Browsing by Author "Ebel, Gregory D."
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- Adventitious viruses persistently infect three commonly used mosquito cell linesWeger-Lucarelli, James; Rückert, Claudia; Grubaugh, Nathan D.; Misencik, Michael J.; Armstrong, Philip M.; Stenglein, Mark D.; Ebel, Gregory D.; Brackney, Doug E. (2018-08)Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.
- Co-Infection Patterns in Individual Ixodes scapularis Ticks Reveal Associations between Viral, Eukaryotic and Bacterial MicroorganismsCross, Shaun T.; Kapuscinski, Marylee L.; Perino, Jacquelyn; Maertens, Bernadette L.; Weger-Lucarelli, James; Ebel, Gregory D.; Stenglein, Mark D. (MDPI, 2018-07-22)Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence.
- Genome Number and Size Polymorphism in Zika Virus Infectious UnitsSexton, Nicole R.; Bellis, Eric D.; Murrieta, Reyes A.; Spangler, Mark Cole; Cline, Parker J.; Weger-Lucarelli, James; Ebel, Gregory D. (2021-03)Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero. It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome. IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero. How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.
- Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineagesOliveira, Glenn; Vogels, Chantal B. F.; Zolfaghari, Ashley; Saraf, Sharada; Klitting, Raphaelle; Weger-Lucarelli, James; Leon, Karla P.; Ontiveros, Carlos O.; Agarwal, Rimjhim; Tsetsarkin, Konstantin A.; Harris, Eva; Ebel, Gregory D.; Wohl, Shirlee; Grubaugh, Nathan D.; Andersen, Kristian G. (Public Library of Science, 2023-02)Zika virus was introduced to the Western Hemisphere, spread rapidly, and led to the 2015-2016 Zika epidemic and a rise in congenital microcephaly. It remains unclear whether Zika virus evolved to become more transmissible directly before or during the epidemic. To investigate whether Zika evolved to become more transmissible, we engineered a library of recombinant viruses that represent twelve major Zika virus lineages that circulated throughout the Americas. We measured the replicative fitness of each of these lineages by infecting live mosquitoes and human cells that are relevant for disease or transmission. We found that two of the lineages, one that dominated Central America and another that existed mostly in the Caribbean, appear to replicate more efficiently in human cells. While the fitness changes do not appear to have significant effects on the 2015-2016 Zika epidemic, our analysis suggests Zika virus evolved at least twice during this outbreak. Monitoring the phenotypic evolution during the course of an outbreak can help control spread and mitigate disease. We believe this framework can be applied to study phenotypic evolution during future epidemics caused by emerging RNA viruses. RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic.
- Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictusMurrieta, Reyes A.; Garcia-Luna, Selene M.; Murrieta, Deedra J.; Halladay, Gareth; Young, Michael C.; Fauver, Joseph R.; Gendernalik, Alex; Weger-Lucarelli, James; Rückert, Claudia; Ebel, Gregory D. (PLoS, 2021-11-09)Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.
- Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of PolyinfectionTalmi-Frank, Dalit; Byas, Alex D.; Murrieta, Reyes; Weger-Lucarelli, James; Rückert, Claudia; Gallichotte, Emily N.; Yoshimoto, Janna A.; Allen, Chris; Bosco-Lauth, Angela M.; Graham, Barbara; Felix, Todd A.; Brault, Aaron C.; Ebel, Gregory D. (MDPI, 2023-05-26)Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.
- Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in miceDuggal, Nisha K.; McDonald, Erin M.; Weger-Lucarelli, James; Hawks, Seth A.; Ritter, Jana M.; Romo, Hannah; Ebel, Gregory D.; Brault, Aaron C. (2019-04)Zika virus (ZIKV) infection can result in neurological disorders including Congenital Zika Syndrome in infants exposed to the virus in utero. Pregnant women can be infected by mosquito bite as well as by sexual transmission from infected men. Herein, the variants of ZIKV within the male reproductive tract and ejaculates were assessed in inoculated mice. We identified two non-synonymous variants at positions E-V330L and NS1-W98G. These variants were also present in the passage three PRVABC59 isolate and infectious clone relative to the patient serum PRVABC59 sequence. In subsequent studies, ZIKV E-330L was less pathogenic in mice than ZIKV E-330V as evident by increased average survival times. In Vero cells, ZIKV E-330L/NS1-98G outcompeted ZIKV E-330V/NS1-98W within 3 passages. These results suggest that the E-330L/NS1-98G variants are attenuating in mice and were enriched during cell culture passaging. Cell culture propagation of ZIKV could significantly affect animal model development and vaccine efficacy studies.
- On the Fly: Interactions Between Birds, Mosquitoes, and Environment That Have Molded West Nile Virus Genomic Structure Over Two DecadesDuggal, Nisha K.; Langwig, Kate E.; Ebel, Gregory D.; Brault, Aaron C. (Oxford University Pres, 2019-09-24)West Nile virus (WNV) was first identified in North America almost 20 yr ago. In that time, WNV has crossed the continent and established enzootic transmission cycles, resulting in intermittent outbreaks of human disease that have largely been linked with climatic variables and waning avian seroprevalence. During the transcontinental dissemination of WNV, the original genotype has been displaced by two principal extant genotypes which contain an envelope mutation that has been associated with enhanced vector competence by Culex pipiens L. (Diptera: Culicidae) and Culex tarsalis Coquillett vectors. Analyses of retrospective avian host competence data generated using the founding NY99 genotype strain have demonstrated a steady reduction in viremias of house sparrows over time. Reciprocally, the current genotype strains WN02 and SW03 have demonstrated an inverse correlation between house sparrow viremia magnitude and the time since isolation. These data collectively indicate that WNV has evolved for increased avian viremia while house sparrows have evolved resistance to the virus such that the relative host competence has remained constant. Intrahost analyses of WNV evolution demonstrate that selection pressures are avian species-specific and purifying selection is greater in individual birds compared with individual mosquitoes, suggesting that the avian adaptive and/or innate immune response may impose a selection pressure on WNV. Phylogenomic, experimental evolutionary systems, and models that link viral evolution with climate, host, and vector competence studies will be needed to identify the relative effect of different selective and stochastic mechanisms on viral phenotypes and the capacity of newly evolved WNV genotypes for transmission in continuously changing landscapes.
- Rapid Evolution of Enhanced Zika Virus Virulence during Direct Vertebrate Transmission ChainsRiemersma, Kasen K.; Jaeger, Anna S.; Crooks, Chelsea M.; Braun, Katarina M.; Weger-Lucarelli, James; Ebel, Gregory D.; Friedrich, Thomas C.; Aliota, Matthew T. (2021-04)Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human to human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here, we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than that in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains is paramount to resist gains in ZIKV virulence. IMPORTANCE We used experimental evolution to model chains of direct and indirect Zika virus (ZIKV) transmission by serially passaging a synthetic swarm of molecularly barcoded ZIKV within and between mosquitoes and mice. We observed that direct mouse transmission chains facilitated a rapid increase in ZIKV replication and enhanced virulence in mice. These findings demonstrate that ZIKV is capable of rapid adaptation to a vertebrate host and indicate that direct human-to-human transmission poses a greater threat to public health than currently realized.
- Small RNA responses of Culex mosquitoes and cell lines during acute and persistent virus infectionRückert, Claudia; Prasad, Abhishek N.; Garcia-Luna, Selene M.; Robison, Alexis; Grubaugh, Nathan D.; Weger-Lucarelli, James; Ebel, Gregory D. (2019-06)RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.