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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.

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.

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these ‘parental’ viruses and the ‘progeny’ poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

Background Transmission dynamics of arboviruses like Zika virus are often evaluated by vector competence (the proportion of infectious vectors given exposure) and the extrinsic incubation period (EIP, the time it takes for a vector to become infectious), but vector age is another critical driver of transmission dynamics. Vectorial capacity (VC) is a measure of transmission potential of a vector-pathogen system, but how these three components, EIP, vector competence and vector age, affect VC in concert still needs study. Methods The interaction of vector competence, EIP, and mosquito age at the time of infection acquisition (Ageacquisition) was experimentally measured in an Aedes aegypti-ZIKV model system, as well as the age-dependence of probability of survival and the willingness to bite. An age-structured vectorial capacity framework (VCage) was then developed using both EIPMin and EIPMax, defined as the time to first observed minimum proportion of transmitting mosquitoes and the time to observed maximum proportion of transmitting mosquitoes. Results The within-mosquito dynamics of vector competence/EIP were not significant among treatments where mosquitoes were exposed at different ages. However, VCage revealed: (i) age-dependence in vector-virus interactions is important for transmission success; (ii) lower vector competence but at shorter EIPs was sufficient for transmission perpetuation; and (iii) R0 may be overestimated by using non-age-structured VC. Conclusions The results indicate that ultimately the temporal component of the virus-vector dynamics is most critical, especially when exposure occurred at advanced mosquito age. While our study is limited to a single virus-vector system, and a multitude of other factors affect both vector competence and mosquito mortality, our methods can be extrapolated to these other scenarios. Results indicate that how ‘highly’ or ‘negligibly’ competent vectors are categorized may need adjustment.

Additional file 1: Figure S1. Illustration of main treatment design for vector competence experimentation. Table S1. Infection and dissemination rates for each day post-infection (dpi) and corresponding mosquito age for each of the three ZIKV treatments. Table S2. Modeled fits of parameters, the type of model, and the parameter values. Figure S2. Observed and predicted daily probabilities of survival for the three treatment groups. Text S1. R packages used in the study. Text S2. Comparison of mortality of Aedes aegypti with respect to bloodmeals. Figure S3. Mortality curves for all treatments and associated controls. Table S3. Mean time to death and sample size for ZIKV-infection treatments and unexposed controls used in the mortality study. Figure S4. Demonstration of the VCage for the two treatments exposed at older ages.

The transmission of vector-borne diseases like Zika virus (ZIKV) is a dynamic process that is defined by intrinsic and extrinsic factors. One of the most important definers of the efficiency of the transmission is vector competence, or the ability of a vector to become infected with and eventually transmit a pathogen. Many things affect vector competence including vector species, discrete populations within species, and environmental factors. Several recent studies have focused on environmental factors such as temperature, and found that temperature not only affects vector competence of ZIKV in Aedes aegypti but also the life traits of the mosquito. Thus, we wanted to determine if the vector competence and extrinsic incubation period (EIP) of ZIKV was altered due to the age at which a mosquito becomes exposed. We found that there were virtually no differences in the proportion of infected, disseminated, or transmitting mosquitoes 5, 8, and 11 days post exposure among those mosquitoes exposed at 5 days post emergence versus those exposed at 12 days post emergence. However, to put this lack of differences into a more biologically relevant context, we investigated the interaction of vector competence, EIP, and age-dependent life traits of lifespan and biting rate. To illustrate the differences, we modified the vectorial capacity (VC) equation, which is a vector-centric equivalent to the basic reproduction number. VC describes the number of secondary cases of vector infection given the introduction of an infectious individual into a naive population. By deriving an age-structure measure (VCage), we are able to demonstrate the effect of age on the transmission of ZIKV by Ae. aegypti. While aging of mosquitoes in the field is limited to pre-parous and parous females, our VCage equation, like age-structured mathematical models, can be used to inform hypothesis regarding the effective density of mosquito vectors, those that are transmitting versus those that are unexposed or exposed but not transmitting due to the effects of age. These results are intuitive. However, our VCage model, like other models with age-structured vector populations, can be used to put this intuition into a quantitative framework and our experimental findings offer more insights into the importance of age-dependence of virus:vector interactions. Our experimental data further offer age-dependent parameterization of several components of VCage that could be used to inform mathematical models where age structure in a mosquito population is important.