Increased Virulence of Culicoides Midge Cell-Derived Bluetongue Virus in IFNAR Mice

Abstract

Bluetongue (BT) is a Culicoides midge-borne hemorrhagic disease affecting cervids and ruminant livestock species, resulting in significant economic losses from animal production and trade restrictions. Experimental animal infections using the α/β interferon receptor knockout IFNAR mouse model and susceptible target species are critical for understanding viral pathogenesis, virulence, and evaluating vaccines. However, conducting experimental vector-borne transmission studies with the vector itself are logistically difficult and experimentally problematic. Therefore, experimental infections are induced by hypodermic injection with virus typically derived from baby hamster kidney (BHK) cells. Unfortunately, for many U.S. BTV serotypes, it is difficult to replicate the severity of the disease seen in natural, midge-transmitted infections by injecting BHK-derived virus into target host animals. Using the IFNAR BTV murine model, we compared the virulence of traditional BHK cell-derived BTV-17 with C. sonorensis midge (W8) cell-derived BTV-17 to determine whether using cells of the transmission vector would provide an in vitro virulence aspect of vector-transmitted virus. At both low and high doses, mice inoculated with W8-BTV-17 had an earlier onset of viremia, earlier onset and peak of clinical signs, and significantly higher mortality compared to mice inoculated with BHK-BTV-17. Our results suggest using a Culicoides W8 cell-derived inoculum may provide an in vitro vector-enhanced infection to more closely represent disease levels seen in natural midge-transmitted infections while avoiding the logistical and experimental complexity of working with live midges.

Keywords: BTV; Culicoides; IFNAR mice; biting midge; bluetongue virus; virulence.

Figure 1

Clinical scores of IFNAR mice following inoculation with (A) 10² PFU and (B) 10⁴ PFU BTV-17 derived from Culicoides W8 cells (red) or baby hamster kidney (BHK) cells (blue). Data points are XY staggered to show individual scores.

Figure 2

Mean body weights, as percent of starting weight, in IFNAR mice following inoculation with (A) 10² PFU and (B) 10⁴ PFU BTV-17 derived from Culicoides W8 cells (red) or baby hamster kidney (BHK) cells (blue). Mean weights of mock-infected negative control mice are shown in gray. Error bars represent the standard error of the mean (SEM; n = 6). Paired t-test was used to determine statistical significance between W8 and BHK treatment groups as indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001).

Figure 3

Survival curves of IFNAR mice following transdermal inoculation with BTV-17 derived from Culicoides W8 cells (red, solid) or baby hamster kidney (BHK) cells (blue, dashed). (A) Mice inoculated with the 10² PFU low dose (Mantel–Haenszel Hazard Ratio = 41.03; Log-rank Mantel–Cox p = 0.0005). (B) Mice inoculated with the 10⁴ PFU high dose (Mantel–Haenszel Hazard Ratio = 39.12; Log-rank Mantel–Cox p = 0.0009).

Figure 4

IFNAR mouse inoculated with 10² PFU W8-BTV-17 euthanized 6 dpi due to a clinical score of 3. Severe edema (over 0.5 mL serosanguinous fluid as shown) in the thoracic cavity and wet abdominal serosal surfaces were observed in all IFNAR mice inoculated with both low (10² PFU) and high (10⁴ PFU) dose W8-BTV-17 and in three inoculated with the high dose of BHK-BTV-17.

Figure 5

Mean virus titers in 50 µL daily blood samples as detected by RT-qPCR in IFNAR mice following challenge with (A) 10² PFU or (B) 10⁴ PFU BTV-17 derived from either Culicoides W8 cells (red with dots) or baby hamster kidney (BHK) cells (blue with triangles). RT-qPCR cycle threshold (Ct; left Y-axis) and Log₁₀ virus particle calculations (right Y-axis) based on RNA concentrations [39]. Error bars represent the standard error of the mean (SEM). Multiple paired t-test was used to determine statistical significance between W8 and BHK treatment groups as indicated (ns, not significant, ** p ≤ 0.01). Missing data for W8-BTV-17 inoculated mice was due to 100% mortality by day 6 (low-dose group) and by day 5 (high-dose group).

Figure 6

BTV-17 as detected by RT-qPCR in IFNAR mice necropsy tissue samples following challenge with (A) 10² PFU or (B) 10⁴ PFU BTV-17 derived from either Culicoides W8 cells (red with dots) or baby hamster kidney (BHK) cells (blue with triangles). Mean RT-qPCR cycle threshold (Ct; left Y-axis) and Log₁₀ virus particle calculations (right Y-axis) based on RNA concentrations [39] per mL of tissue homogenate. MLN; mesenteric lymph node. One way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance as indicated (* p ≤ 0.05). Error bars represent the standard error of the mean (SEM, n = 6).

Figure 7

Infectious virus titers in tissues of IFNAR mice as detected by plaque assay following transdermal inoculation with (A) 10² PFU and (B) 10⁴ PFU BTV-17 derived from either Culicoides W8 cells (red with dots) or baby hamster kidney (BHK) cells (blue with triangles). MLN; mesenteric lymph node. One-way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance as indicated (* p ≤ 0.05).