Biology subjects

Rasgon, J. L.

Publications and source records attributed to Rasgon, J. L..

34 records · Page 2Linked to original sources

bioRxiv2022

Signatures of adaptive decreased virulence of deformed wing virus in an isolated population of wild honey bees (Apis mellifera)

Understanding the ecological and evolutionary processes that drive host-pathogen interactions is critical for combating epidemics and conserving species. The Varroa destructor mite and deformed wing virus (DWV) are two synergistic threats to Western honey bee (Apis mellifera) populations across the globe. Distinct honey bee populations have been found to self-sustain despite Varroa infestations, including colonies within the Arnot Forest outside Ithaca, NY, USA. We hypothesized that in these honey bee populations, DWV has been selected to produce an avirulent infection phenotype, allowing for the persistence of both host and disease-causing agents. To investigate this, we assessed the presence and titer of viruses in bees from the Arnot Forest and managed apiaries, and assessed genomic variation and virulence differences between DWV isolates. Across groups, we found viral abundance was similar, but viral genotypes were distinct. We also found that infections with viral isolates from the Arnot Forest resulted in higher survival and lower rates of symptomatic deformed wings, compared to analogous isolates from managed colonies, providing preliminary evidence to support the hypothesis of adaptive decreased viral virulence. Overall, this multi-level investigation of virus genotype and phenotype across different contexts reveals critical insight into global bee health and the ecological and evolutionary processes that drive host-pathogen interactions.

evolutionary biology↗

bioRxiv2022

Culex tarsalis is a competent host of the insect-specific alphavirus Eilat virus (EILV)

Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here we fill this gap by investigating EILVs host competence and tissue tropism in five mosquito species: Aedes aegypti, Culex tarsalis, Anopheles gambiae, Anopheles stephensi, and Anopheles albimanus. Of the tested species, Cx. tarsalis was the most competent host for EILV. The virus was found in Cx. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cells lines were not competent for EILV infection. We tested a potential invertebrate host (Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCEThe recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis--a vector of harmful human pathogens including West Nile Virus--is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission--a crucial step in discerning how Eilat virus maintains itself in nature.

microbiology↗

bioRxiv2022

Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycle, ticks face significant challenges related to water homeostasis. When blood-feeding they must excrete water and ions, but when off-host (for stretches lasting several months) they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. We find that most arachnid AQP-like sequences (including those of I. scapularis) form a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. We next sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. We additionally examined IsAQP1 expression across different life stages and adult female tissues. We found IsAQP1 to be an efficient water channel with salivary gland-specific expression, implying its function may be closely tied to osmoregulation during blood-feeding. Its functional properties were unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance during blood feeding and may hold promise as a target for novel vector control efforts. Graphical TOC/Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY eTOCIsAQP1, an aquaporin found in the black legged tick (Ixodes scapularis), is a functional water channel with low permeability to glycerol. In a phylogenetic analysis of 176 sequences from 48 arachnid species, most tick and other arachnid AQP-like genes formed a monophyletic clade nested within aquaglyceroporins (GLPs). IsAQP1 shows salivary-gland specific expression in adult females--with highest expression in unfed females--suggesting a role in water balance during blood feeding.

genetics↗

bioRxiv2022

Anopheles albimanus is a potential alphavirus vector in the Americas

Despite its ecological flexibility and geographical co-occurrence with human pathogens, little is known about the ability for Anopheles albimanus to transmit arboviruses. To address this gap, we challenged An. albimanus females with four alphaviruses and one flavivirus and monitored progression of infections. We found this species is an efficient vector of the alphaviruses Mayaro virus, Onyong-nyong virus, and Sindbis virus, though the latter two do not currently exist in its habitat range. An. albimanus was able to become infected with Chikungunya virus, but virus dissemination was rare (indicating the presence of a midgut escape barrier), and no mosquito transmitted. Mayaro virus rapidly established disseminated infections in An. albimanus females and was detected in the saliva of a substantial proportion of infected mosquitoes. Consistent with previous work in other anophelines, we find that An. albimanus is refractory to infection with flaviviruses, a phenotype that did not depend on midgut-specific barriers. Our work demonstrates that An. albimanus may be a vector of neglected emerging human pathogens and adds to recent evidence that anophelines are competent vectors for diverse arboviruses.

microbiology↗

bioRxiv2022

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes

Multiple Wolbachia strains can block pathogen infection, replication, and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase 2 (Man2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed Man2 knockouts and examined effects on both Wolbachia and virus levels, using both dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titers were significantly elevated in Man2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the Man2 KO mutation was associated with decreased DENV titers, but in a Wolbachia-infected background, the Man2 KO mutation significantly modulated virus blocking. In contrast, the Man2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that Man2 modulates arbovirus infection in Ae. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

microbiology↗

bioRxiv2022

Sexual transmission of Anopheles gambiae densovirus (AgDNV) leads to disseminated infection in mated females

BackgroundAnopheles gambiae densovirus (AgDNV) is an insect-specific, single-stranded DNA virus that infects An. gambiae, the major mosquito species responsible for transmitting malaria parasites throughout Sub-Saharan Africa. AgDNV is a benign virus that is very specific to its mosquito host and therefore has potential to serve as a vector control tool via paratransgenesis (genetic modification of mosquito symbionts) to limit transmission of human pathogens. Prior to being engineered into a control tool, the natural transmission dynamics of AgDNV between An. gambiae mosquitoes needs to be fully understood. Additionally, improved knowledge of AgDNV infection in male mosquitoes is needed. In this study, we examine the tissue tropism of AgDNV in the male reproductive tract and investigate both venereal and vertical transmission dynamics of the virus. MethodsAn. gambiae adult males were infected with AgDNV via micro-injection and reproductive tissues collected and assayed for AgDNV using qPCR. Next, uninfected females were introduced to AgDNV-infected or control males and, after several nights of mating, both the spermatheca and female carcass were assessed for venereally transmitted AgDNV. Finally, F1s from this cross were collected and assayed to quantify vertical transmission of the virus. ResultsAgDNV infected the reproductive tract of male mosquitoes, including the testes and male accessory glands (MAGs), without affecting mating rates. AgDNV-infected males venereally transmitted virus to females, and these venereally-infected females developed disseminated infection throughout the body. However, AgDNV was not vertically transmitted to F1s resulting from this cross. ConclusionsInfected male releases could be an effective strategy to introduce AgDNV-based paratransgenic tools into naive populations of An. gambiae females.

microbiology↗

bioRxiv2021

Identification of microRNAs in the West Nile virus vector Culex tarsalis

BackgroundmicroRNAs (miRNAs) represent a group of small non-coding RNAs that are crucial gene regulators of important biological functions including development and pathogen defense in most living organisms. Presently, there is a lack of availability of information regarding the miRNAs in the mosquito Culex tarsalis, which is one of the most important vectors of West Nile virus (WNV) in the United States. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate the presence of a repertoire of miRNAs in Cx. tarsalis mosquitoes. ResultsUsing bioinformatic approaches we analyzed small RNA sequencing data from the Cx. tarsalis CT embryonic cell line to discover 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Cx. quinquefasciatus, mi-184 was found to be the most abundant miRNA in Cx tarsalis. We also identified an additional 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both cell line and adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. ConclusionsCx. tarsalis is an important vector of many medically important pathogens including WNV and Western Equine encephalitis. Here we report a detailed insight into the miRNA population in Cx. tarsalis mosquitoes. These results will open new avenues of research deciphering the role of miRNAs in different Cx. tarsalis biological events such as development, metabolism, immunity and pathogen infection.

genomics↗

bioRxiv2021

Novel genome assemblies and Evolutionary Dynamics of North American Anopheles mosquitoes

Anopheles mosquitoes are the principal vectors for malaria and lymphatic filariasis, and evidence for arboviral transmission under laboratory and natural contexts has been demonstrated. Vector management approaches require an understanding of the ecological, epidemiological, and biological context of the species in question, and increased interest in gene drive systems for vector control applications has resulted in an increased need for genome assemblies from understudied mosquito species. While the genomes for many Anopheles species have been sequenced, North American Anopheles taxa have been neglected. In this study we present novel genome assemblies for the North American species An. crucians, An. freeborni, An. albimanus, and An. quadrimaculatus, and examine the evolutionary relationship between these species. We identified 790 shared single copy orthologs between the newly sequenced genomes and created a phylogeny using 673 of the orthologs, identifying 525 orthologs with evidence for positive selection on at least one branch of the phylogeny. Gene ontology terms such as calcium ion signaling, histone binding, and protein acetylation were identified as being biased in the set of selected genes. These novel genome sequences will be useful in developing our understanding of the diverse biological traits that drive vectorial capacity in anophelines.

genomics↗

bioRxiv2021

In vitro and in vivo co-infection and super-infection dynamics of Mayaro and Zika viruses in mosquito and vertebrate backgrounds

Factors related to increasing globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Co-circulation of multiple arboviruses in the same geographic regions provides impetus to study the impacts of multiple arbovirus infections in a single vector. In the present study we describe co-infection and super-infection with Mayaro virus (Family Togaviridae, genus Alphavirus) and Zika virus (family Flaviviridae, genus Flavivirus) in vertebrate cells, mosquito cells, and Aedes aegypti mosquitoes to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination and transmission. In Aedes aegypti mosquitoes, co-infection has a negative impact on infection and dissemination rates for Zika virus, but not Mayaro virus, when compared to single infection scenarios, and super-infection of Mayaro virus with a previous Zika virus infection resulted in increased Mayaro virus infection rates. We found that co-infection and super-infection negatively affected Zika viral replication in vertebrate cells (Vero and Huh), resulting in the complete blockage of Zika virus replication in some scenarios. At the cellular level, we demonstrate that single vertebrate and insect cells can be simultaneously infected with Zika and Mayaro viruses. This study highlights the dynamics of arboviral co- and super-infections and emphasizes the importance of considering these dynamics during risk assessment in epidemic areas.

microbiology↗

bioRxiv2020

Everything you wanted to know about Mayaro virus but were afraid to ask: Characterization and lifecycle of Mayaro virus in vertebrate and invertebrate cellular backgrounds

Mayaro virus (MAYV) is an emerging new world alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis, and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, life cycle, and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron dense clathrin-coated pits in infected cells, and reduced viral production in the presence of dynasore, ammonium chloride, and bafilomycin, indicates that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production, and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation of biological understanding valuable for therapeutic and preventive interventions.

microbiology↗

bioRxiv2020

Mayaro Virus Infection Elicits an Innate Immune Response and Represses Autophagy in Anopheles stephensi

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naive regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint suggested activation of the Toll pathway at 7 dpi and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes. AUTHOR SUMMARYMayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discover novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species.

microbiology↗

bioRxiv2020

Vectorchip: Microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Mosquito bites transmit a number of human pathogens resulting in potentially fatal diseases including malaria, dengue, chikungunya, West Nile encephalitis, and Zika. Although female mosquitoes transmit pathogens via salivary droplets deposited during blood feeding on a host, very little is known about the genomic content of these nanoliter scale droplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform for molecular interrogation of individual mosquito bites in a high-throughput fashion. An ultra-thin PDMS membrane coupled to a microfluidic chip acts as a biting interface, through which freely-behaving mosquitoes deposit saliva droplets by biting into isolated arrayed micro-wells enabling molecular interrogation of individual bites. By modulating membrane thickness, the device enables on-chip comparison of biting capacity and provides a mechanical filter allowing selection of a specific mosquito species. Utilizing Vectorchip, we show on-chip simultaneous detection of mosquito DNA as well as viral RNA from Zika infected Aedes aegypti mosquitoes - demonstrating multiplexed high-throughput screening of vectors and pathogens. Focus-forming assays performed on-chip quantify number of infectious viral particles transmitted during mosquito bites, enabling assessment of active virus transmission. The platform presents a promising approach for single-bite-resolution laboratory and field characterization of vector pathogen communities, to reveal the intricate dynamics of pathogen transmission, and could serve as powerful early warning artificial "sentinel" for mosquito-borne diseases.

bioengineering↗

bioRxiv2020

CRISPR/Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci)

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest, and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject, and which have high mortality post-injection. We developed a CRISPR/Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.

genetics↗

bioRxiv2019

Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

Innovative tool development is essential for continued advancement in malaria control and depends on a deeper understanding of the molecular mechanisms that govern transmission of malaria parasites by Anopheles mosquitoes. Targeted disruption of genes in mosquito vectors is a powerful method to uncover the underlying biology of vector-pathogen interactions, and genome manipulation technologies can themselves form the basis of mosquito and pathogen control strategies. However, the embryo injection methods used to genetically manipulate mosquitoes, and in particular Anopheles species, are difficult and inefficient, particularly for non-specialist laboratories. We have adapted a strategy called ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) to deliver the Cas9 ribonucleoprotein complex to adult mosquito ovaries and generate targeted and heritable mutations in the malaria vector Anopheles stephensi. We found that gene editing by ReMOT Control in Anopheles mosquitoes was comparable to the technique in Ae. aegypti and as efficient in editing as standard embryo injections. The adaptation of this technology to Anopheles mosquitoes opens up the power of reverse genetics to malaria vector labs that do not have the equipment or technical expertise to perform embryo injections and establishes the flexibility of ReMOT Control for gene-editing in non-Aedes species.

genetics↗

bioRxiv2018

Anopheles Mosquitoes May Drive Invasion and Transmission of Mayaro Virus across Geographically Diverse Regions

The Togavirus (Alphavirus) Mayaro virus (MAYV) was initially described in 1954 from Mayaro County (Trinidad) and has been responsible for outbreaks in South America and the Caribbean. Imported MAYV cases are on the rise, leading to invasion concerns similar to Chikungunya and Zika viruses. Little is known about the range of mosquito species that are competent MAYV vectors. We tested vector competence of 2 MAYV genotypes for six mosquito species (Aedes aegypti, Anopheles gambiae, An. stephensi, An. quadrimaculatus, An. freeborni, Culex quinquefasciatus). Ae. aegypti and Cx. quinquefasciatus were poor MAYV vectors, and either were poorly infected or poorly transmitted. In contrast, all Anopheles species were able to transmit MAYV, and 3 of the 4 species transmitted both genotypes. The Anopheles species tested are divergent and native to widely separated geographic regions, suggesting that Anopheles may be important in the invasion and spread of MAYV across diverse regions of the world.

epidemiology↗

bioRxiv2017

Heat Shock Protein 70 (Hsp70) Is Involved In The Zika Virus Cellular Infection Process

Zika virus (ZIKV) is a historically neglected flavivirus that has recently caused epidemics in the western hemisphere. ZIKV has been associated with severe symptoms including infant microcephaly and Guillain Barre syndrome, stimulating interest in understanding factors governing ZIKV infection. Heat shock protein 70 (Hsp70) has been shown to be an infection factor for multiple viruses. We investigated the role of Hsp70 in the ZIKV infection process. We localized Hsp70 protein to the Vero cell membrane surface by confocal microscopy and demonstrated that, inside the cell, there is significant co-localization between Hsp70 and ZIKV E protein. Inducing and suppressing Hsp70 expression increased and decreased ZIKV production, respectively. Antibody blocking cell surface-localized Hsp70 decreased ZIKV cell infection rates and production of infectious virus particles, as did competition with recombinant Hsp70 protein. Our data suggest that Hsp70 is an important factor in the ZIKV infection process. Understanding the interactions between Hsp70 and ZIKV may lead to novel therapeutics for ZIKV infection, particularly for pregnant women and fetuses.

microbiology↗