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ARS Home Plains Area Manhattan, Kansas Center for Grain and Animal Health Research ABADRU Research





- Drolet, Barbara
- Nayduch, Dana
- McGregor, Bethany
- Scroggs, Stacey
- Hudson, Amy
- Rusch, Travis
- Shults, Phillip
- Molik, David
- 2023
- 2022
- Publications
- Animal Health (103)
- Veterinary, Medical and Urban Entomology (104)

Research Project: Predicting and Mitigating Vesicular Stomatitis Virus (VSV) in North America **Location:
                            Arthropod-borne Animal Diseases Research** 2023 Annual Report **Objectives**
                                

Objective 1:  Ascertain the viral ecology and factors mediating the introduction and expansion of VSV in the U.S. 
Objective 1A. Identify viral genetic determinants mediating emergence of epidemic VSV in the U.S. as well as adaptation to insect and animal hosts. 
Objective 1B. Characterize epidemiological, biotic and abiotic factors 
associated with vectorial capacity, emergence, incursion, and expansion of VSV from endemic areas into the U.S. 
Objective 2.  Develop intervention strategies to minimize the impact of VSV disease outbreaks.
Objective 2A. Develop model-based early warning systems to predict future incursions of VSV from Mexico to the U.S.
Objective 2B. Identify vector transmission control strategies based on our understanding of virus-vector-host interactions. **Approach**
                                

Vesicular stomatitis (VS) is a vector-borne, zoonotic disease caused by the RNA virus, vesicular stomatitis virus (VSV). Disease in cattle and pigs is clinically indistinguishable from foot-and-mouth disease (FMD), one of the most devastating exotic diseases in the U.S. which was eradicated in 1929. For the past 100 years, incursions of VS have occurred in the U.S. at 8–10-year intervals. Viral incursions originating in endemic regions of southern Mexico start in western border states (NM, TX, AZ) and expand northward with outbreaks often covering over a million square kilometers. Recent outbreaks occurred in 2004-05, 2014-15 and 2019-20, causing thousands of cases across 12 states, and suggesting shorter intervals (5-10 y) may be the new normal. This Project Plan is proposed by two ARS Units, with complementary VSV expertise, to conduct research under two overarching objectives or goals: 1) to identify ecological and virus-vector-host factors that mediate incursion and expansion of VS in the US; and 2) to develop countermeasures including rapid assessment, early warning models and vector control strategies, to reduce the impact of VS disease to US agriculture. This project integrates molecular biology, virology, pathology, entomology, phylogeography, and ecology to better understand the viral, vector, host, and environmental drivers of VS epidemiology across its spatiotemporal domain. Our multidisciplinary approach spans from basic research to applied, and from molecular and organismal (biotic) levels to environmental (abiotic) levels. The proposed project also involves mutually beneficial collaborations with the ARS VSV-Grand Challenge project "Vesicular Stomatitis as a Model for a Predictive Disease Ecology" and three other CRIS Project Plans across three National Programs. **Progress Report**
                                

Objective 1: Research continued to ascertain the viral ecology and factors mediating the introduction and expansion of vesicular stomatitis virus (VSV) in the U.S.
Progress was made toward characterizing the epidemiological, biotic, and abiotic factors associated with vectorial capacity, emergence, incursion, and expansion of VSV from endemic areas into the U.S. One of these factors is the maintenance and transmission of VSV by Culicoides biting midges. Progress was made toward understanding the efficiency of venereal transmission of VSV between midges during mating. When VSV infects a midge, it undergoes changes that make it more efficient at infecting and replicating in midge cells. This increased fitness facilitates the midge-to-midge transmission that occurs during mating. Genome sequences are being analyzed to determine whether this is due to genetic changes in the virus. This research may help explain the highly efficient venereal transmission seen in midges and highlights the importance of cell line specificity and limitations in investigating VSV-vector interactions. 
In characterizing abiotic factors that affect vectorial capacity, progress was made towards understanding the effects of global warming on the health, longevity, and behavior of biting midges, and their ability to transmit VSV. Higher temperatures correlated with shorter lifespans, shorter egg-laying cycles, and higher rates of viral replication including dissemination to the midge’s salivary glands for transmission. Lower temperatures correlated to longer lifespans, more days in their egg-laying cycles, and lower viral replication in the midge. Most midges preferred to rest in areas that fall within their preferred physiological range regardless of the environmental temperatures at which they were being maintained. These preferred temperatures maximized their survival, the number of egg-laying cycles, and the likelihood of VSV transmission. This suggests that higher global temperatures may mean higher virus transmission rates, and that even when temperatures get above the ideal conditions for midges, they will find areas to rest that optimize their health and that of the virus. 
To evaluate VSV transmission competence in all experimental studies progress was made toward adapting an embryonated chicken egg (ECE) transmission model for Culicoides midges and VSV. Shell removal has been 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 VSV inoculation must occur by injecting virus into the yolk or vasculature of the ECE and minimum infectious doses for positive controls have been established. VSV has been isolated from embryonic livers following needle inoculation of positive controls. VSV has been isolated from midges after they feed on inoculated ECEs, therefore ECE-to-midge VSV transmission has been demonstrated. Initial trials are now being conducted to determine whether midge-to-ECE VSV transmission can be demonstrated which will be the critical last step for using ECEs as a transmission model for VSV.   
Progress was also made in predicting outbreaks for 2023 based on a generalized linear model of climate anomalies in Mexico at different time lags. The research highlights our abilities to forecast vesicular stomatitis outbreak occurrence and spread in the U.S. and helps prioritize future work. Forecasting disease spread by identifying data layers, creating iterative workflows, and conveying uncertainty and ultimately risk to stakeholders is ongoing. 
Progress was made toward developing a model for early warning forecasting for vesicular stomatitis disease outbreaks. Researchers currently only have access to county-level case counts in non-machine-readable formats during outbreaks of vesicular stomatitis, both in the U.S. and Mexico. However, for modeling of disease spread, the exact GPS coordinates of infected premises provide much finer spatial data set. ARS is combining county-level situational reports that are available from APHIS weekly during outbreaks, and the GPS information available only after outbreaks, to improve with-in outbreak situational reports and use in future forecast modeling and early warning systems of VS spread.
Phylogeographic and environmental factors associated with the 2014-2015 VSV outbreak were analyzed using whole genome sequence data to reconstruct the phylogenetic history of viruses isolated from both the incursion and overwintering years as well as map any genetic changes. Models were also used to predict the factors that best explain the differences in transmission between these two years.
Virus transmission by Culicoides biting midges is linked to blood feeding which is required for females to produce eggs. Due to the relationship between egg development, blood feeding, and pathogen transmission, there is a need to understand gene expression patterns related to egg development and pathogen infection. MicroRNAs (miRNAs) degrade messenger RNAs in a sequence-specific manner which stops protein production. To determine the Culicoides miRNA catalog, small RNA molecules were sequenced and analyzed using bioinformatics (miRDeep2) of whole female midges and digestive tissues before and after blood feeding. Our analyses characterized 76 miRNAs within C. sonorensis. Based on our findings, we suggest an interesting evolutionary relationship between miRNA expression and blood meal requirements across blood-feeding insects. We also identified miRNAs with expression patterns regulated by blood meal ingestion and/or tissue and others regulated by virus infection. 
Objective 2: Research continued to develop intervention strategies to minimize the impact of VSV disease outbreaks.  
Progress was made toward identifying vector transmission control strategies based on our understanding of virus-vector-host interactions. VSV infects the sensory tissues of biting midges, including the eye and brain. The effect of VSV infection on midge movement in response to light (phototaxis) was examined. Virus harvesting and purification from a midge cell line was optimized to create a VSV stock for conducting infection and behavior trials in colony midges. 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 VSV-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. Previously developed immunohistochemical staining protocols will be used to correlate phototaxic behavior with virus presence in eye tissue. This data will be critical for improving or developing efficient trapping strategies for VSV-infected biting midges. 
Progress was made toward optimizing the procedure for extracting VSV RNA from a single midge as well as quantitative polymerase chain reaction (qPCR) for detection and quantification of VSV. This highly sensitive and specific technique is used for confirming VSV is present in midges infected for behavioral trials, and it gives insight into the level of infection between individual midges. 
Progress was made in determining the level of infection in midges that were fed VSV with a bloodmeal compared to those that were microinjected with pure VSV. During bloodmeal infection, VSV enters the midge through the natural feeding process. This closely mimics the natural route of VSV transmission in the wild. However, with this method, it can be challenging to control the exact virus dose and ensure consistent infection rates across midges. Microinjection involves injecting VSV directly into the midge’s body with a fine needle. This method bypasses the natural transmission route but offers precise control over the virus dose and allows for more consistent infection rates and the creation of positive controls. 
Progress was also made toward determining what genes are consistently expressed across midge life stages and during VSV infection. Infection rates after oral feeding, as detected by qPCR, were determined to be too variable to conduct differential expression reliably. Therefore, microinjection was optimized. Time course experiments were conducted to track VSV viral RNA levels daily for seven days to generate viral replication curve. High-titer VSV stocks were produced, midge mRNA extraction protocols were optimized to isolate quality RNA for sequencing from individual specimens, and mRNA sequencing is ongoing. 
Gene expression is being assessed under several conditions including midge cells, larvae, male and female pupae, male and female adults, and females infected with VSV. An initial study was conducted to determine the level of gene expression stability for each treatment. These results will be used as a reliable baseline for comparing gene expression levels and will give insight into how different treatments, such as infection with VSV, affect gene expression and cellular processes in a midge. 
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. **Accomplishments**
                                

1. Small molecules can make a big difference in biting midge biology. Culicoides biting midges are tiny flies capable of transmitting diseases to livestock and wildlife that result in significant global economic losses. Understanding the biology of these insects is crucial in developing strategies to reduce disease spread. MicroRNAs (miRNAs) are small molecules that regulate gene expression and play a pivotal role in the midge’s development, egg production, immune system, and competency for transmitting viruses. However, knowledge of specific miRNAs in Culicoides midges remains limited. In a collaborative study, ARS scientists in Manhattan, Kansas, and University of South Carolina-Aiken researchers identified 76 different miRNAs in whole female insects and midgut tissues after blood feeding. Some miRNAs were like those found in other insects, while others were unique. Some were more abundant in specific tissues associated with digestion, reproduction, and viral infections. This study establishes a foundation for future research on miRNAs in biting midge biology and their role in virus transmission, aiding in the identification of gene targets and the development of new strategies to control virus transmission by biting midges. **Review Publications**
                            *Mills, M.K., Rozo-Lopez, P., Bryant, W.B., Drolet, B.S. 2023. MicroRNA expression dynamics in Culicoides sonorensis biting midges following blood-feeding. Insects. 14:611-625. https://doi.org/10.3390/insects14070611.*


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