Research Project: Ecology of Hemorrhagic Orbiviruses in North America

Location: Arthropod-borne Animal Diseases Research

2023 Annual Report

Objectives
Objective 1. Determine biotic and abiotic factors mediating orbivirus transmission in the United States. • Identify molecular targets for orbivirus cell surface attachment to inform host range susceptibility. • Determine physiological effects of orbivirus infection of C. sonorensis biting midges on vectorial capacity. • Assess effects of increasing global temperatures on orbivirus transmission by C. sonorensis biting midges to inform predictive biology and disease ecology. • Identify and characterize habitats of immature Culicoides to determine species distributions and densities. Objective 2. Develop intervention strategies to minimize the impact of disease outbreaks caused by Orbiviruses. • Identify vector transmission control strategies based on our understanding of vector-Orbivirus interactions.

Approach
While there are many critical aspects of orbivirus transmission that are poorly understood for assessing disease risk and developing mitigation strategies, it is clear that the multi-host transmission dynamics and maintenance of orbiviruses in nature requires susceptible, viremic animals, and competent Culicoides biting midge vectors. Identifying biotic and abiotic factors affecting these orbivirus-vector-host interactions from a molecular to organismal scale, and from basic science to applied, is key to understanding orbiviral emergence, spread, adaptation to new environments, and to developing effective control strategies. In Objective 1, we will investigate biotic factors including virus-host molecular interactions, to better understand host range susceptibility (Obj. 1A), and virus-vector interactions, to better understand effects of viral infection on midge physiology (Obj. 1B) and inform how infected vector population densities are affected during outbreaks. We will investigate abiotic factors including the effects of environmental temperature on vector competence, allowing us to determine how rising global temperatures may alter vector-borne transmission dynamics (Obj. 1C), and how ecological conditions alter suitability of midge breeding habitats, affecting population distributions, densities, and transmission potential to nearby animals (Obj. 1D). In Objective 2, we will investigate virus-vector interactions to inform novel methods for disease surveillance, midge management, and identify targets to block virus transmission. We will investigate the effects of virus infection on midge sensory responses and exploit virus-induced physiological changes to improve midge management strategies using light traps (Obj. 2A), specifically targeted to orbivirus-infected Culicoides vectors. Outcomes of this research will 1) address key knowledge gaps in virus-vector-host-environment interactions which underlie orbivirus transmission dynamics and our ability to predict risk to livestock and wildlife, and 2) lead to improved, novel intervention strategies to minimize the impact of these devastating hemorrhagic diseases in the US.

Progress Report
Objective 1: Research continued on determining biotic and abiotic factors mediating orbivirus transmission in the U.S. Progress was made toward identifying the molecular targets for orbivirus cell surface attachment to inform host range susceptibility. Codon usage bias analysis was conducted for insect cells and viral genes of the two attachment proteins, VP2 and VP5, and were cloned into a baculovirus vector for expression in SF9 insect cells. Cellular membrane protein preparations were made from sheep and Culicoides midge cells which will be used for far-western blotting with the expressed proteins to target the receptor. Progress was made toward determining the physiological effects of bluetongue virus (BTV) infection on Culicoides sonorensis biting midges. Baseline reproductive trials for blood fed midges were completed. Three longevity trials for BTV-infected midges were conducted. A baseline BTV susceptibility study was conducted on a new Kansas colony of biting midges. Progress was made toward determining the dynamics of simultaneous or staggered co-infection of midges with BTV serotypes. Full genome sequencing was used to select two serotypes with the most significant differences in the majority of segments for the co-infection study. Virus stocks were made for BTV-13 and 17 in Culicoides W8 cells. A co-infection procedure in midges with two BTV serotypes was developed. Primers and probes were designed and validated to detect BTV by quantitative polymerase chain reaction (PCR). Capacity for genome sequence analyses was increased by establishing accounts with USDA ARS’s scientific computing initiative, SCINet, and ‘Ceres, a high-performance computer cluster. Progress was made toward understanding biotic factors in BTV infection dynamics with the initiation of an RNA interference (RNAi) study to determine whether small interfering RNAs (siRNA) that target BTV-17 nonstructural protein 1 (NS1) could reduce viral replication in midge cells. Low dose infections at early time points showed the most viral replication inhibition by siRNAs. Progress was made toward adapting an embryonated chicken egg (ECE) transmission model for Culicoides midges and orbiviruses. Shell removal was optimized for midge access to microvasculature while ensuring embryo longevity. A midge-secure feeding cage was designed to allow midges free access to vasculature. It has been determined that BTV infection results after virus delivery to the yolk, white, or vasculature of the ECE and the minimum infectious doses for positive controls has been established. BTV has been isolated from embryonic livers of positive controls. BTV has been isolated from midges after they feed on inoculated ECEs, therefore ECE-to-midge BTV transmission has been demonstrated. Initial trials are now being conducted to determine whether midge-to-ECE BTV transmission can be demonstrated which will be the critical last step for using ECEs as a transmission model for BTV. To understand how increasing environmental temperatures affect the lifespan of midges, baseline survival studies with uninfected midges was completed. This will be compared to BTV infected midges to determine whether viral infection alters longevity at different environmental temperatures. Progress was also made towards evaluating thermal resting temperatures of BTV infected midges. A second thermal gradient was purchased, set up, and a midge-secure lid and an arena has been constructed. Midge injections are being optimized and a pilot study was conducted to test the thermal arena. Progress was made toward obtaining long term, geo-referenced BTV and epizootic hemorrhagic disease virus (EHDV) outbreak records from multiple organizations (government and university) and performing initial quality assessments to determine their use in predictive outbreak modeling for these orbiviruses. Progress was made toward identifying the impacts of microbiome, soil chemistry, and water chemistry of Culicoides larval habitats to determine species distributions and characterize favored conditions. Monthly collections of midge larval habitat sites were conducted from 12 sites including five at the Kansas State University (KSU) Animal Science Units (agricultural sites) and seven at the Konza Prairie Biological Station (sylvatic sites). At each site, larval emergence assays were conducted to determine the species and sexes of the midge community present. Mud and water samples were collected and prepared for chemical analysis by KSU Soil Chemistry Laboratory and microbiome samples were prepared for sequence analysis by the University of Kansas. A preliminary analysis of a few Konza sites representing bison, cattle, and non-grazed habitats was also conducted. Modeling of the North American distribution of Culicoides sonorensis was conducted, based on habitat suitability predictions and georeferenced specimens from the Smithsonian Institute in a maximum entropy (MaxEnt) model. Data were compiled to produce the most likely distributions of the vector species, as well as several closely related species, to help improve vector surveillance efforts. Objective 2: Research continued toward developing intervention strategies to minimize the impact of disease outbreaks caused by Orbiviruses. Progress was made toward identifying vector transmission control strategies based on our understanding of vector-orbivirus interactions. BTV infects sensory tissues, including the eye, of biting midges. Studies were conducted to determine the effects of BTV infection on Culicoides midge movement in response to light (phototaxis). Three midge-secure light arenas with interchangeable light-emitting diodes (LEDs) were designed and initial trials were conducted to measure and compare phototaxis behavior between BTV-infected and uninfected midges. Baseline wavelength preferences were determined by counting the number of midges that fly into the corresponding collection cups. Protocols and methods were optimized to microdissect tissue from the midge eye for staining to evaluate pathological changes in eye architecture, and for extracting BTV RNA from a single midge for quantitative polymerase chain reaction (qPCR) detection and quantification. This behavioral and infection data will be critical for improving or developing efficient trapping strategies that target BTV-infected biting midges. The baseline gene expression in midge cells and across midge life stages (egg, larvae, pupae, adults) was determined. This baseline will be compared to BTV-infected midges to identify the effects of viral infection on gene expression levels and cellular processes. Initial oral feeding infection studies were conducted and infection rates were confirmed by qPCR. Due to the inherent variability in oral feeding infection rates, microinjection methods were optimized to conduct differential expression reliably. High-titer BTV stocks were produced for use in midge infections. Time course experiments were conducted to track daily BTV viral RNA levels for seven days to generate a viral replication curve. Midge mRNA extraction protocols are being optimized to isolate quality RNA for sequencing from individual specimens. A computational pipeline for assembly, quality control, and assembly reduction of midge sequencing data was developed. Workflows were coded for genome assembly, contaminant filtering, and data quality control. Testing and optimizing code using test datasets is ongoing and awaiting mRNA sequences. Additional progress was made toward developing strategies to minimize the impact of orbivirus disease outbreaks by using zoo animals as sentinels for early disease detection, and surveying surrounding midge populations to determine risk to nearby livestock. Two biting midge species, transmitting three epizootic hemorrhagic disease virus (EHDV) serotypes, were responsible for the outbreak. Utilizing established infrastructure, such as zoological parks, to investigate animal disease outbreaks and conduct midge surveillance is one way to address surveillance deficiencies in the U.S.

Accomplishments
1. Microbes, soil, and nearby animals determine when and where biting midges will be found. Biting midges are tiny insects that spread diseases to livestock and wildlife, causing significant economic losses and trade restrictions. Midge larvae are in semi-aquatic habitats like the shorelines of ponds and springs. But what exactly attracts adult midges to these places to lay their eggs is not known. Understanding what influences where midges emerge can help scientists and livestock owners develop better, targeted, more cost-effective control strategies to reduce midge populations. This, in turn, reduces the risk of disease in their livestock. ARS researchers in Manhattan, Kansas, in collaboration with Kansas State University, studied the microbes and chemical compositions of the soil midges prefer and determined whether their presence in ponds and springs was associated with different animal grazing activities. The types of habitats, grazing by animals like cattle or bison, and soil properties such as nitrogen, carbon, and organic matter, all influenced the types of microbes in the soil and the presence of midges. Certain groups of microbes were found to either increase or decrease depending on whether midges were present, which provides important clues about the interrelatedness between microbes and midge survival. This information can be used to further explore how manipulating the microbes or habitat can lessen or even inhibit usage by midges, thereby reducing transmission of midge-borne viruses.

2. Zoos can be used to detect animal viruses that pose a risk to U.S. livestock and wildlife. Insect-transmitted viral diseases of humans and animals are on the rise, yet insect surveillance in the U.S. is declining. This reduces our ability to detect virus early and identify geographic areas at highest risk for disease. One way to address this deficiency is by utilizing our nation’s zoos as valuable monitoring sites. This allows for strategic insect trapping with simultaneous close observation of animals susceptible to disease. An outbreak of midge-transmitted epizootic hemorrhagic disease (EHD) at the Minnesota Zoo in fall 2020 exemplifies the effectiveness of this approach. ARS researchers in Manhattan, Kansas, collaborated with zoo officials to identify local biting midges and determine which species were transmitting virus. After collecting, identifying, and testing nearly 2000 midges, two different species carrying three EHD virus types were identified as being responsible for the outbreak. This study highlights the importance and utility of using public animal holding facilities, such as zoos, as sentinels to better understand when and where viruses may emerge.

Review Publications
Scroggs, S.L., Offerdahl, D.K., Stewart, P.E., Shaia, C., Griffin, A.J., Bloom, M.E. 2023. Of murines and humans: Modeling persistent Powassan disease in C57BL/6 mice. mBio. Article e03606-22. https://doi.org/10.1128/mbio.03606-22.
McGregor, B.L., Reister-Hendricks, L.M., Nordmeyer, C., Stapleton, S., Davis, T.M., Drolet, B.S. 2023. Using zoos as sentinels for re-emerging arboviruses: Vector surveillance during an outbreak of epizootic hemorrhagic disease at the Minnesota Zoo. Pathogens. 12(1):140-149. https://doi.org/10.3390/pathogens12010140.
Neupane, S., Davis, T.M., Nayduch, D., McGregor, B.L. 2023. Habitat type and host grazing regimen influence the soil microbial diversity and communities within potential biting midge larval habitats. Environmental Microbiome. 18(1):5-21. https://doi.org/10.1186/s40793-022-00456-8.