Identification of a BTV-Strain-Specific Single Gene That Increases Culicoides Vector Infection Rate

Abstract

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-14S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-41S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-14S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo

“…The role of VP2 in the binding, entry, and infection of Culicoides mesenteron gut cells, the barrier to infection ( 52 ), is as yet undetermined. The ability of BTV-4 MOR2004/04 derived segment -2 (VP 2) to confer the ability of efficient virus replication in C. sonorensis has recently been confirmed using reverse-engineered BTV reassortant strains ( 53 ).…”

Visualisation of bluetongue virus in the salivary apparatus of Culicoides biting midges highlights the accessory glands as a primary arboviral infection site

“…The bodies of all dissected Culicoides were homogenised, total RNA was extracted and BTV genome detected and quantified using a BTV-specific qRT-PCR (Hofmann et al, 2008). As observed in studies of Culicoides vector competence for BTV (Guimera Ropiak et al, 2021;Sanders et al, 2022), in groups infected by oral feeding, insects separate into three defined populations: those with no viral RNA (vRNA) detected (no Ct); individuals with lower amounts of vRNA (≤ 4.00E+07 genome copies/body, equivalent to a Ct value of ≈25 in our assay) and deemed to not support virus transmission (hence not vector competent), and those with greater quantities of vRNA (≥4.00E+07 genome copies/insect) and deemed vector competent (Figure 6…”

“…Six µl of each extracted RNA was tested by qRT-PCR targeting Segment 10 (Seg-10) of BTV as described (Hofmann et al, 2008) but adapted for the SuperScript III platinum one-step qRT-34 PCR (Invitrogen™, Life Technologies, Inchinnan, UK). Viral genome copies were quantified using a 10-fold dilution series of BTV-1 Seg-10 RNA transcript as standard (Ropiak et al, 2021). qRT-PCR results were plotted using GraphPad Prism software version 9.3.1 (GraphPad Software, San Diego, USA).…”

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus

“…In addition, a considerable number of viruses in this genus are important animal pathogens; for instance, midge-associated bluetongue virus (BTV), African horse sickness virus, and epizootic hemorrhagic disease virus (EHDV) cause acute disease with high mortality in domestic animals, leading to huge economic losses in the livestock industry ( 4 , 5 ). The emergence of orbiviruses depends on the distribution, activity, and seasonal abundance of competent vectors, such as the adults of certain midge ( Culicoides ) species ( 6 ). In addition, orbiviruses have been found in a wide host range including ticks, mosquitoes, midges, ruminants, birds, and humans ( 7 – 11 ).…”