Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses.
OBJECTIVE
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.
Studdert
M J
MJ
Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010.
Azuolas
J K
JK
Vasey
J R
JR
Hall
R A
RA
Ficorilli
N
N
Huang
J-A
JA
D018354:Alphavirus Infections / Q000175:diagnosis / Q000662:veterinary; D000595:Amino Acid Sequence; D000818:Animals; D017931:DNA Primers; D018068:Encephalitis Virus, Murray Valley / Q000235:genetics / Q000302:isolation & purification; D004671:Encephalitis, Arbovirus / Q000175:diagnosis / Q000662:veterinary; D006734:Horse Diseases / Q000175:diagnosis*; D006736:Horses; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012398:Ross River virus / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014901:West Nile Fever / Q000175:diagnosis / Q000662:veterinary; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D012367:RNA, Viral