Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses.
Abstract: To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results: The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. Conclusions: Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RT-PCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.
Publication Date: 2004-04-16 PubMed ID: 15084017DOI: 10.1111/j.1751-0813.2003.tb11438.xGoogle Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research focuses on the development and validation of diagnostic tests for detecting infections caused by Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) in horses. These tests use the method of reverse transcription polymerase chain reaction (RT-PCR) for their identification and have showcased specificity and sensitivity.
Research Methods
- The researchers devised primer sets based on the nucleotide sequence that encodes the envelope glycoprotein E2 of RRV and the nonstructural protein 5 (NS5) of KV and MVEV.
- The primer sets were used in single round polymerase chain reactions to test for the presence of these viruses in infected cell cultures, horse blood, and synovial fluid samples.
- The tests were applied to detect the presence of RRV in horses which showed clinical symptoms aligned with RRV infection.
Research Results
- The outcomes indicated that the primer pairs developed for each of the three viruses were able to amplify a product of expected size from prototype viruses grown in cell culture.
- Nucleotide sequencing validated the identity of each of the products, confirming the RT-PCRs were specific to each virus.
- Test sensitivity was demonstrated through the successful identification of RRV in the blood samples of 8 horses showing symptoms consistent with the virus. It detected as little as 50 TCID50 of RRV per milliliter of serum.
- No cross-reactions were noted, confirming the specificity of the tests. The primer pairs did not confuse or misinterpret other products present in the blood samples as the virus being tested for.
- The RRV primers also detected the virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture.
- However, due to the unavailability of horses suspected to be infected with KV and MVEV, validation of tests for those viruses could not be completed.
Research Conclusions
- The research showed that the developed RT-PCR tests were specifically effective for detecting RRV, thus enhancing the ability to confirm the presence of the virus in clinical samples.
- Though the RT-PCR primers showed specificity for diagnosing KV and MVEV infections in cell cultures, further validation for these tests requires the availability of the appropriate clinical samples from horses infected with these viruses.
Cite This Article
APA
Studdert MJ, Azuolas JK, Vasey JR, Hall RA, Ficorilli N, Huang JA.
(2004).
Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses.
Aust Vet J, 81(1-2), 76-80.
https://doi.org/10.1111/j.1751-0813.2003.tb11438.x Publication
Researcher Affiliations
- Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010.
MeSH Terms
- Alphavirus Infections / diagnosis
- Alphavirus Infections / veterinary
- Amino Acid Sequence
- Animals
- DNA Primers
- Encephalitis Virus, Murray Valley / genetics
- Encephalitis Virus, Murray Valley / isolation & purification
- Encephalitis, Arbovirus / diagnosis
- Encephalitis, Arbovirus / veterinary
- Horse Diseases / diagnosis
- Horses
- Molecular Sequence Data
- RNA, Viral / analysis
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Ross River virus / genetics
- Ross River virus / isolation & purification
- Sensitivity and Specificity
- Sequence Alignment
- West Nile Fever / diagnosis
- West Nile Fever / veterinary
- West Nile virus / genetics
- West Nile virus / isolation & purification
Citations
This article has been cited 4 times.- Yuen KY, Bielefeldt-Ohmann H. Ross River Virus Infection: A Cross-Disciplinary Review with a Veterinary Perspective. Pathogens 2021 Mar 17;10(3).
- Stephenson EB, Peel AJ, Reid SA, Jansen CC, McCallum H. The non-human reservoirs of Ross River virus: a systematic review of the evidence. Parasit Vectors 2018 Mar 19;11(1):188.
- Tan CH, Wong PS, Li MZ, Yang HT, Chong CS, Lee LK, Yuan S, Leo YS, Ng LC, Lye DC. Membrane feeding of dengue patient's blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction. Parasit Vectors 2016 Apr 15;9:211.
- Fu JYL, Chua CL, Abu Bakar AS, Vythilingam I, Wan Sulaiman WY, Alphey L, Chan YF, Sam IC. Susceptibility of Aedes albopictus, Ae. aegypti and human populations to Ross River virus in Kuala Lumpur, Malaysia. PLoS Negl Trop Dis 2023 Jun;17(6):e0011423.
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