Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.
BACKGROUND
Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection.


OBJECTIVE
To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV.


METHODS
TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT-PCR were verified by virus isolation and ERBV positive samples were verified by rRT-PCR using a different set of primers.


RESULTS
The detection limit of the rRT-PCR for both viruses was 10-100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co-circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT-PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp).


CONCLUSIONS
The rRT-PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV.


CONCLUSIONS
These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.
Virology Unit, The Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland.
M
M
Quinlivan
G
G
Maxwell
P
P
Lyons
S
S
Arkins
A
A
Cullinane
D000818:Animals; D005537:Aphthovirus / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002460:Cell Line; D052698:Erbovirus / Q000235:genetics / Q000302:isolation & purification*; D006734:Horse Diseases / Q000821:virology*; D006736:Horses; D010850:Picornaviridae Infections / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
D012367:RNA, Viral