Dynamics of epizootic hemorrhagic disease virus serotype 2 infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae)

Authors: MILLS, MARY - Kansas State University; RUDER, MARK - University Of Georgia; Nayduch, Dana; MICHEL, KRISTIN - Kansas State University; Drolet, Barbara

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2017
Publication Date: 11/27/2017
Citation: Mills, M.K., Ruder, M.G., Nayduch, D., Michel, K., Drolet, B.S. 2017. Dynamics of epizootic hemorrhagic disease virus serotype 2 infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae). PLoS One. https://doi.org/10.1371/journal.pone.0188865.
DOI: https://doi.org/10.1371/journal.pone.0188865

Interpretive Summary: Culicoides sonorensis biting midges transmit epizootic hemorrhagic disease virus (EHDV) during blood feeding, causing fatal disease in white-tailed deer. There are seven different types of EHDV, three of which are endemic in the USA. To date, little is known about the virus infection in the transmitting midge. To characterize EHDV type 2 infection within the midge, a multidisciplinary detection approach was used to examine infectious virus, viral nucleic acid and virus infection of specific midge organs over the course of infection to determine infection prevalence, virus dissemination, and viral load. Overall, results suggest that EHDV type 2 infects approximately 50% of midges that ingest virus, and that it disseminates from the midgut to secondary infection sites throughout the midge as early as five days post infection.

Technical Abstract: Culicoides sonorensis biting midge is a confirmed vector of epizootic hemorrhagic disease virus (EHDV), which causes mortality in white-tailed deer and ruminant populations. Currently, of the seven EHDV serotypes, only 1, 2, and 6 are present in the USA, and very few studies have focused on the infection time course of these serotypes within the midge. The objective of this current research was to characterize EHDV-2 infection within the midge by measuring infection prevalence, virus dissemination, and viral load over the course of infection. Midges were fed a blood meal containing 106.9 PFU/ml EHDV-2, collected every 12 h from 0-2 days post feeding (dpf) and daily from 3-10 dpf, and processed using several techniques. Cytopathic effect assays and qPCR were used to determine infection prevalence, revealing a 50 % infection rate by 10 dpf using both methods. Using immunohistochemistry, EHDV-2 infection was detectable at 5 dpf, and shown to disseminate from the midgut to other tissues, including fat body, eyes, and salivary glands by 5 dpf. Stain intensity increased from 5-8 dpf, indicating replication of EHDV-2 in secondary infection sites after dissemination. This finding is also supported by trends in viral load over time, determined by plaque assays and qPCR. An increase in titer between 4-5 dpf correlated with viral replication in the midgut as seen with staining at day 5, while the subsequent gradual increase in viral load from 8-10 dpf suggested viral replication in midges with disseminated infection. Overall, the data presented herein suggest that EHDV-2 is likely to disseminate via the hemolymph to secondary infection sites throughout the midge and that virus transmission by C. sonorensis may occur as early as five days post infection.