A Method for Repeated, Longitudinal Sampling of Individual Aedes aegypti for Transmission Potential of Arboviruses

SIMPLE SUMMARY: Mosquito-borne viruses, such as Zika virus (ZIKV), remain a major public health concern worldwide. Vector competence is defined by the ability of a vector (mosquito) to become infected by and subsequently transmit a virus. Not all species of mosquitoes will transmit the same viruses; therefore, it is imperative that we continue to study mosquito–virus pairings in order to assess risk of transmission in different areas. Traditionally, a competent vector is determined by a high proportion of infectious saliva at terminal time points. However, a multitude of factors, such as mosquito biting habits and time, will have an impact on vector competence. We herein present a novel method for measuring biting habits and ZIKV transmission over time. To do this, we offered individual mosquitoes a bloodmeal (180 μL) every other day from 9 to 24 days post-exposure. Biting behavior was recorded as either probing, blood fed, or no bite; the bloodmeal was then collected and tested for the presence of ZIKV. Our results were successful in measuring behavior and viral transmission over time, and demonstrated variation among individual mosquitoes for both biting behavior and the amount of virus expectorated over time. Our results highlight the need for continued investigation into the complexity of vector competence, and we offer a method to aid in such investigations. ABSTRACT: Mosquito-borne viruses are the cause of significant morbidity and mortality worldwide, especially in low- and middle-income countries. Assessing risk for viral transmission often involves characterization of the vector competence of vector–virus pairings. The most common determination of vector competence uses discreet, terminal time points, which cannot be used to investigate variation in transmission aspects, such as biting behavior, over time. Here, we present a novel method to longitudinally measure individual biting behavior and Zika virus (ZIKV) transmission. Individual mosquitoes were exposed to ZIKV, and from 9 to 24 days post-exposure, individuals were each offered a 180 μL bloodmeal every other day. Biting behavior was observed and characterized as either active probing, feeding, or no bite. The bloodmeal was then collected, spun down, serum collected, and tested for ZIKV RNA via qRT-PCR to determine individuals’ vector competence over time. This included whether transmission to the bloodmeal was successful and the titer of expectorated virus. Additionally, serum was inoculated onto Vero cells in order to determine infectiousness of positive recovered sera. Results demonstrate heterogeneity in not only biting patterns but expectorated viral titers among individual mosquitoes over time. These findings demonstrate that the act of transmission is a complex process governed by mosquito behavior and mosquito–virus interaction, and herein we offer a method to investigate this phenomenon.