In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Culicoides Sonorensis and Viruses
2.2. C. sonorensis Infection and Maintenance
2.3. In Situ Hybridization: RNAscope 2.5 HD Chromogenic Duplex Detection
2.4. Image Capture
3. Results
3.1. Identification of C. sonorensis Structures in Hematoxylin-and-Eosin-Stained Slides
3.2. Control Probes Indicate Successful RNAscope Hybridization and Probe Detection
3.3. Detection and Distribution of BTV-10 and BTV-17 in Single Infection Treatment Groups
3.4. Detection and Distribution of BTV-10 and BTV-17 in Co-Infection Treatment Group
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Infection Treatment Group | Detection of BTV-10 Segment 2 Only | Detection Of BTV-17 Segment 2 Only | Detection of BTV-10 and BTV-17 Segment 2 | No Detection |
---|---|---|---|---|
BTV-10 | 2/15 (13.3%) | 0/15 (0%) | 0/15 (0%) | 13/15 (86.7%) |
BTV-17 | 0/13 (0%) | 8/13 (61.5%) | 0/13 (0%) | 5/13 (38.5%) |
BTV-10 and BTV-17 | 1/14 (7.1%) | 3/14 (21.4%) | 3/14 (21.4%) | 7/14 (50.0%) |
BTV-10 Infection Treatment Group | BTV-17 Infection Treatment Group | ||
---|---|---|---|
Detection of BTV-10 | Detection of BTV-17 | ||
Number of Midges with BTV Detection | 2 Out of 15 Midges | 8 Out of 13 Midges | |
Dissemination Pattern | 1 Midge with BTV-10 Dissemination | 1 Midge with BTV-10 Localized to Midgut | 8 Midges with BTV-17 Dissemination |
Tissue | |||
Johnston’s organ | n/a | n/a | 3/3 (++) to (+++) |
ommatidia | n/a | n/a | 7/7 (+++) to (++++) |
cerebral ganglion | n/a | n/a | 7/7 (++) to (+++) (periphery) |
salivary gland | 1/1 (+) | 0/1 | 5/6 (++) to (+++) |
dorsal longitudinal muscle | 1/1 (+) | 0/1 | 5/8 (+) |
tracheal cuticle | n/a | n/a | 8/8 (++) to (+++) |
midgut | 1/1 (++++) | 1/1 (++++) | 6/6 (+++) to (++++) |
ovarian follicles | 0/1 | 0/1 | 0/8 |
follicular epithelium | 1/1 (+++) | 0/1 | 6/8 (++) to (+++) |
fat body | 1/1 (++++) | 0/1 | 8/8 (+++) |
cuticle | 1/1 (+++) | 0/1 | 7/8 (++) to (++++) |
BTV-10 and BTV-17 Infection Treatment Group | ||||
---|---|---|---|---|
Detection of BTV-10 | Detection of BTV-17 | Detection of BTV-10 and BTV-17 | ||
Number of Midges with BTV Detection | 1 Out of 14 Midges | 3 Out of 14 Midges | 3 Out of 14 Midges | |
Dissemination Pattern | 1 Midge with BTV-10 Dissemination | 3 Midges with BTV-17 Dissemination | 2 Midges with BTV-10 and 17 Dissemination | 1 Midge with BTV-17 Dissemination and BTV-10 Localized to Midgut |
Tissue/Organ | ||||
Johnston’s organ | n/a | 1/1 (++) | n/a | n/a |
ommatidia | n/a | 2/2 (++) to (++++) | 2/2 (BTV-10 and 17) (++) to (+++) | n/a |
cerebral ganglion | n/a | 2/2 (++) to (+++) | 2/2 (BTV-10 and 17) (++) to (+++) | n/a |
salivary gland | n/a | 2/2 (+) | 2/2 (BTV-10 and 17) (++) | n/a |
dorsal longitudinal muscle | 1/1 (+) | 3/3 (+) | 2/2 (BTV-10 and 17) (+) | 1/1 (BTV-17 only) (+) |
tracheal cuticle | n/a | 3/3 (++) to (+++) | 2/2 (BTV-10 and 17) (++) | 1/1 (BTV-17 only) (++) |
midgut | 1/1 (+++) | 2/2 (+++) to (++++) | 1/2 (BTV-10 and 17) (+++) to (++++) | 1/1 (BTV-10 and 17) (++++) |
ovarian follicles | n/a | 0/3 | 0/2 | 0/1 |
follicular epithelium | n/a | 3/3 (++) to (+++) | 2/2 (BTV-10 and 17) (++) | 1/1 (BTV-17 only) (+) |
fat body | 1/1 (+++) | 3/3 (++) to (++++) | 2/2 (BTV-10 and 17) (+++) | 1/1 (BTV-17 only) (+++) |
cuticle | 1/1 (+++) | 3/3 (++) to (++++) | 2/2 (BTV-10 and 17) (++) to (+++) | 1/1 (BTV-17 only) (++) |
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Carpenter, M.; Benavides Obon, A.; Kopanke, J.; Lee, J.; Reed, K.; Sherman, T.; Rodgers, C.; Stenglein, M.; McDermott, E.; Mayo, C. In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis. Pathogens 2023, 12, 1207. https://doi.org/10.3390/pathogens12101207
Carpenter M, Benavides Obon A, Kopanke J, Lee J, Reed K, Sherman T, Rodgers C, Stenglein M, McDermott E, Mayo C. In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis. Pathogens. 2023; 12(10):1207. https://doi.org/10.3390/pathogens12101207
Chicago/Turabian StyleCarpenter, Molly, AnaMario Benavides Obon, Jennifer Kopanke, Justin Lee, Kirsten Reed, Tyler Sherman, Case Rodgers, Mark Stenglein, Emily McDermott, and Christie Mayo. 2023. "In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis" Pathogens 12, no. 10: 1207. https://doi.org/10.3390/pathogens12101207
APA StyleCarpenter, M., Benavides Obon, A., Kopanke, J., Lee, J., Reed, K., Sherman, T., Rodgers, C., Stenglein, M., McDermott, E., & Mayo, C. (2023). In Situ Hybridization (RNAscope) Detection of Bluetongue Virus Serotypes 10 and 17 in Experimentally Co-Infected Culicoides sonorensis. Pathogens, 12(10), 1207. https://doi.org/10.3390/pathogens12101207