rates are provided in S4 Table. Asterisks (*) indicate statistically significant differences among mosquito strains at each variable level, based on logistic regression with likelihood ratio tests (p < 0.05). Panel labels (A1–C4) correspond to comparisons across (1) strain, (2) genotype, (3) incubation period in cell culture, and (4) extrinsic incubation period. Marker shape and color indicate mosquito strain: blue circle = CXQU, purple square = AEAE Orlando, and pink triangle = AEAE Lower Keys. Missing data points reflect treatment combinations not tested due to mosquito availability constraints.