Location: Arthropod-borne Animal Diseases Research
2024 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 evaluating viral genetic determinants mediating emergence of epidemic VSV in the U.S. and transmission by Culicoides midges. Site-directed mutagenesis was used to target the coding sequence of five amino acids in a highly pathogenic VSV strain to determine if these sites were involved in virulence and transmissibility. Studies conducted by collaborators at ARS Plum Island, New York, showed no difference in pathogenesis in pigs between the wildtype and the mutant. Midge studies at ARS Manhattan, Kansas, showed no differences in infection rate of midges following ingestion of virus but did show a decrease in dissemination of the mutant to the salivary glands compared to the wildtype, suggesting these five sites in the viral genome may play a role in vector-borne transmissibility of the virus.
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 to be characterized is the population dynamics of virus within a host that ultimately determine the fitness of a virus to infect and be transmitted by the midge vector. Progress was made toward characterizing the molecular evolution of VSV in Culicoides midge and mammalian host cells. Five serial passages of virus were completed, infectious virus and viral RNA have been isolated and quantified. As black flies are also a vector of VSV, development of a black fly cell line has been initiated. A protocol was also developed for serial passaging of VSV in adult midges.
Another important epidemiological factor of VSV is how it overwinters between vector seasons to cause outbreaks in consecutive years in the absence of infected animals. Previously we showed that VSV is transmitted with extremely high efficiency between midges during mating which may contribute to the ability of the virus to overwinter. To understand this midge-to-midge transmission efficiency, the infection dynamics of virus from insects and virus from animals was compared. Results showed that VSV multiplying in insects is highly adapted to insect cells. Therefore, even though there is only a small amount of virus in the insect, virus transferred during mating is primed for infecting the mate resulting in a very efficient transmission route.
In understanding when, where, and how VSV enters the U.S. from endemic regions of Mexico and then spreads, progress was made toward characterizing the virus strain that recently caused a severe outbreak in California in an area with no history of previous outbreaks. Field-caught midges and black flies were tested for VSV RNA and infectious virus. Whole genome sequences of virus isolated from infected horses were used to conduct a phylogenetic analysis to look for relatedness to endemic Mexico strains. The sequences are also being used to examine the within-host diversity of VSV genomes.
To better understand how midges respond to VSV when they are infected, a transcriptome analysis was conducted to see what genes turn on or off in response to infection using midge cell cultures. Results showed an early immune response to VSV infection which then wanes, resulting in a productive, persistent infection.
Objective 2: Research continued on developing intervention strategies to minimize the impact of VSV disease outbreaks.
Progress was made toward determining the effects of VSV infection on phototactic and circadian rhythm behaviors in midges. The goal is to improve vector control strategies with light traps that specifically target VSV-infected midges by optimizing the timing of trapping based on circadian rhythm studies, and specific light wavelengths used based on phototaxis studies. Monthly circadian rhythm recordings have been conducted with infected and uninfected midges to assess changes in daily activity patterns as a result of changes in season as well as infection status. Recordings continue and data analysis is ongoing. For the phototaxis studies, thus far, no observable differences in light attraction have been detected in midges following infection with VSV in the current light arena design. A redesign/optimization of the light arena is ongoing.
Progress was made toward examining gene expression in midges infected with VSV across tissues and treatments. The goal of this work is to use transcriptome analysis to correlate gene expression with observed sensory behaviors (such as phototaxis, as above) that may be impacted by virus infection. A bioinformatic analysis pipeline for analyzing transcriptome data in midges (infected, controls) has been constructed, and sample preparation protocols have been created and validated. In addition, reference genes were optimized and tested, and candidate genes were identified that can be used to compare gene expression across various conditions or tissues in real-time quantitative polymerase chain reaction (PCR) assays.
Progress was made toward minimizing the impact of VSV disease outbreaks in real-time. County-level reports in Mexico and the U.S. have been compiled to the present day, to improve early warning of outbreaks in the U.S. A preliminary risk framework for targeting potential overwintering locations from the 2023 VSV outbreak in California was developed. Locations surrounding positive 2023 premises were assigned higher risk, especially when seasonal precipitation was higher and temperatures were cooler. Other variables linked with vector capacity and disease spread, such as vegetation greenness and soil properties, will be included moving forward. VSV outbreak model development is ongoing.
Accomplishments
1. Livestock viruses may hide in insect populations during the winter. Vesicular stomatitis virus (VSV) infects cows, horses, and pigs and causes disease resulting in animal production loss, quarantines, and movement/trade restrictions. Typically, VSV is transmitted from animal to animal by direct contact, but the virus can also be transmitted by insects such as Culicoides biting midges. These tiny flies ingest just a few virus particles when blood feeding on an infected animal, which then multiply inside their bodies, and are transmitted to other animals the next time they feed. Midges also pass the virus very efficiently to other midges during mating. The maintenance of virus via midge-to-midge transmission may be how VSV persists over the winter, when midges aren’t feeding on animals, then reemerges in livestock the next summer once the insects start blood feeding again. ARS scientists in Manhattan, Kansas, demonstrated that one reason midges can transmit VSV to other midges so efficiently is that viruses that come from insect cells have an increased ability to infect more insect cells. This phenomenon bolsters the idea of midge-to-midge infection efficiency and highlights the importance of Culicoides midges in VSV maintenance and transmission, which can help scientists better understand the epidemiology of vesicular disease.
2. Insects don’t mount a full defense response to livestock viruses, allowing them to quickly multiply and spread. Culicoides midges are tiny biting flies capable of transmitting vesicular stomatitis virus (VSV) which can cause disease in horses, cattle, and swine. Midges acquire VSV by feeding on infected animals and can mount immune responses to the virus. Characterizing the midge’s response to VSV can help researchers uncover factors that either promote or inhibit virus infection success in the midge, which ultimately affects their ability to transmit the virus among animal hosts. ARS researchers in Manhattan, Kansas, in collaboration with Kansas State University, examined whether cell cultures derived from midges turned on or off certain genes in response to infection with VSV. Genes involved in the midge’s immune response were activated quickly after infection, but that response only lasted for one hour. This suggests that by one hour, either the virus suppresses the midge’s immune response, or the midge downregulates its response, both mechanisms allowing the virus to multiply and spread to other cells. This work provides the first insights into the genetic interactions between VSV and cells from an insect that is known to carry and transmit it. Because responses observed in a whole insect may differ from just cells, future studies are aimed at understanding this complex interaction of virus and midges in live insects.
Review Publications
Scroggs, S.L., Bird, E.J., Molik, D.C., Nayduch, D. 2023. Vesicular stomatitis virus elicits early transcriptome response in Culicoides sonorensis cells. Viruses. 15(10). Article 2108. https://doi.org/10.3390/v15102108.
Rozo-Lopez, P., Drolet, B.S. 2023. Culicoides-specific fitness increase of vesicular stomatitis virus in insect-to-insect infections. Insects. 15(1):34-47. https://doi.org/10.3390/insects15010034.