Biology subjects

Gallichotte, E. N.

Publications and source records attributed to Gallichotte, E. N..

6 recordsLinked to original sources

bioRxiv2026

Temperature alters Culex tarsalis West Nile virus vector competence, tissue bottlenecks, and transcriptional responses

West Nile virus is an arbovirus transmitted by Culex mosquitoes. Temperature is frequently the strongest abiotic factor impacting transmission in many virus-vector pairings, including WNV and Culex. However, it is unknown how temperature impacts WNV population bottlenecks and transcriptional responses to infection in Culex tarsalis. We found that elevated temperatures led to increased likelihood of transmission of rare virus variants due to increased vector competence and less stringent tissue barriers. Analysis of Cx. tarsalis transcriptional responses confirmed that WNV infection had minimal impacts on transcriptional activity. Temperature, however, induced significant changes to mosquito gene expression, whereas canonical immune gene expression minimally changed in response to infection. Thus, temperature impacts bottlenecks in the Culex-WNV system independent of known antiviral pathways, possibly by altering virus replication dynamics. While temperature had a dramatic impact on bottleneck stringency, this is likely independent of antiviral pathways and instead some other change to mosquito biology. Together, these results shed light on the relationships between temperature, virus evolutionary dynamics and antiviral immunity.

microbiology↗

bioRxiv2025

Development of ENTV reverse genetics system and phenotypic evaluation of rescued virus reveals host-specific replication patterns in mosquitoes

Entebbe bat virus (ENTV) is a bat-associated flavivirus with no known vector. Research into the biology of this virus, including assessment of the possibility that it may be vector-transmitted, is hindered by a lack of molecular tools and robust genetic systems. Therefore, we sequenced the complete 3 untranslated region, which was not previously available, and developed an infectious clone of ENTV to facilitate further investigation of the virus. Virus derived from the clone replicated similarly to the parental virus isolate in various vertebrate cells. Surprisingly, ENTV replicated to high titers in Aedes aegypti and Aedes albopictus mosquito cell lines, but there was no replication or infection in Culex tarsalis cells. In addition, phylogenetic and bioinformatics analyses strongly suggested that ENTV may be associated with a mosquito host. Given the bioinformatics support and efficient growth in Aedes cells, we orally exposed Ae. aegypti and Ae. albopictus to ENTV to evaluate infection. The ENTV blood-fed mosquitoes were all negative for infection; however, when ENTV was intrathoracically inoculated, bypassing the initial midgut infection and escape barriers, it replicated to high levels in the body, without dissemination of infectious virus into the saliva. These findings suggest that, despite demonstrating high molecular compatibility at the cellular level in Aedes mosquitoes, Ae. aegypti and Ae. albopictus are unlikely to serve as competent vectors for ENTV transmission due to strong midgut infection barriers. The clone presented in this manuscript should help to clarify the mechanisms for transmission and maintenance of ENTV, which remain poorly understood.

microbiology↗

bioRxiv2024

SARS-CoV-2 within-host population expansion, diversification and adaptation in zoo tigers, lions and hyenas

SARS-CoV-2 rapidly adapts to new hosts following cross-species transmission; this is highly relevant as novel within-host variants have emerged following infection of susceptible wild and domestic animal species. Furthermore, SARS-CoV-2 transmission from animals (e.g., white-tailed deer, mink, domestic cats, and others) back to humans has also been observed, documenting the potential of novel animal-derived variants to infect humans. We investigated SARS-CoV-2 evolution and host-specific adaptation during an outbreak in Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) at Denver Zoo in late 2021. SARS-CoV-2 genomes from longitudinal samples collected from 16 individuals were evaluated for within-host variation and genomic signatures of selection. The outbreak was likely initiated by a single spillover of a rare Delta sublineage subsequently transmitted from tigers to lions to hyenas. Within-host virus populations rapidly expanded and diversified. We detected signatures of purifying and positive selection, including strong positive selection in hyenas and in the nucleocapsid (N) gene in all animals. Four candidate species-specific adaptive mutations were identified: N A254V in lions and hyenas, and ORF1a E1724D, spike T274I, and N P326L in hyenas. These results reveal accelerated SARS-CoV-2 adaptation following host shifts in three non-domestic species in daily contact with humans.

evolutionary biology↗

bioRxiv2024

SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo

In late 2019, SARS-CoV-2 spilled-over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions, and compare species, sex, age, virus and antibody detection assay, and types, frequency and length of clinical signs, demonstrating broad heterogeneity amongst infections. We then describe a SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for four months. Lions had significantly more viral RNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health. ImportanceSurveillance and experimental testing have shown many animal species, including companion, wildlife, and conservatory, are susceptible to SARS-CoV-2. Early in the COVID-19 pandemic, big cats at zoological institutions were among the first documented cases of naturally infected animals; however, challenges in the ability to collect longitudinal samples in zoo animals have limited our understanding of SARS-CoV-2 kinetics and clearance in these settings. We measured SARS-CoV-2 infections over three months in lions, tigers and hyenas at Denver Zoo, and detected viral RNA, infectious virus, neutralizing antibodies, and recrudescence after initial clearance. We found lions had longer and higher levels of virus compared to the other species. All animals were infected by a rare viral lineage circulating in the human population, suggesting a single spillover followed by interspecies transmission. These data are important in better understanding natural SARS-CoV-2 spillover, spread and infection kinetics within multiple species of zoo animals.

microbiology↗

bioRxiv2024

A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection

The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells, suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level. Author SummaryWest Nile virus is the leading cause of mosquito-borne disease in N. America. Cx. tarsalis is a highly competent vector of WNV that plays a central role in the transmission and maintenance of WNV in nature. It is hypothesized that the permissibility of mosquito midgut cells contributes to the midgut infection barrier and thus impacts the ability of pathogens to establish infection in a mosquito. Additionally, it is postulated that the midgut is the most important organ with respect to determining vector competence. The recent publication of the full Cx. tarsalis genome, in conjunction with the growing body of work demonstrating the successful application of single-cell RNA sequencing methodologies in insect models made it possible for us to examine the cellular composition of the Cx. tarsalis midgut, and WNV infection dynamics therein, at single-cell resolution. We found cell-type-specific differences in viral RNA levels suggesting variability in WNV replication efficiency in specific cell types, identified patterns of differential expression associated with WNV infection in specific cell populations, and characterized aspects of the innate immune response to WNV infection at the tissue and cellular level.

microbiology↗

bioRxiv2022

Impact of prior infection on SARS-CoV-2 antibody responses in vaccinated long-term care facility staff

SARS-CoV-2 emerged in 2019 and has resulted in millions of deaths worldwide. Certain populations are at higher risk for infection, especially staff and residents at long term care facilities (LTCF), due to the congregant living setting, and residents with many comorbidities. Prior to vaccine availability, these populations represented a large fraction of total COVID-19 cases and deaths in the U.S. Due to the high-risk setting and outbreak potential, staff and residents were among the first groups to be vaccinated. To define the impact of prior infection on response to vaccination, we measured antibody responses in a cohort of staff members at a LTCF, many of whom were previously infected by SARS-CoV-2. We found that neutralizing, receptor-binding-domain (RBD) and nucleoprotein (NP) binding antibody levels were significantly higher post-full vaccination course in individuals that were previously infected, and NP antibody levels could discriminate individuals with prior infection from vaccinated individuals. While an anticipated antibody titer increase was observed after vaccine booster dose in naive individuals, boost response was not observed in individuals with previous COVID-19 infection. We observed a strong relationship between neutralizing antibodies and RBD-binding antibodies post-vaccination across all groups, suggesting RBD-binding antibodies may be used as a correlate of neutralization. One individual with high levels of neutralizing and binding antibodies experienced a breakthrough infection (prior to the introduction of Omicron), demonstrating that the presence of antibodies is not always sufficient for complete protection against infection. These results highlight that history of COVID-19 exposure significantly increases SARS-CoV-2 antibody responses following vaccination. ImportanceLong-term care facilities (LTCFs) have been disproportionately impacted by COVID-19, due to their communal nature, high-risk profile of residents and vulnerability to respiratory pathogens. In this study, we analyzed the role of prior natural immunity to SARS-CoV-2 on post-vaccination antibody responses. The LTCF in our cohort experienced a large outbreak with almost 40% of staff becoming infected. We found that individuals that were infected prior to vaccination, had higher levels of neutralizing and binding antibodies post-vaccination. Importantly, the second vaccine dose significantly boosted antibody levels in those that were immunologically naive prior to vaccination, but not those that had prior immunity. Regardless of pre-vaccination immune status, levels of binding and neutralizing antibodies were highly correlated. The presence of NP-binding antibodies can be used to identify individuals that were previously infected when pre-vaccination immune status is not known. Our results reveal that vaccination antibody responses differ depending on prior natural immunity.

immunology↗