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. 2020 Mar 18;14(3):e0008047.
doi: 10.1371/journal.pntd.0008047. eCollection 2020 Mar.

Impact of Temperature on the Extrinsic Incubation Period of Zika Virus in Aedes Aegypti

Free PMC article

Impact of Temperature on the Extrinsic Incubation Period of Zika Virus in Aedes Aegypti

Olivia C Winokur et al. PLoS Negl Trop Dis. .
Free PMC article


Since Zika virus (ZIKV) emerged as a global human health threat, numerous studies have pointed to Aedes aegypti as the primary vector due to its high competence and propensity to feed on humans. The majority of vector competence studies have been conducted between 26-28°C, but arboviral extrinsic incubation periods (EIPs), and therefore transmission efficiency, are known to be affected strongly by temperature. To better understand the relationship between ZIKV EIPs and temperature, we evaluated the effect of adult mosquito exposure temperature on ZIKV infection, dissemination, and transmission in Ae. aegypti at four temperatures: 18°C, 21°C, 26°C, and 30°C. Mosquitoes were exposed to viremic mice infected with a 2015 Puerto Rican ZIKV strain, and engorged mosquitoes were sorted into the four temperatures with 80% RH and constant access to 10% sucrose. ZIKV infection, dissemination, and transmission rates were assessed via RT-qPCR from individual mosquito bodies, legs and wings, and saliva, respectively, at three to five time points per temperature from three to 31 days, based on expectations from other flavivirus EIPs. The median time from ZIKV ingestion to transmission (median EIP, EIP50) at each temperature was estimated by fitting a generalized linear mixed model for each temperature. EIP50 ranged from 5.1 days at 30°C to 24.2 days at 21°C. At 26°C, EIP50 was 9.6 days. At 18°C, only 15% transmitted by day 31 so EIP50 could not be estimated. This is among the first studies to characterize the effects of temperature on ZIKV EIP in Ae. aegypti, and the first to do so based on feeding of mosquitoes on a live, viremic host. This information is critical for modeling ZIKV transmission dynamics to understand geographic and seasonal limits of ZIKV risk; it is especially relevant for determining risk in subtropical regions with established Ae. aegypti populations and relatively high rates of return travel from the tropics (e.g. California or Florida), as these regions typically experience cooler temperature ranges than tropical regions.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Study design workflow.
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
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.
Fig 3
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
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
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.

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