Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

Assessing Reassortment between Bluetongue Virus Serotypes 10 and 17 at Different Coinfection Ratios in Culicoides sonorenesis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct-cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.

Integrating Veterinary Diagnostic Laboratories for Emergency Use Testing during Pandemics1.

The SARS-CoV-2 pandemic showed limitations in human outbreak testing. Veterinary diagnostic laboratories (VDLs) possess capabilities to bolster emergency test capacity. Surveys from 26 participating VDLs found human SARS-CoV-2 testing was mutually beneficial, including One Health benefits. VDLs indicated testing >3.8 million human samples during the pandemic, which included some challenges.

Recovery of multireassortant bluetongue virus serotype 6 sequences from a mule deer (Odocoileus hemionus) and Dorset sheep (Ovis aries) in Colorado.

We report the discovery of two bluetongue virus serotype 6 (BTV-6) reassortants recovered from a domestic sheep and a free-ranging mule deer in northern Colorado. At the time of this publication, whole-genome sequencing of BTV-6 isolates in the Western U.S. have not been undertaken. These findings reflect the incursive movement of geographically distinct BTV serotypes into important agricultural areas of the U.S. and demonstrate reassortment with regionally circulating serotypes.

SARS-CoV-2 surveillance in a veterinary health system provides insight into transmission risks.

OBJECTIVE: To investigate the prevalence and seropositivity of SARS-CoV-2 in companion and exotic animals in a veterinary healthcare system. SAMPLE: A total of 341 animals were sampled by a combination of oral and nasal swabs. Serum from whole blood was collected from a subset of animals (86 canines, 25 felines, and 6 exotic animals). METHODS: After informed owner consent, convenience samples from client-owned animals and the pets of students and staff members associated with Colorado State University's Veterinary Health System were collected between May 2021 and September 2022. Study samples were collected by trained veterinarians, Veterinary Health System staff, and veterinary students. RESULTS: SARS-CoV-2 RNA was detected by reverse transcription PCR in 1.6% (95% CI, 0.5% to 4.6%) of domestic canines and 1.1% (95% CI, 0.2% to 6.1%) of domestic felines. No RNA was detected in any of the exotic animal species tested (n = 66). Plaque reduction neutralization tests indicated that 12.8% (95% CI, 7.3% to 21.5%) of canines and 12.0% (95% CI, 4.2% to 30.0%) of felines had neutralizing antibodies against SARS-CoV-2. CLINICAL RELEVANCE: This study provides insight regarding SARS-CoV-2 spillover in domestic companion and exotic animals and contributes to our understanding of transmission risk in the veterinary setting.

In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in a devastating disease and significant economic losses. As a virus with a segmented genome, reassortment among the BTV serotypes that have co-infected a host may increase genetic diversity, which can alter BTV transmission dynamics and generate epizootic events. The objective of this study was to determine the extent of dissemination and characterize the tropism of BTV serotypes 10 and 17 in co-infected Culicoides sonorensis. Midges were exposed to both BTV serotypes via blood meal and processed for histologic slides 10 days after infection. An in situ hybridization approach was employed using the RNAscope platform to detect the nucleic acid segment 2 of both serotypes. Observations of the mosaic patterns in which serotypes did not often overlap suggest that co-infection at the cellular level may not be abundant with these two serotypes in C. sonorensis. This could be a consequence of superinfection exclusion. Understanding BTV co-infection and its biological consequences will add an important dimension to the modeling of viral evolution and emergence.

Culicoides-borne Orbivirus epidemiology in a changing climate.

Orbiviruses are of significant importance to the health of wildlife and domestic animals worldwide; the major orbiviruses transmitted by multiple biting midge (Culicoides) species include bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus. The viruses, insect vectors, and hosts are anticipated to be impacted by global climate change, altering established Orbivirus epidemiology. Changes in global climate have the potential to alter the vector competence and extrinsic incubation period of certain biting midge species, affect local and long-distance dispersal dynamics, lead to range expansion in the geographic distribution of vector species, and increase transmission period duration (earlier spring onset and later fall transmission). If transmission intensity is associated with weather anomalies such as droughts and wind speeds, there may be changes in the number of outbreaks and periods between outbreaks for some regions. Warmer temperatures and changing climates may impact the viral genome by facilitating reassortment and through the emergence of novel viral mutations. As the climate changes, Orbivirus epidemiology will be inextricably altered as has been seen with recent outbreaks of bluetongue, epizootic hemorrhagic disease, and African horse sickness outside of endemic areas, and requires interdisciplinary teams and approaches to assess and mitigate future outbreak threats.

Role of Spillover and Spillback in SARS-CoV-2 Transmission and the Importance of One Health in Understanding the Dynamics of the COVID-19 Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is notable both for its impact on global public health as well as its well-publicized transmissibility to other species of animals. Infection of incidental animal hosts is concerning because of possible emergence of novel variants due to viral mutation. Species that are susceptible to SARS-CoV-2 include domestic and nondomestic cats, domestic dogs, white-tailed deer, mink, and golden hamsters, among others. We detail possible origins of transmission of SARS-CoV-2 to humans, as well as the ecological and molecular mechanisms needed for the virus to establish infection in humans from animals. We highlight examples of SARS-CoV-2 spillover, spillback, and secondary spillover, demonstrating the breadth in the variability of hosts and current transmission events that have been documented in domestic, captive, and wild animals. Lastly, we turn our focus to the importance of animal hosts as potential reservoirs and sources of variant emergence that can have profound effects on the human population. We note that a One Health approach emphasizing surveillance of animals and humans in certain environments using interdisciplinary collaboration is encouraged to manage disease surveillance, regulation on animal trade and testing, and animal vaccine development that will mitigate further disease outbreaks. These efforts will minimize the spread of SARS-CoV-2 and advance knowledge to prevent the spread of future emerging infectious diseases.

Modeling cellular co-infection and reassortment of bluetongue virus in Culicoides midges.

When related segmented RNA viruses co-infect a single cell, viral reassortment can occur, potentially leading to new strains with pandemic potential. One virus capable of reassortment is bluetongue virus (BTV), which causes substantial health impacts in ruminants and is transmitted via Culicoides midges. Because midges can become co-infected by feeding on multiple different host species and remain infected for their entire life span, there is a high potential for reassortment to occur. Once a midge is co-infected, additional barriers must be crossed for a reassortant virus to emerge, such as cellular co-infection and dissemination of reassortant viruses to the salivary glands. We developed three mathematical models of within-midge BTV dynamics of increasing complexity, allowing us to explore the conditions leading to the emergence of reassortant viruses. In confronting the simplest model with published data, we estimate that the average life span of a bluetongue virion in the midge midgut is about 6 h, a key determinant of establishing a successful infection. Examination of the full model, which permits cellular co-infection and reassortment, shows that small differences in fitness of the two infecting strains can have a large impact on the frequency with which reassortant virions are observed. This is consistent with experimental co-infection studies with BTV strains with different relative fitnesses that did not produce reassortant progeny. Our models also highlight several gaps in existing data that would allow us to elucidate these dynamics in more detail, in particular the times it takes the virus to disseminate to different tissues, and measurements of viral load and reassortant frequency at different temperatures.

Increased Ifng and Il10 Expression Correlate with Disease in Rodent Models Experimentally Infected with Modoc Virus.

Flaviviruses present an ongoing threat to global public health, although the factors that contribute to the disease remain incompletely understood. We examined an acute Modoc virus (MODV) infection of two rodent models. Viral RNA was detected in the kidneys, spleen, liver, brain, urine, and sera of experimentally infected deer mice, a reservoir host of MODV, and Syrian hamsters, a known disease model. As expected, clinical outcomes differed between species, and the levels of viral RNA recovered from various tissues demonstrated signs of differential replication and tissue tropism. Multivariate analysis indicated significance in the profile of expressed genes between species when analyzed across tissues and over time (p = 0.02). Between-subject effects with corrected models revealed a significance specific to the expression of Ifng (p = 0.01). the expression of Ifng was elevated in hamsters as compared to deer mice in brain tissues at all timepoints. As the over-expression of Ifng has been shown to correlate with decreased vascular integrity, the findings presented here offer a potential mechanism for viral dissemination into the CNS. The expression of IL10 also differed significantly between species at certain timepoints in brain tissues; however, it is uncertain how increased expression of this cytokine may influence the outcome of MODV-induced pathology.

Assessment of Local and Systemic Changes in Plant Gene Expression and Aphid Responses during Potato Interactions with Arbuscular Mycorrhizal Fungi and Potato Aphids.

This research examined aphid and plant responses to distinct levels (none, low, and high) of arbuscular mycorrhizal (AM) fungal root colonization by studying the association between potato aphids (Macrosiphum euphorbiae), potatoes (Solanum tuberosum), and AM fungi (Rhizophagus intraradices). It extends knowledge on gene expression changes, assessed by RT-qPCR, of ten defense-related genes at two time-points post-herbivory (24 h and 10 days), focusing on aphid-infested local leaves, non-infested systemic leaves, and roots. The results showed that aphid fitness was not altered by AM symbiosis. At 24 h, ETHYLENE RECEPTOR 1 gene expression was repressed in roots of aphid-infested non-mycorrhizal plants and aphid-infested plants with a high level of AM fungal root colonization, but not on aphid-infested plants with a low level of AM fungal root colonization. At 10 days, ALLENE OXIDE CYCLASE and POTATO TYPE I PROTEASE INHIBITOR were upregulated exclusively in local leaves of aphid-infested plants with a low level of AM fungal root colonization. In addition, local and systemic changes in plant gene expression appeared to be regulated exclusively by AM status and aphid herbivory. In summary, the gene expression data provide insights on mycorrhizal potato responses to aphid herbivory and serve as a starting point for future studies using this system.

Transcriptomic Signatures of Tacaribe Virus-Infected Jamaican Fruit Bats.

Tacaribe virus (TCRV) is a mammalian arenavirus that was first isolated from artibeus bats in the 1950s. Subsequent experimental infection of Jamaican fruit bats (Artibeus jamaicensis) caused a disease similar to that of naturally infected bats. Although substantial attention has focused on bats as reservoir hosts of viruses that cause human disease, little is known about the interactions between bats and their pathogens. We performed a transcriptome-wide study to illuminate the response of Jamaican fruit bats experimentally infected with TCRV. Differential gene expression analysis of multiple tissues revealed global and organ-specific responses associated with innate antiviral responses, including interferon alpha/beta and Toll-like receptor signaling, activation of complement cascades, and cytokine signaling, among others. Genes encoding proteins involved in adaptive immune responses, such as gamma interferon signaling and costimulation of T cells by the CD28 family, were also altered in response to TCRV infection. Immunoglobulin gene expression was also elevated in the spleens of infected bats, including IgG, IgA, and IgE isotypes. These results indicate an active innate and adaptive immune response to TCRV infection occurred but did not prevent fatal disease. This de novo assembly provides a high-throughput data set of the Jamaican fruit bat and its host response to TCRV infection, which remains a valuable tool to understand the molecular signatures involved in antiviral responses in bats. IMPORTANCE As reservoir hosts of viruses associated with human disease, little is known about the interactions between bats and viruses. Using Jamaican fruit bats infected with Tacaribe virus (TCRV) as a model, we characterized the gene expression responses to infection in different tissues and identified pathways involved with the response to infection. This report is the most detailed gene discovery work in the species to date and the first to describe immune gene expression responses in bats during a pathogenic viral infection.

Lack of pharmacokinetic or pharmacodynamic interaction between memantine and donepezil.

BACKGROUND: Memantine, a low- to moderate-affinity, uncompetitive N-methyl-D-aspartate receptor antagonist, was approved in the US for treatment of moderate to severe Alzheimer's disease in October 2003. OBJECTIVE: To determine whether an in vivo pharmacokinetic interaction exists between memantine and the acetylcholinesterase (AChE) inhibitor donepezil. METHODS: In this open-label, multiple-dose study, 24 healthy subjects (aged 18-35 y) received oral administration of memantine 10 mg on day 1. Following a 14-day washout period, subjects were orally administered donepezil 5 mg once daily for 7 days on an outpatient basis. Beginning on day 22, the donepezil dosage was doubled for 22 days to the target dose of 10 mg once daily, with the last donepezil dose concomitantly administered with memantine 10 mg on day 43. Assessments included pharmacokinetic as well as safety parameters. In addition, AChE inhibition was measured in red blood cells by radiolabeled-enzyme assay following administration of donepezil alone and after a single memantine dose. RESULTS: Data from 19 subjects who completed the study indicated no significant pharmacokinetic interactions between a single dose of memantine and multiple doses of donepezil. Percent maximum inhibition of AChE activity (mean +/- SD) by donepezil was 77.8 +/- 7.3% and not significantly different upon coadministration of a single dose of memantine (81.1 +/- 5.7%). Two subjects withdrew due to adverse events while taking donepezil alone. Single memantine doses administered with multiple donepezil doses were well tolerated. CONCLUSIONS: The pharmacokinetic and pharmacodynamic data from this study indicated a lack of interaction between memantine and donepezil, suggesting that memantine and donepezil may be safely and effectively used in combination.