Advances in understanding bat infection dynamics across biological scales.

Over the past two decades, research on bat-associated microbes such as viruses, bacteria and fungi has dramatically increased. Here, we synthesize themes from a conference symposium focused on advances in the research of bats and their microbes, including physiological, immunological, ecological and epidemiological research that has improved our understanding of bat infection dynamics at multiple biological scales. We first present metrics for measuring individual bat responses to infection and challenges associated with using these metrics. We next discuss infection dynamics within bat populations of the same species, before introducing complexities that arise in multi-species communities of bats, humans and/or livestock. Finally, we outline critical gaps and opportunities for future interdisciplinary work on topics involving bats and their microbes.

How Should a One Health Perspective Promote Cross-Disciplinary Research About Bat-Associated Viruses in Uganda?

Bats are diverse mammals that are globally distributed and ecologically critical, yet some bat species are associated with disease agents that have severe consequences for human health. Disease outbreak responses require interdisciplinary knowledge of bat-associated pathogens and microbial transmission patterns. Health promotion requires close, collaborative attention to the needs, vulnerabilities, and interests of diverse stakeholders, including the public and professionals in public health, conservation, ecology, social science, communication, and policy. This article describes a successful One Health engagement among such stakeholders and partners looking to motivate both bat-human ecology preservation and viral disease management in Uganda.

Corrigendum: Intrinsic factors driving mosquito vector competence and viral evolution: a review.

[This corrects the article DOI: 10.3389/fcimb.2023.1330600.].

On the Origin and Evolution of the Mosquito Male-determining Factor Nix.

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus, 2 important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males.

Dishevelled Has Anti-Viral Activity in Rift Valley Fever Virus Infected Aedes aegypti.

Mosquitoes in the genera Aedes and Culex are vectors of Rift Valley fever virus (RVFV), which emerges in periodic epidemics in Africa and Saudi Arabia. Factors that influence the transmission dynamics of RVFV are not well characterized. To address this, we interrogated mosquito host-signaling responses through analysis of differentially expressed genes (DEGs) in two mosquito species with marked differences in RVFV vector competence: Aedes aegypti (Aae, low competence) and Culex tarsalis (Cxt, high competence). Mosquito-host transcripts related to three different signaling pathways were investigated. Selected genes from the Wingless (Wg, WNT-beta-catenin) pathway, which is a conserved regulator of cell proliferation and differentiation, were assessed. One of these, dishevelled (DSH), differentially regulates progression/inhibition of the WNT and JNK (c-Jun N-terminal Kinase) pathways. A negative regulator of the JNK-signaling pathway, puckered, was also assessed. Lastly, Janus kinase/signal transducers and activators of transcription (JAK-STAT) are important for innate immunity; in this context, we tested domeless levels. Here, individual Aae and Cxt were exposed to RVFV MP-12 via oral bloodmeals and held for 14 days. Robust decreases in DEGs in both Aae and Cxt were observed. In particular, Aae DSH expression, but not Cxt DSH, was correlated to the presence/absence of viral RNA at 14 days post-challenge (dpc). Moreover, there was an inverse relationship between the viral copy number and aaeDSH expression. DSH silencing resulted in increased viral copy numbers compared to controls at 3 dpc, consistent with a role for aaeDSH in antiviral immunity. Analysis of cis-regulatory regions for the genes of interest revealed clues to upstream regulation of these pathways.

Micro‒Global Positioning Systems for Identifying Nightly Opportunities for Marburg Virus Spillover to Humans by Egyptian Rousette Bats.

Marburg virus disease, caused by Marburg and Ravn orthomarburgviruses, emerges sporadically in sub-Saharan Africa and is often fatal in humans. The natural reservoir is the Egyptian rousette bat (ERB), which sheds virus in saliva, urine, and feces. Frugivorous ERBs discard test-bitten and partially eaten fruit, potentially leaving infectious virus behind that could be consumed by other susceptible animals or humans. Historically, 8 of 17 known Marburg virus disease outbreaks have been linked to human encroachment on ERB habitats, but no linkage exists for the other 9 outbreaks, raising the question of how bats and humans might intersect, leading to virus spillover. We used micro‒global positioning systems to identify nightly ERB foraging locations. ERBs from a known Marburg virus‒infected population traveled long distances to feed in cultivated fruit trees near homes. Our results show that ERB foraging behavior represents a Marburg virus spillover risk to humans and plausibly explains the origins of some past outbreaks.

Bartonella Infection in Fruit Bats and Bat Flies, Bangladesh.

Bats harbor diverse intracellular Bartonella bacteria, but there is limited understanding of the factors that influence transmission over time. Investigation of Bartonella dynamics in bats could reveal general factors that control transmission of multiple bat-borne pathogens, including viruses. We used molecular methods to detect Bartonella DNA in paired bat (Pteropus medius) blood and bat flies in the family Nycteribiidae collected from a roost in Faridpur, Bangladesh between September 2020 and January 2021. We detected high prevalence of Bartonella DNA in bat blood (35/55, 64%) and bat flies (59/60, 98%), with sequences grouping into three phylogenetic clades. Prevalence in bat blood increased over the study period (33% to 90%), reflecting an influx of juvenile bats in the population and an increase in the prevalence of bat flies. Discordance between infection status and the clade/genotype of detected Bartonella was also observed in pairs of bats and their flies, providing evidence that bat flies take blood meals from multiple bat hosts. This evidence of bat fly transfer between hosts and the changes in Bartonella prevalence during a period of increasing nycteribiid density support the role of bat flies as vectors of bartonellae. The study provides novel information on comparative prevalence and genetic diversity of Bartonella in pteropodid bats and their ectoparasites, as well as demographic factors that affect Bartonella transmission and potentially other bat-borne pathogens.

Exploring the Mosquito-Arbovirus Network: A Survey of Vector Competence Experiments.

Arboviruses receive heightened research attention during major outbreaks or when they cause unusual or severe clinical disease, but they are otherwise undercharacterized. Global change is also accelerating the emergence and spread of arboviral diseases, leading to time-sensitive questions about potential interactions between viruses and novel vectors. Vector competence experiments help determine the susceptibility of certain arthropods to a given arbovirus, but these experiments are often conducted in real time during outbreaks, rather than with preparedness in mind. We conducted a systematic review of reported mosquito-arbovirus competence experiments, screening 570 abstracts to arrive at 265 studies testing in vivo arboviral competence. We found that more than 90% of potential mosquito-virus combinations are untested in experimental settings and that entire regions and their corresponding vectors and viruses are undersampled. These knowledge gaps stymie outbreak response and limit attempts to both build and validate predictive models of the vector-virus network.

Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications.

Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous research groups have promoted DNA as an information storage medium, less attention has been devoted to the design of economical, scalable DNA barcode libraries. Here, we present an alternative modular approach to sequence design. Barcode sequences were constructed from smaller, interchangeable blocks, allowing for the combinatorial assembly of numerous distinct tags. We demonstrated the design and construction of first-generation (N = 256) and second-generation (N = 512) modular barcode libraries, from fewer than 50 total single-stranded oligonucleotides for each library. To avoid contamination during experimental validation, a liquid-handling robot was employed for oligonucleotide mixing. Generating barcode sequences in-house reduces dependency upon external entities for unique tag generation, increasing flexibility in barcode generation and deployment. Next generation sequencing (NGS) detection of 256 different samples in parallel highlights the multiplexing afforded by the modular barcode design coupled with high-throughput sequencing. Deletion variant analysis of the first-generation library informed sequence design for enhancing barcode assembly specificity in the second-generation library.

Intrinsic factors driving mosquito vector competence and viral evolution: a review.

Mosquitoes are responsible for the transmission of numerous viruses of global health significance. The term "vector competence" describes the intrinsic ability of an arthropod vector to transmit an infectious agent. Prior to transmission, the mosquito itself presents a complex and hostile environment through which a virus must transit to ensure propagation and transmission to the next host. Viruses imbibed in an infectious blood meal must pass in and out of the mosquito midgut, traffic through the body cavity or hemocoel, invade the salivary glands, and be expelled with the saliva when the vector takes a subsequent blood meal. Viruses encounter physical, cellular, microbial, and immunological barriers, which are influenced by the genetic background of the mosquito vector as well as environmental conditions. Collectively, these factors place significant selective pressure on the virus that impact its evolution and transmission. Here, we provide an overview of the current state of the field in understanding the mosquito-specific factors that underpin vector competence and how each of these mechanisms may influence virus evolution.

Exposure of Egyptian Rousette Bats (Rousettus aegyptiacus) and a Little Free-Tailed Bat (Chaerephon pumilus) to Alphaviruses in Uganda.

The reservoir for zoonotic o'nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats.

Correction: Laboratory demonstration of the vertical transmission of Rift Valley fever virus by Culex tarsalis mosquitoes.

[This corrects the article DOI: 10.1371/journal.pntd.0009273.].

A Novel Glucocorticoid and Androgen Receptor Modulator Reduces Viral Entry and Innate Immune Inflammatory Responses in the Syrian Hamster Model of SARS-CoV-2 Infection.

Despite significant research efforts, treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain limited. This is due in part to a lack of therapeutics that increase host defense to the virus. Replication of SARS-CoV-2 in lung tissue is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses that amplify tissue injury. Antagonists of the androgen (AR) and glucocorticoid (GR) receptors have shown efficacy in models of COVID-19 and in clinical studies because the cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2), are transcriptionally regulated by these receptors. We postulated that the GR and AR modulator, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID-19 by down-regulating expression of critical genes regulated through these receptors. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were examined at 3, 5 and 7 days post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the progression of SARS-CoV-2 infection. Results indicated that oral administration of PT150 caused a dose-dependent decrease in replication of SARS-CoV-2 in lung, as well as in expression of ACE2 and TMPRSS2. Lung hypercellularity and infiltration of macrophages and CD4+ T-cells were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular docking studies suggest that PT150 binds to the co-activator interface of the ligand-binding domain of both AR and GR, thereby acting as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR revealed a high degree of sequence identity maintained across multiple species, including humans, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID-19 across variants of SARS-CoV-2.

Safety study of Rift Valley Fever human vaccine candidate (DDVax) in mosquitoes.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen with significant human and veterinary health consequences that periodically emerges in epizootics. RVFV causes fetal loss and death in ruminants and in humans can lead to liver and renal disease, delayed-onset encephalitis, retinitis, and in some cases severe haemorrhagic fever. A live attenuated vaccine candidate (DDVax), was developed by the deletion of the virulence factors NSs and NSm from a clinical isolate, ZH501, and has proven safe and immunogenic in rodents, pregnant sheep and non-human primates. Deletion of NSm also severely restricted mosquito midgut infection and inhibited vector-borne transmission. To demonstrate environmental safety, this study investigated the replication, dissemination and transmission efficiency of DDVax in mosquitoes following oral exposure compared to RVFV strains MP-12 and ZH501. Infection and dissemination profiles were also measured in mosquitoes 7 days after they fed on goats inoculated with DDvax or MP-12. We hypothesized that DDVax would infect mosquitoes at significantly lower rates than other RVFV strains and, due to lack of NSm, be transmission incompetent. Exposure of Ae. aegypti and Cx. tarsalis to 8 log10 plaque forming units (PFU)/ml DDVax by artificial bloodmeal resulted in significantly reduced DDVax infection rates in mosquito bodies compared to controls. Plaque assays indicated negligible transmission of infectious DDVax in Cx. tarsalis saliva (1/140 sampled) and none in Ae. aegypti saliva (0/120). Serum from goats inoculated with DDVax or MP-12 did not harbour detectable infectious virus by plaque assay at 1, 2 or 3 days post-inoculation. Infectious virus was, however, recovered from Aedes and Culex bodies that fed on goats vaccinated with MP-12 (13.8% and 4.6%, respectively), but strikingly, DDvax-positive mosquito bodies were greatly reduced (4%, and 0%, respectively). Furthermore, DDVax did not disseminate to legs/wings in any of the goat-fed mosquitoes. Collectively, these results are consistent with a beneficial environmental safety profile.

Mosquito tagging using DNA-barcoded nanoporous protein microcrystals.

Conventional mosquito marking technology for mark-release-recapture (MRR) is quite limited in terms of information capacity and efficacy. To overcome both challenges, we have engineered, lab-tested, and field-evaluated a new class of marker particles, in which synthetic, short DNA oligonucleotides (DNA barcodes) are adsorbed and protected within tough, crosslinked porous protein microcrystals. Mosquitoes self-mark through ingestion of microcrystals in their larval habitat. Barcoded microcrystals persist trans-stadially through mosquito development if ingested by larvae, do not significantly affect adult mosquito survivorship, and individual barcoded mosquitoes are detectable in pools of up to at least 20 mosquitoes. We have also demonstrated crystal persistence following adult mosquito ingestion. Barcode sequences can be recovered by qPCR and next-generation sequencing (NGS) without detectable amplification of native mosquito DNA. These DNA-laden protein microcrystals have the potential to radically increase the amount of information obtained from future MRR studies compared to previous studies employing conventional mosquito marking materials.

Susceptibility and barriers to infection of Colorado mosquitoes with Rift Valley fever virus.

Rift Valley fever virus (RVFV) causes morbidity and mortality in humans and domestic ungulates in sub-Saharan Africa, Egypt, and the Arabian Peninsula. Mosquito vectors transmit RVFV between vertebrates by bite, and also vertically to produce infectious progeny. Arrival of RVFV into the United States by infected mosquitoes or humans could result in significant impacts on food security, human health, and wildlife health. Elucidation of the vectors involved in the post-introduction RVFV ecology is paramount to rapid implementation of vector control. We performed vector competence experiments in which field-collected mosquitoes were orally exposed to an epidemic strain of RVFV via infectious blood meals. We targeted floodwater Aedes species known to feed on cattle, and/or deer species (Aedes melanimon Dyar, Aedes increpitus Dyar, Aedes vexans [Meigen]). Two permanent-water-breeding species were targeted as well: Culiseta inornata (Williston) of unknown competence considering United States populations, and Culex tarsalis Coquillett as a control species for which transmission efficiency is known. We tested the potential for midgut infection, midgut escape (dissemination), ovarian infection (vertical transmission), and transmission by bite (infectious saliva). Tissues were assayed by plaque assay and RT-qPCR, to quantify infectious virus and confirm virus identity. Tissue infection data were analyzed using a within-host model under a Bayesian framework to determine the probabilities of infection outcomes (midgut-limited infection, disseminated infection, etc.) while estimating barriers to infection between tissues. Permanent-water-breeding mosquitoes (Cx. tarsalis and Cs. inornata) exhibited more efficient horizontal transmission, as well as potential for vertical transmission, which is contrary to the current assumptions of RVFV ecology. Barrier estimates trended higher for Aedes spp., suggesting systemic factors in the differences between these species and Cx. tarsalis and Cs. inornata. These data indicate higher potential for vertical transmission than previously appreciated, and support the consensus of RVFV transmission including a broad range of potential vectors.

Investigations on Vector-Borne and Aerosol Transmission Potential of Kaeng Khoi Virus in Cave-Dwelling Wrinkle-Lipped Free-Tailed Bats (Chaerephon plicatus) in Thailand.

Kaeng Khoi virus (KKV; Order: Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus), is an endemic viral infection of the wrinkle-lipped free-tailed bat (Chaerephon plicatus; also known as Tadarida plicata plicata). Viral isolates from bat bugs (Family: Cimicidae) suggest vector-borne transmission, but in general little is known about the ecology of KKV and seroprevalence in the local human and animal populations. Transmission studies and a serosurvey were carried out in Kaeng Khoi cave, Saraburi province, Thailand, during 1973-1974. Experimental transmission studies were performed with bat bugs captured within the cave to determine the potential for vector-borne transmission, and sentinel laboratory mice placed inside arthropod-proof cages within the cave to assess the potential for aerosolized transmission. Antibodies to KKV were detected in roof rats (Rattus rattus) inhabiting the cave, in dogs living in the valley, and in humans. Freshly collected cimicids were positive for KKV, but the virus did not replicate in laboratory-inoculated bugs. Sentinel mice placed in Kaeng Khoi cave in open cages consistently became infected with KKV, as determined by the development of neutralizing antibodies. Mice placed in arthropod-proof cages also developed antibodies, indicating the possibility of airborne transmission of KKV.

Viral Ecology and Natural Infection Dynamics of Kaeng Khoi Virus in Cave-Dwelling Wrinkle-Lipped Free-Tailed Bats (Chaerephon plicatus) in Thailand.

Kaeng Khoi virus (KKV; Order: Bunyavirales), is an endemic viral infection of the wrinkle-lipped free-tailed bat (Chaerephon plicatus aka Tadarida plicata plicata). Little is known about the ecology and maintenance of KKV within the bat population, nor the infection dynamics and transmission among bats or between bats and other vertebrates. Therefore, KKV was studied in Kaeng Khoi cave, Saraburi province, Thailand, during 1973-1974 with the objectives to (1) characterize the seasonal infection rates of KKV in the context of the bat population ecology, and (2) describe the infection dynamics and viral shedding by naturally- and experimentally-infected bats. To this end, the free-tailed bat population was estimated by a series of timed photographs taken during the evening exodus. The case population of 900,000 adult bats doubled at the time of weaning of the young and returned to its previous level soon thereafter. The newborn bats had neutralizing antibodies to KKV that were likely to be maternal in origin. The KKV antibody prevalence in adult bats was high (69-91%) in March-May and low (29-40%) in August and September. Kaeng Khoi virus was isolated from 75% of dead and 50% of moribund bats, but was not found in nearly 400 apparently healthy bats. Virus was present in saliva, urine and blood of most of the naturally-moribund bats tested. Consistent with observations from naturally-infected bats, experimental infection of bats with KKV revealed significant liver pathology, also suggestive that this is not a benign infection. Kaeng Khoi virus is an endemic, year-round infection maintained by the annual recruitment of a large number of immunologically-naïve juvenile bats. Moreover, it produces an acute infection in the bat, either leading to death by hepatitis, or immunity.

Setting the Terms for Zoonotic Diseases: Effective Communication for Research, Conservation, and Public Policy.

Many of the world's most pressing issues, such as the emergence of zoonotic diseases, can only be addressed through interdisciplinary research. However, the findings of interdisciplinary research are susceptible to miscommunication among both professional and non-professional audiences due to differences in training, language, experience, and understanding. Such miscommunication contributes to the misunderstanding of key concepts or processes and hinders the development of effective research agendas and public policy. These misunderstandings can also provoke unnecessary fear in the public and have devastating effects for wildlife conservation. For example, inaccurate communication and subsequent misunderstanding of the potential associations between certain bats and zoonoses has led to persecution of diverse bats worldwide and even government calls to cull them. Here, we identify four types of miscommunication driven by the use of terminology regarding bats and the emergence of zoonotic diseases that we have categorized based on their root causes: (1) incorrect or overly broad use of terms; (2) terms that have unstable usage within a discipline, or different usages among disciplines; (3) terms that are used correctly but spark incorrect inferences about biological processes or significance in the audience; (4) incorrect inference drawn from the evidence presented. We illustrate each type of miscommunication with commonly misused or misinterpreted terms, providing a definition, caveats and common misconceptions, and suggest alternatives as appropriate. While we focus on terms specific to bats and disease ecology, we present a more general framework for addressing miscommunication that can be applied to other topics and disciplines to facilitate more effective research, problem-solving, and public policy.

Uganda Mountain Community Health System-Perspectives and Capacities towards Emerging Infectious Disease Surveillance.

In mountain communities like Sebei, Uganda, which are highly vulnerable to emerging and re-emerging infectious diseases, community-based surveillance plays an important role in the monitoring of public health hazards. In this survey, we explored capacities of village health teams (VHTs) in Sebei communities of Mount Elgon in undertaking surveillance tasks for emerging and re-emerging infectious diseases in the context of a changing climate. We used participatory epidemiology techniques to elucidate VHTs' perceptions on climate change and public health and assessed their capacities to conduct surveillance for emerging and re-emerging infectious diseases. Overall, VHTs perceived climate change to be occurring with wider impacts on public health. However, they had inadequate capacities in collecting surveillance data. The VHTs lacked transport to navigate through their communities and had insufficient capacities in using mobile phones for sending alerts. They did not engage in reporting other hazards related to the environment, wildlife, and domestic livestock that would accelerate infectious disease outbreaks. Records were not maintained for disease surveillance activities and the abilities of VHTs to analyze data were also limited. However, VHTs had access to platforms that could enable them to disseminate public health information. The VHTs thus need to be retooled to conduct their work effectively and efficiently through equipping them with adequate logistics and knowledge on collecting, storing, analyzing, and relaying data, which will improve infectious disease response and mitigation efforts.