Impacts of Infectious Dose, Feeding Behavior, and Age of Culicoides sonorensis Biting Midges on Infection Dynamics of Vesicular Stomatitis Virus

Abstract

Culicoides sonorensis biting midges are biological vectors of vesicular stomatitis virus (VSV) in the U.S. Yet, little is known regarding the amount of ingested virus required to infect midges, nor how their feeding behavior or age affects viral replication and vector competence. We determined the minimum infectious dose of VSV-New Jersey for C. sonorensis midges and examined the effects of multiple blood-feeding cycles and age at the time of virus acquisition on infection dynamics. A minimum dose of 3.2 logs of virus/mL of blood resulted in midgut infections, and 5.2 logs/mL resulted in a disseminated infection to salivary glands. For blood-feeding behavior studies, ingestion of one or two non-infectious blood meals (BM) after a VSV infectious blood meal (VSV-BM) resulted in higher whole-body virus titers than midges receiving only the single infectious VSV-BM. Interestingly, this infection enhancement was not seen when a non-infectious BM preceded the infectious VSV-BM. Lastly, increased midge age at the time of infection correlated to increased whole-body virus titers. This research highlights the epidemiological implications of infectious doses, vector feeding behaviors, and vector age on VSV infection dynamics to estimate the risk of transmission by Culicoides midges more precisely.

Surveillance along the Rio Grande during the 2020 Vesicular Stomatitis Outbreak Reveals Spatio-Temporal Dynamics of and Viral RNA Detection in Black Flies

“…The rRT-PCR assay used in this study was recently utilized to detect infection after experimental exposure of biting midges to VSV [80,81]; midges were considered positive for VSV with Ct values as high as 35.6 when orally infected, and 34.8 when intrathoracically infected. In the current study, the highest Ct value detected for a black fly deemed to be positive for VSV was 32.5.…”

Detection of Vesicular Stomatitis Virus Indiana from Insects Collected during the 2020 Outbreak in Kansas, USA

“…Based on positive and negative controls, samples with a minimum of two RT-qPCR reactions with Ct ≤ 36.5 were considered positive for VSV-IN RNA. This cutoff value was based on our previous work with Culicoides midges and VSV-NJ [ 31 , 68 ]. Standard curves and calculation of Ct values were carried out with the 7500 Fast Dx software (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA).…”

Comparison of Endemic and Epidemic Vesicular Stomatitis Virus Lineages in Culicoides sonorensis Midges

“…Orally and intrathoracically infected midges were maintained in environmental chambers at 25 ± 1 • C and 70-80% relative humidity with a 13:11 light: dark cycle and offered 10% sucrose solution ad libitum. Ten midges injected with each VSV lineage were collected at 3-and 10-days post-injection (dpi) in either 300 µL of TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or 500 µL of antibiotic medium [28,37] and stored at −80 • C until further processing for RT-qPCR and virus isolation, respectively. To test midgut infection (decapitated bodies) and dissemination to salivary glands (heads with salivary glands), 60 midges infected orally with each lineage were collected at 7-and 10-days post-feeding (dpf) in 300 µL of TRIzol or 500 µL of antibiotic medium and stored at −80 • C until further processing for RT-qPCR and virus isolation, respectively.…”

“…The midgut is the most critical organ in determining vector competence [27]. To infect the midgut epithelium, a threshold level of VSV infective particles in a blood meal is required [28]. However, this infection threshold can be different for specific virus and vector populations [27].…”

“…The importance of C. sonorensis as a VSV vector has been well established [25,[34][35][36][37]. Previous research has shown a detailed description of the vector competence and the temporal progression of VSV infection in the midge [25,28,34], bite transmission [35,36], non-conventional routes of transmission [37], and vector capacity variation [38]. However, the influence of viral genetic factors on Culicoides-virus interactions and their potential impact on transmission are understudied.…”