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Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.
Abstract: Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope(®) and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope(®)) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis.
Publication Date: 2016-08-19 PubMed ID: 27541817DOI: 10.1007/s00705-016-3014-5Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Evaluation Study
- Journal Article
Summary
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This article outlines the development of two tests for detecting the equine arteritis virus (EAV) in tissues from aborted horse fetuses, and compares their performance against widely-used techniques. The researchers found that one of the new tests had comparable sensitivity to the existing methods, and will be invaluable in investigating how EAV causes disease.
Development of RNA in situ hybridization Assays
- The research focused on the creation of two different RNA in situ hybridization (ISH) assays. These assays are designed to spot the presence of equine arteritis virus (EAV) RNA in formalin-fixed paraffin-embedded (FFPE) tissues.
- The EAV is responsible for equine viral arteritis which causes respiratory and reproductive disorders in equids. It can trigger abortion in pregnant mares and induce fulminant bronchointerstitial pneumonia in foals.
Comparison with Existing Techniques
- The team compared the performance of the newly designed ISH assays with virus isolation (VI) techniques, which are considered as the gold standard.
- The team also compared the tests with a technique known as nucleocapsid-specific immunohistochemistry (IHC). This method detects the presence of virus proteins in a tissue sample.
- Testing was performed on 80 FFPE tissue samples collected from 16 aborted fetuses infected with the EAV.
Assessment of the RNA in situ hybridization Assays
- The team found that the performance of the conventional RNA ISH assay was significantly less sensitive compared to the newly developed RNAscope® ISH and the traditional IHC assay.
- On the other hand, the RNAscope® assay’s sensitivity was comparable to that of the IHC assay.
- This indicates that the RNAscope® ISH assay is a highly sensitive technique for detecting the presence of EAV RNA in FFPE tissues.
Implications of the Study
- The study demonstrates that the use of oligonucleotide probes along with a signal amplification system (in this case, RNAscope®) can dramatically enhance the detection of EAV RNA in FFPE tissues.
- Notably, it shows that the sensitivity of such a system is similar to that of IHC techniques.
- The availability of these assays offers the potential for better understanding the mechanisms of EAV pathogenesis, thus providing valuable tools for EAV disease research.
Cite This Article
APA
Carossino M, Loynachan AT, James MacLachlan N, Drew C, Shuck KM, Timoney PJ, Del Piero F, Balasuriya UB.
(2016).
Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.
Arch Virol, 161(11), 3125-3136.
https://doi.org/10.1007/s00705-016-3014-5 Publication
Researcher Affiliations
- 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
- University of Kentucky Veterinary Diagnostic Laboratory, University of Kentucky, Lexington, KY, USA.
- Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA.
- Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA.
- 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
- 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
- Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
- 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA. ubalasuriya.@uky.edu.
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Equartevirus / genetics
- Equartevirus / isolation & purification
- Female
- Fetus / virology
- Horse Diseases / diagnosis
- Horses
- Immunohistochemistry
- In Situ Hybridization / methods
- Molecular Diagnostic Techniques / methods
- RNA, Viral / analysis
- RNA, Viral / genetics
- Sensitivity and Specificity
- Virology / methods
Citations
This article has been cited 13 times.- Gao W, Liu M, Nurdaly K, Caidan D, Sun Y, Duan J, Zhao J, Gong X, Zhou J, Zhang Y, Chen Q. Unveiling Equine Abortion Pathogens: A One Health Perspective on Prevalence and Resistance in Northwest China. Pathogens 2025 Dec 11;14(12).
- Gaide N, Crispo M, Jbenyeni A, Bleuart C, Delverdier M, Vergne T, Le Loc'h G, Guérin JL. Validation of an RNAscope assay for the detection of avian influenza A virus. J Vet Diagn Invest 2023 Sep;35(5):500-506.
- Carossino M, Del Piero F, Lee J, Needle DB, Levine JM, Riis RR, Maes R, Wise AG, Mullaney K, Ferracone J, Langohr IM. Relationship between Uveal Inflammation and Viral Detection in 30 Cats with Feline Infectious Peritonitis. Pathogens 2022 Aug 5;11(8).
- Mungenast F, Fernando A, Nica R, Boghiu B, Lungu B, Batra J, Ecker RC. Next-Generation Digital Histopathology of the Tumor Microenvironment. Genes (Basel) 2021 Apr 7;12(4).
- De Biase D, Prisco F, Piegari G, Ilsami A, d'Aquino I, Baldassarre V, Zito Marino F, Franco R, Papparella S, Paciello O. RNAScope in situ Hybridization as a Novel Technique for the Assessment of c-KIT mRNA Expression in Canine Mast Cell Tumor. Front Vet Sci 2021;8:591961.
- Giessler KS, Samoilowa S, Soboll Hussey G, Kiupel M, Matiasek K, Sledge DG, Liesche F, Schlegel J, Fux R, Goehring LS. Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses. Front Vet Sci 2020;7:621.
- Carossino M, Ip HS, Richt JA, Shultz K, Harper K, Loynachan AT, Del Piero F, Balasuriya UBR. Detection of SARS-CoV-2 by RNAscope(®) in situ hybridization and immunohistochemistry techniques. Arch Virol 2020 Oct;165(10):2373-2377.
- Costigliolo F, Lombardo K, Arend LJ, Rosenberg AZ, Matoso A, Carter-Monroe N, Bagnasco SM. BK Virus RNA in Renal Allograft Biopsies. J Histochem Cytochem 2020 May;68(5):319-325.
- Liu D, Tedbury PR, Lan S, Huber AD, Puray-Chavez MN, Ji J, Michailidis E, Saeed M, Ndongwe TP, Bassit LC, Schinazi RF, Ralston R, Rice CM, Sarafianos SG. Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus. Viruses 2019 Nov 8;11(11).
- Carossino M, Dini P, Kalbfleisch TS, Loynachan AT, Canisso IF, Cook RF, Timoney PJ, Balasuriya UBR. Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis. PLoS Pathog 2019 Jul;15(7):e1007950.
- Munganyinka E, Margaria P, Sheat S, Ateka EM, Tairo F, Ndunguru J, Winter S. Localization of cassava brown streak virus in Nicotiana rustica and cassava Manihot esculenta (Crantz) using RNAscope® in situ hybridization. Virol J 2018 Aug 14;15(1):128.
- Carossino M, Dini P, Kalbfleisch TS, Loynachan AT, Canisso IF, Shuck KM, Timoney PJ, Cook RF, Balasuriya UBR. Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus. J Virol 2018 May 1;92(9).
- Carossino M, Loynachan AT, Canisso IF, Cook RF, Campos JR, Nam B, Go YY, Squires EL, Troedsson MHT, Swerczek T, Del Piero F, Bailey E, Timoney PJ, Balasuriya UBR. Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8(+) T and CD21(+) B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract. J Virol 2017 Jul 1;91(13).
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