Fig 1.
Five five-week-old interferon knockout mice were inoculated with 105 Vero PFU of ZIKV PR15 via subcutaneous injection and held for two days to reach peak viremia. After two days, mice were anesthetized, blood was collected via each mouse’s submandibular vein (i.e., cheek punch) and blood serum ZIKV titer was assessed using Vero cell plaque assay, and 1,200 female Aedes aegypti mosquitoes aged 3–5 days were exposed to all five mice. Bloodfed mosquitoes were randomly sorted into four half-gallon cartons held at one of four constant temperatures: 18, 21, 26, or 30°C. At days chosen based on published extrinsic incubation studies of other flaviviruses, mosquitoes were cold-anesthetized in cohorts of 20 females and then legs and wings were removed and saliva was collected by inserting the proboscis into a capillary tube containing fetal bovine serum for 20 minutes. Individual bodies, legs/wings, and the saliva sample from each mosquito were placed separately in tubes containing cell culture media and homogenized. Viral RNA was extracted and ZIKV viral RNA (vRNA) titers were determined for each body, legs/wings, and saliva sample using RT-qPCR. A cycle threshold (Ct) value ≤ 38 was considered positive for ZIKV RNA. Positive bodies indicated ZIKV infection, positive legs and wings indicated ZIKV dissemination, and positive saliva indicated ZIKV transmission.
Fig 2.
Percent of mosquitoes with infected, disseminated, and transmitted ZIKV vRNA of 20 tested at each temperature and time point combination tested.
Samples were processed in duplicate by RT-qPCR. Ct values < 38 in at least one of the duplicates indicated positive vRNA.
Table 1.
ZIKV vRNA infection, dissemination, and transmission rates by temperature and time point.
Samples were processed in duplicate by RT-qPCR. Ct values < 38 in at least one of the duplicates indicated positive vRNA.
Table 2.
Coefficients from the logistic model for the probability of ZIKV transmission as a function of time and temperature.
Fig 3.
Fitted logistic curves showing the proportions of Ae. aegypti transmitting ZIKV vRNA over time by temperature.
Each point represents the observed proportion of mosquitoes (of 20 tested) that transmitted at each temperature and time-point. The estimated EIP50 is indicated for each temperature.
Fig 4.
(A) Fitted logistic curves showing the relationship between dissemination titer (log10(genomes)) and proportion transmitting ZIKV RNA for each temperature. Dot size indicates the number of mosquitoes within ±.5 log10(genomes) of plotted number. (B) Fitted line showing the relationship between dissemination titer (log10(genomes)) and proportion transmitting ZIKV RNA for 26 and 30°C combined. Dot size indicates the number of mosquitoes within ±.5 log10(genomes) of plotted number.
Fig 5.
Fitted curves showing the median EIP of the flaviviruses ZIKV, dengue virus (DENV), and West Nile virus (WNV) over a range of temperatures.
ZIKV–PR is the Puerto Rico strain used in this study, whereas ZIKV–MX is a Mexican strain used by Tesla et al. 2018.