Temperate Conditions Limit Zika Virus Genome Replication.

Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29°C, it was limited at cool temperatures (<22°C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28°C) and cool (20°C) temperatures. We found that the cool temperature did not alter Zika virus entry or translation, but it affected genome replication and reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28°C and the cells were subsequently shifted to 20°C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian lineage of Zika virus, while the African lineage Zika virus was less restricted at 20°C. IMPORTANCE With half of the human population at risk, arboviral diseases represent a substantial global health burden. Zika virus, previously known to cause sporadic infections in humans, emerged in the Americas in 2015 and quickly spread worldwide. There was an urgent need to better understand the disease pathogenesis and develop therapeutics and vaccines, as well as to understand, predict, and control virus transmission. In order to efficiently predict the seasonality and geography for Zika virus transmission, we need a deeper understanding of the host-pathogen interactions and how they can be altered by environmental factors such as temperature. Identifying the step in the virus replication cycle that is inhibited under cool conditions can have implications in modeling the temperature suitability for arbovirus transmission as global environmental patterns change. Understanding the link between pathogen replication and environmental conditions can potentially be exploited to develop new vector control strategies in the future.

Warming temperatures could expose more than 1.3 billion new people to Zika virus risk by 2050.

In the aftermath of the 2015 pandemic of Zika virus (ZIKV), concerns over links between climate change and emerging arboviruses have become more pressing. Given the potential that much of the world might remain at risk from the virus, we used a previously established temperature-dependent transmission model for ZIKV to project climate change impacts on transmission suitability risk by mid-century (a generation into the future). Based on these model predictions, in the worst-case scenario, over 1.3 billion new people could face suitable transmission temperatures for ZIKV by 2050. The next generation will face substantially increased ZIKV transmission temperature suitability in North America and Europe, where naïve populations might be particularly vulnerable. Mitigating climate change even to moderate emissions scenarios could significantly reduce global expansion of climates suitable for ZIKV transmission, potentially protecting around 200 million people. Given these suitability risk projections, we suggest an increased priority on research establishing the immune history of vulnerable populations, modeling when and where the next ZIKV outbreak might occur, evaluating the efficacy of conventional and novel intervention measures, and increasing surveillance efforts to prevent further expansion of ZIKV.

Temperature drives Zika virus transmission: evidence from empirical and mathematical models.

Temperature is a strong driver of vector-borne disease transmission. Yet, for emerging arboviruses we lack fundamental knowledge on the relationship between transmission and temperature. Current models rely on the untested assumption that Zika virus responds similarly to dengue virus, potentially limiting our ability to accurately predict the spread of Zika. We conducted experiments to estimate the thermal performance of Zika virus (ZIKV) in field-derived Aedes aegypti across eight constant temperatures. We observed strong, unimodal effects of temperature on vector competence, extrinsic incubation period and mosquito survival. We used thermal responses of these traits to update an existing temperature-dependent model to infer temperature effects on ZIKV transmission. ZIKV transmission was optimized at 29°C, and had a thermal range of 22.7°C-34.7°C. Thus, as temperatures move towards the predicted thermal optimum (29°C) owing to climate change, urbanization or seasonality, Zika could expand north and into longer seasons. By contrast, areas that are near the thermal optimum were predicted to experience a decrease in overall environmental suitability. We also demonstrate that the predicted thermal minimum for Zika transmission is 5°C warmer than that of dengue, and current global estimates on the environmental suitability for Zika are greatly over-predicting its possible range.

Effects of changes in temperature on Zika dynamics and control.

When a rare pathogen emerges to cause a pandemic, it is critical to understand its dynamics and the impact of mitigation measures. We use experimental data to parametrize a temperature-dependent model of Zika virus (ZIKV) transmission dynamics and analyse the effects of temperature variability and control-related parameters on the basic reproduction number (R0) and the final epidemic size of ZIKV. Sensitivity analyses show that these two metrics are largely driven by different parameters, with the exception of temperature, which is the dominant driver of epidemic dynamics in the models. Our R0 estimate has a single optimum temperature (≈30°C), comparable to other published results (≈29°C). However, the final epidemic size is maximized across a wider temperature range, from 24 to 36°C. The models indicate that ZIKV is highly sensitive to seasonal temperature variation. For example, although the model predicts that ZIKV transmission cannot occur at a constant temperature below 23°C (≈ average annual temperature of Rio de Janeiro, Brazil), the model predicts substantial epidemics for areas with a mean temperature of 20°C if there is seasonal variation of 10°C (≈ average annual temperature of Tampa, Florida). This suggests that the geographical range of ZIKV is wider than indicated from static R0 models, underscoring the importance of climate dynamics and variation in the context of broader climate change on emerging infectious diseases.

Temperature Dramatically Shapes Mosquito Gene Expression With Consequences for Mosquito-Zika Virus Interactions.

Vector-borne flaviviruses are emerging threats to human health. For successful transmission, the virus needs to efficiently enter mosquito cells and replicate within and escape several tissue barriers while mosquitoes elicit major transcriptional responses to flavivirus infection. This process will be affected not only by the specific mosquito-pathogen pairing but also by variation in key environmental variables such as temperature. Thus far, few studies have examined the molecular responses triggered by temperature and how these responses modify infection outcomes, despite substantial evidence showing strong relationships between temperature and transmission in a diversity of systems. To define the host transcriptional changes associated with temperature variation during the early infection process, we compared the transcriptome of mosquito midgut samples from mosquitoes exposed to Zika virus (ZIKV) and non-exposed mosquitoes housed at three different temperatures (20, 28, and 36°C). While the high-temperature samples did not show significant changes from those with standard rearing conditions (28°C) 48 h post-exposure, the transcriptome profile of mosquitoes housed at 20°C was dramatically different. The expression of genes most altered by the cooler temperature involved aspects of blood-meal digestion, ROS metabolism, and mosquito innate immunity. Further, we did not find significant differences in the viral RNA copy number between 24 and 48 h post-exposure at 20°C, suggesting that ZIKV replication is limited by cold-induced changes to the mosquito midgut environment. In ZIKV-exposed mosquitoes, vitellogenin, a lipid carrier protein, was most up-regulated at 20°C. Our results provide a deeper understanding of the temperature-triggered transcriptional changes in Aedes aegypti and can be used to further define the molecular mechanisms driven by environmental temperature variation.

Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and R0 of Zika in Aedes aegypti.

Zika virus (ZIKV) is an arbovirus primarily transmitted by Aedes mosquitoes. Like most viral infections, ZIKV viremia varies over several orders of magnitude, with unknown consequences for transmission. To determine the effect of viral concentration on ZIKV transmission risk, we exposed field-derived Ae. aegypti mosquitoes to four doses (10(3), 10(4), 10(5), 10(6) PFU/mL) representative of potential variation in the field. We demonstrate that increasing ZIKV dose in the blood-meal significantly increases the probability of mosquitoes becoming infected, and consequently disseminating virus and becoming infectious. Additionally, we observed significant interactions between dose and days post-infection on dissemination and overall transmission efficiency, suggesting that variation in ZIKV dose affects the rates of midgut escape and salivary gland invasion. We did not find significant effects of dose on mosquito mortality. We also demonstrate that detecting virus using RT-qPCR approaches rather than plaque assays potentially over-estimates key transmission parameters, including the time at which mosquitoes become infectious and viral burden. Finally, using these data to parameterize an R0 model, we showed that increasing viremia from 10(4) to 10(6) PFU/mL increased relative R0 3.8-fold, demonstrating that variation in viremia substantially affects transmission risk.

Zika and chikungunya: mosquito-borne viruses in a changing world.

The reemergence and growing burden of mosquito-borne virus infections have incited public fear and growing research efforts to understand the mechanisms of infection-associated health outcomes and to provide better approaches for mosquito vector control. While efforts to develop therapeutics, vaccines, and novel genetic mosquito-control technologies are underway, many important underlying ecological questions remain that could significantly enhance our understanding and ability to predict and prevent transmission. Here, we review the current knowledge about the transmission ecology of two recent arbovirus invaders, the chikungunya and Zika viruses. We introduce the viruses and mosquito vectors, highlighting viral biology, historical routes of transmission, and viral mechanisms facilitating rapid global invasion. In addition, we review factors contributing to vector global invasiveness and transmission efficiency. We conclude with a discussion of how human-induced biotic and abiotic environmental changes facilitate mosquito-borne virus transmission, emphasizing critical gaps in understanding. These knowledge gaps are tremendous; much of our data on basic mosquito ecology in the field predate 1960, and the mosquitoes themselves, as well as the world they live in, have substantially changed. A concerted investment in understanding the basic ecology of these vectors, which serve as the main drivers of pathogen transmission in both wildlife and human populations, is now more important than ever.

Fine-scale variation in microclimate across an urban landscape shapes variation in mosquito population dynamics and the potential of Aedes albopictus to transmit arboviral disease.

Most statistical and mechanistic models used to predict mosquito-borne disease transmission incorporate climate drivers of disease transmission by utilizing environmental data collected at geographic scales that are potentially coarser than what mosquito populations may actually experience. Temperature and relative humidity can vary greatly between indoor and outdoor environments, and can be influenced strongly by variation in landscape features. In the Aedes albopictus system, we conducted a proof-of-concept study in the vicinity of the University of Georgia to explore the effects of fine-scale microclimate variation on mosquito life history and vectorial capacity (VC). We placed Ae. albopictus larvae in artificial pots distributed across three replicate sites within three different land uses-urban, suburban, and rural, which were characterized by high, intermediate, and low proportions of impervious surfaces. Data loggers were placed into each larval environment and in nearby vegetation to record daily variation in water and ambient temperature and relative humidity. The number of adults emerging from each pot and their body size and sex were recorded daily. We found mosquito microclimate to significantly vary across the season as well as with land use. Urban sites were in general warmer and less humid than suburban and rural sites, translating into decreased larval survival, smaller body sizes, and lower per capita growth rates of mosquitoes on urban sites. Dengue transmission potential was predicted to be higher in the summer than the fall. Additionally, the effects of land use on dengue transmission potential varied by season. Warm summers resulted in a higher predicted VC on the cooler, rural sites, while warmer, urban sites had a higher predicted VC during the cooler fall season.

Zika Virus Exhibits Lineage-Specific Phenotypes in Cell Culture, in Aedes aegypti Mosquitoes, and in an Embryo Model.

Zika virus (ZIKV) has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in Aedes aegypti mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes.

Zika Virus Induced Mortality and Microcephaly in Chicken Embryos.

The explosive spread of the Zika virus (ZIKV) through South and Central America has been linked to an increase in congenital birth defects, specifically microcephaly. Representative rodent models for investigating infections include direct central nervous system (CNS) injections late in pregnancy and transplacental transmission in immunodeficient mice. Microcephaly in humans may be the result of infection occurring early in pregnancy, therefore recapitulating that the human course of ZIKV infection should include normal embryo exposed to ZIKV during the first trimester. In ovo development of the chicken embryo closely mirrors human fetal neurodevelopment and, as a comparative model, could provide key insights into both temporal and pathophysiological effects of ZIKV. Chick embryos were directly infected early and throughout incubation with ZIKV isolated from a Mexican mosquito in January 2016. High doses of virus caused embryonic lethality. In a subset of lower dosed embryos, replicating ZIKV was present in various organs, including the CNS, throughout development. Surviving ZIKV-infected embryos presented a microcephaly-like phenotype. Chick embryos were longitudinally monitored by magnetic resonance imaging that documented CNS structural malformations, including enlarged ventricles (30% increase) and stunted cortical growth (decreased telencephalon by 18%, brain stem by 32%, and total brain volume by 18%), on both embryonic day 15 (E15) and E20 of development. ZIKV-induced microcephaly was observed with inoculations of as few as 2-20 viral particles. The chick embryo model presented ZIKV embryonic lethal effects and progressive CNS damage similar to microcephaly.