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Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.ImportanceOutbreaks of Chikungunya virus (CHIKV) have occurred throughout Africa, Asia, and Europe. Climate change permits the expansion of Aedes mosquito vectors into more temperate regions, broadening the geographic range and increasing the frequency of future human outbreaks. The molecular basis underlying the broad host and cellular tropism of CHIKV remains unresolved. While several host molecules have been implicated in CHIKV viral attachment and entry, the role of lipid-mediated attachment (viral apoptotic mimicry) is unclear. We observed that higher levels of externalized phosphatidylserine (PS) in the viral lipid bilayer correlated with enhanced CHIKV infectivity in mammalian cells abundant with PS receptors and lacking alternative attachment factors. Interestingly, CHIKV infection in mosquito Aag2 cells was also affected by viral PS accessibility. This study further delineates the role of virus-cell attachment molecules in CHIKV infection. Viral apoptotic mimicry has potential to influence CHIKV dynamics in vivo in both the human and mosquito host.

Vector-borne viruses (arboviruses) are emerging threats to both human and animal health. The global expansion of dengue virus, West Nile virus, chikungunya, and most recently Zika virus are prominent examples of how quickly mosquito-transmitted viruses can emerge and spread. We currently lack high quality data from a diversity of mosquito-arbovirus systems on the specific mosquito and viral traits that drive disease transmission. Further, the factors that contribute to variation in these traits and disease transmission remain largely unidentified. In this chapter, we outline and explore the following: 1. the specific mechanisms governing the outcome of vector-virus interactions 2. how genetic variation across mosquito populations and viral strains, as well as environmental variation in abiotic and biotic factors shape the mosquito-virus interaction and 3. the implications of these interactions for understanding and predicting arbovirus transmission, as well as for control of mosquito species that transmit human pathogens.

Summary Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4., Graphical Abstract, Highlights • MERS-CoV infected cells expressing DPP4 from 16 bat species • MERS-CoV spike rapidly adapted to species variation in DPP4 • Viral adaptations modified the surface charge of viral spike • Different routes of spike adaptation enhanced entry with the same DPP4 variant, MERS-CoV is a zoonotic pathogen capable of infecting numerous species. However, our understanding of how this virus adapts to new species remains unclear. Letko et al. experimentally observe several different routes in the stepwise, adaptive evolution of MERS-CoV to a unique host-species variant of the viral receptor.

Models predicting disease transmission are a vital tool in the control of mosquito populations and malaria reduction as they can target intervention efforts. We compared the performance of temperature-dependent transmission models when mosquito life history traits were allowed to change across the lifespan of Anopheles stephensi, the urban malaria mosquito, to models parameterized with commonly derived estimates of lifetime trait values. We conducted an experiment on adult female An. stephensi to generate daily per capita values for lifespan, egg production, and biting rate at six constant temperatures. Both temperature and age significantly affected trait values. Further, we found quantitative and qualitative differences between temperature-trait relationships estimated based on daily rates versus directly observed lifetime values. Incorporating these temperature-trait relationships into an expression governing transmission suitability, relative R0(T), model resulted in minor differences in the breadth of suitable temperatures for Plasmodium falciparum transmission between the two models constructed from only An. stephensi trait data, but a substantial increase in breadth compared to a previously published model consisting of trait data from multiple mosquito species. Overall this work highlights the importance of considering how mosquito trait values vary with mosquito age and mosquito species when generating temperature-based environmental suitability predictions of transmission.

Models predicting disease transmission are vital tools for long-term planning of malaria reduction efforts, particularly for mitigating impacts of climate change. We compared temperature-dependent malaria transmission models when mosquito life-history traits were estimated from a truncated portion of the lifespan (a common practice) versus traits measured across the full lifespan. We conducted an experiment on adult female Anopheles stephensi , the Asian urban malaria mosquito, to generate daily per capita values for mortality, egg production and biting rate at six constant temperatures. Both temperature and age significantly affected trait values. Further, we found quantitative and qualitative differences between temperature–trait relationships estimated from truncated data versus observed lifetime values. Incorporating these temperature–trait relationships into an expression governing the thermal suitability of transmission, relative R 0 ( T ), resulted in minor differences in the breadth of suitable temperatures for Plasmodium falciparum transmission between the two models constructed from only An. stephensi trait data. However, we found a substantial increase in thermal niche breadth compared with a previously published model consisting of trait data from multiple Anopheles mosquito species. Overall, this work highlights the importance of considering how mosquito trait values vary with mosquito age and mosquito species when generating temperature-based suitability predictions of transmission.

We evaluated the stability of Ebola virus on surfaces and in fluids under simulated environmental conditions for the climate of West Africa and for climate-controlled hospitals. This virus remains viable for a longer duration on surfaces in hospital conditions than in African conditions and in liquid than in dried blood.

The ongoing Ebola virus outbreak in West Africa has highlighted questions regarding stability of the virus and detection of RNA from corpses. We used Ebola virus–infected macaques to model humans who died of Ebola virus disease. Viable virus was isolated

On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions.

Recent epidemics of Zika, dengue, and chikungunya have heightened the need to understand the seasonal and geographic range of transmission by Aedes aegypti and Ae. albopictus mosquitoes. We use mechanistic transmission models to derive predictions for how the probability and magnitude of transmission for Zika, chikungunya, and dengue change with mean temperature, and we show that these predictions are well matched by human case data. Across all three viruses, models and human case data both show that transmission occurs between 18–34°C with maximal transmission occurring in a range from 26–29°C. Controlling for population size and two socioeconomic factors, temperature-dependent transmission based on our mechanistic model is an important predictor of human transmission occurrence and incidence. Risk maps indicate that tropical and subtropical regions are suitable for extended seasonal or year-round transmission, but transmission in temperate areas is limited to at most three months per year even if vectors are present. Such brief transmission windows limit the likelihood of major epidemics following disease introduction in temperate zones., Author summary Understanding the drivers of recent Zika, dengue, and chikungunya epidemics is a major public health priority. Temperature may play an important role because it affects virus transmission by mosquitoes, through its effects on mosquito development, survival, reproduction, and biting rates as well as the rate at which mosquitoes acquire and transmit viruses. Here, we measure the impact of temperature on transmission by two of the most common mosquito vector species for these viruses, Aedes aegypti and Ae. albopictus. We integrate data from several laboratory experiments into a mathematical model of temperature-dependent transmission, and find that transmission peaks at 26–29°C and can occur between 18–34°C. Statistically comparing model predictions with recent observed human cases of dengue, chikungunya, and Zika across the Americas suggests an important role for temperature, and supports model predictions. Using the model, we predict that most of the tropics and subtropics are suitable for transmission in many or all months of the year, but that temperate areas like most of the United States are only suitable for transmission for a few months during the summer (even if the mosquito vector is present).

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir. IMPORTANCE The ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) has caused 701 laboratory-confirmed cases to date, with 249 fatalities. Although bats and dromedary camels have been identified as potential MERS-CoV hosts, the virus has so far not been isolated from any species other than humans. The inability of MERS-CoV to infect commonly used animal models, such as hamster, mice, and ferrets, indicates the presence of a species barrier. We show that the MERS-CoV receptor DPP4 plays a pivotal role in the observed species tropism of MERS-CoV and subsequently identified the amino acids in DPP4 responsible for this restriction. Using a combined modeling and experimental approach, we predict that, based on the ability of MERS-CoV to utilize the DPP4 of common Middle East livestock species, such as camels, goats, sheep, and cows, these form a potential MERS-CoV intermediate host reservoir species.

On September 20, 2012, a Saudi Arabian physician reported the isolation of a novel coronavirus from a patient with pneumonia on ProMED-mail. Within a few days, the same virus was detected in a Qatari patient receiving intensive care in a London hospital, a situation reminiscent of the role air travel played in the spread of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. SARS-CoV originated in China's Guangdong Province and affected more than 8000 patients in 26 countries before it was contained 6 months later. Over a year after the emergence of this novel coronavirus--Middle East respiratory syndrome coronavirus (MERS-CoV)--it has caused 178 laboratory-confirmed cases and 76 deaths. The emergence of a second highly pathogenic coronavirus within a decade highlights the importance of a coordinated global response incorporating reservoir surveillance, high-containment capacity with fundamental and applied research programs, and dependable communication pathways to ensure outbreak containment. Here, we review the current state of knowledge on the epidemiology, ecology, molecular biology, clinical features, and intervention strategies of the novel coronavirus, MERS-CoV.

Tacaribe virus (TCRV) was isolated in the 1950s from artibeus bats captured on the island of Trinidad. The initial characterization of TCRV suggested that artibeus bats were natural reservoir hosts. However, nearly 60 years later experimental infections of Jamaican fruit bats (Artibeus jamaicensis) resulted in fatal disease or clearance, suggesting artibeus bats may not be a reservoir host. To further evaluate the TCRV reservoir host status of artibeus bats, we captured bats of six species in Trinidad for evidence of infection. Bats of all four fruigivorous species captured had antibodies to TCRV nucleocapsid, whereas none of the insectivore or nectarivore species did. Many flat-faced fruit-eating bats (A. planirostris) and great fruit-eating bats (A. literatus) were seropositive by ELISA and western blot to TCRV nucleocapsid antigen, as were two of four Seba’s fruit bats (Carollia perspicillata) and two of three yellow-shouldered fruit bats (Sturnira lilium). Serum neutralization tests failed to detect neutralizing antibodies to TCRV from these bats. TCRV RNA was not detected in lung tissues or lung homogenates inoculated onto Vero cells. These data indicate that TCRV or a similar arenavirus continues to circulate among fruit bats of Trinidad but there was no evidence of persistent infection, suggesting artibeus bats are not reservoir hosts.

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., Author summary Environmental factors influence the dynamics of mosquito-borne disease transmission. Most models used to predict mosquito-borne disease transmission incorporate climate data collected at coarser scales than mosquitoes typically experience. Climate conditions can vary greatly between indoor and outdoor environments, and are influenced by landscape features. We conducted a field experiment with the Asian tiger mosquito to explore how microclimate variation across an urban landscape affects mosquito life history and potential to transmit arboviruses, like dengue. We demonstrate that climate conditions captured by weather stations do not reflect relevant mosquito microclimate, and that subtle variation in mean and diurnal ranges of temperature and relative humidity can lead to appreciable variation in key mosquito / pathogen traits that are important for transmission. Our results have implications for statistical and mechanistic models used to predict variation in transmission across seasons, regions, and land uses, but also for building in realistic environmental variation in laboratory work on mosquito-pathogen interactions. Finally, the variation in microclimate we observed across land use was subtle; likely because our study site is a relatively small city. Nevertheless, these translated into considerable differences in mosquito traits and dengue transmission potential, suggesting these effects could be much larger in more expansive cities.

Most statistical and mechanistic models used to predict mosquito borne disease transmission incorporate climate drivers of disease transmission by utilizing environmental data collected at scales that are potentially coarser than what mosquito vectors 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 theAedes albopictussystem, 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 placedAe. albopictuslarvae 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.

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the zoonotic potential of Betacoronaviruses. Investigations into the origin of MERS-CoV have focused on two potential reservoirs: bats and camels. Here, we investigated the role of bats as a potential reservoir for MERS-CoV. In vitro, the MERS-CoV spike glycoprotein interacted with Jamaican fruit bat (Artibeus jamaicensis) dipeptidyl peptidase 4 (DPP4) receptor and MERS-CoV replicated efficiently in Jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for MERS-CoV. To shed light on the intrinsic host-virus relationship, we inoculated 10 Jamaican fruit bats with MERS-CoV. Although all bats showed evidence of infection, none of the bats showed clinical signs of disease. Virus shedding was detected in the respiratory and intestinal tract for up to 9 days. MERS-CoV replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. Analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. Our results indicate that MERS-CoV maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for MERS-CoV.

A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.