Natural reassortment of a segmented RNA arbovirus illustrates plasticity of phenotype in the arthropod vector and mammalian hostin vivo

Abstract

Segmented RNA viruses are a taxonomically diverse group of 11 families that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during co-infection of a host by a process termed 'reassortment'. Reassortment enables rapid evolutionary change, but in the case of segmented RNA viruses utilising an arthropod vector is set against the constraint of purifying selection and genetic bottlenecks imposed by replication in two evolutionarily distant hosts. In this study, we use an in vivo host: arbovirus: vector model to investigate the impact of reassortment on two phenotypic traits: vector competence and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV is comprised of 10 linear segments of dsRNA and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4 and BTV-8) were isolated from naturally infected ruminants in Europe and parental/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to infect Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the parental virus strains which allowed inferences of the genome segments conferring this phenotypic trait.

Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus

“…BTV-4 MOR2009/07 [KC1], a strain of moderate clinical severity with a high infection rate of (AA) C. sonorensis (42), was obtained from the Orbivirus Reference Collection (The Pirbright Institute). Serial passage on KC cells (7 days, 28°C) generated BTV-4 MOR2009/07 [KC2] infectious tissue culture supernatant (TCS) for in vivo studies.…”

“…Serial passage on KC cells (7 days, 28°C) generated BTV-4 MOR2009/07 [KC2] infectious tissue culture supernatant (TCS) for in vivo studies. Harvest, storage, and quantification (end-point titration to determine 50% tissue culture infective dose (TCID 50 )) of the TCS was performed as previously described (39,42), except that KC cells were incubated at 28°C, with secondary antibody diluted at 1:250. Serotype specificity was confirmed by qRT-PCR targeting BTV segment 2 (43).…”

“…One sheep in each experimental replicate (n = 5) remained uninfected (and un-depleted) to monitor for contact transmission (Figure 1A). The infection rates of these blood-fed C. sonorensis individuals were then determined as previously described (42). Uninfected C. sonorensis (two pots per sheep) were blood-fed on each sheep at peak viraemia (as above) to investigate the BTV infection rate as an indicator for onwards transmission to a susceptible Culicoides vector.…”

“…Uninfected C. sonorensis (two pots per sheep) were blood-fed on each sheep at peak viraemia (as above) to investigate the BTV infection rate as an indicator for onwards transmission to a susceptible Culicoides vector. Between eight and 16 fully engorged, blood-fed individuals were collected from each sheep under controlled CO 2 anaesthesia and immediately processed (as previously described (42)) to determine the initial quantity of Schematic diagram of experimental design and timeline. (A) Adult Culicoides sonorensis were fed on a defibrinated horse blood-virus mixture using the Hemotek feeding system to enable uptake of BTV-4 MOR2009/07.…”

“…We next determined if the observed BTV viraemia dynamics were influenced by the number of TI Culicoides midges feeding on the sheep to establish infection. Here, we inferred TI midges as those with a BTV segment 10 qRT-PCR Cq value of less than 25 (42). Comparing the viraemia profile in sheep infected by a low (n < 18) and high (n ≥ 18) number of TI midges at 0 dpi (Figure 4B), we observed a significant difference in the viraemia profiles of WC1 + gd T cell-depleted sheep (p = 0.016), but not mock, CD4 + , or CD8 + T cell-depleted sheep (p = 0.744, 0.779, and 0.106, respectively).…”