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Temperature shapes the distribution, seasonality, and magnitude of mosquito-borne disease outbreaks. Mechanistic models predicting transmission often use mosquito and pathogen thermal responses from constant temperature experiments. However, mosquitoes live in fluctuating environments. Rate summation (nonlinear averaging) is a common approach to infer performance in fluctuating environments, but its accuracy is rarely validated. We measured three mosquito traits that impact transmission (bite rate, survival, fecundity) in a malaria mosquito ( Anopheles stephensi ) across temperature gradients with three diurnal temperature ranges (0, 9 and 12°C). We compared thermal suitability models with temperature-trait relationships observed under constant temperatures, fluctuating temperatures, and those predicted by rate summation. We mapped results across An. stephenesi 's native Asian and invasive African ranges. We found: 1) daily temperature fluctuation significantly altered trait thermal responses; 2) rate summation partially captured decreases in performance near thermal optima, but also incorrectly predicted increases near thermal limits; and 3) while thermal suitability characterized across constant temperatures did not perfectly capture suitability in fluctuating environments, it was more accurate for estimating and mapping thermal limits than predictions from rate summation. Our study provides insight into methods for predicting mosquito-borne disease risk and emphasizes the need to improve understanding of organismal performance under fluctuating conditions.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production., Importance: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing., Competing Interests: The authors declare no conflict of interest.

Chikungunya virus (CHIKV) is the causative agent of the human disease chikungunya fever, characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals are currently available for CHIKV. Therefore, the prevention of attachment of viral particles to host cells is a potential intervention strategy. As an arbovirus, CHIKV infects a wide variety of cells in both its mammalian and mosquito host. This broad cell tropism might stem from CHIKV's ability to bind to a variety of entry factors in the host cell including phosphatidylserine receptors (PSRs), glycosaminoglycans (GAGs), and the proteinaceous receptor Mxra8, among others. In this study, we aimed to determine the relevance of each attachment factor during CHIKV entry into a panel of mammalian and mosquito cells. Our data suggest that the importance of particular binding factors during CHIKV infection is highly cell line dependent. Entry into mammalian Vero cells was mediated through attachment to PSRs, mainly T-cell immunoglobulin mucin domain-1 (TIM-1). Conversely, CHIKV infection into HAP1 and NIH3T3 was predominantly mediated by heparan sulfate (HS) and Mxra8, respectively. Entry into mosquito cells was independent of PSRs, HS, and Mxra8. Although entry into mosquito cells remains unclear, our data denotes the importance of careful evaluation of reagents used to identify receptor use in invertebrate cells. While PSRs, GAGs, and Mxra8 all enhance entry in a cell line dependent manner, none of these factors are necessary for CHIKV entry, suggesting additional host factors are involved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Reyes Ballista, Miazgowicz, Acciani, Jimenez, Belloli, Havranek and Brindley.)

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.

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.

Unlabelled: The novel emerging coronavirus Middle East respiratory syndrome coronavirus (MERS-CoV) binds to its receptor, dipeptidyl peptidase 4 (DPP4), via 14 interacting amino acids. We previously showed that if the five interacting amino acids which differ between hamster and human DPP4 are changed to the residues found in human DPP4, hamster DPP4 does act as a receptor. Here, we show that the functionality of hamster DPP4 as a receptor is severely decreased if less than 4 out of 5 amino acids are changed., Importance: The novel emerging coronavirus MERS-CoV has infected >1,600 people worldwide, and the case fatality rate is ∼36%. In this study, we show that by changing 4 amino acids in hamster DPP4, this protein functions as a receptor for MERS-CoV. This work is vital in the development of new small-animal models, which will broaden our understanding of MERS-CoV and be instrumental in the development of countermeasures., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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.

Unlabelled: 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Published 2014. This article is a US Government work and is in the public domain in the USA.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)