What have we learned from 7 years of equine rhinitis B virus qPCR testing in nasal secretions from horses with respiratory signs.

BACKGROUND: Equine rhinitis B virus (ERBV) has been given little attention by practitioners compared to other respiratory viruses, mainly because of the lack of diagnostic modalities and association with clinical disease. The objective of the study was to determine the frequency of detection of ERBV in nasal secretions from 6568 horses with acute onset of respiratory signs. METHODS: ERBV-positive qPCR results from nasal secretions submitted to a molecular diagnostic laboratory from 2013 to 2019 were reviewed. RESULTS: A total of 333 ERBV qPCR-positive samples (5.1%) were detected with increasing yearly frequency since the introduction of the assay in 2013. In comparison, only three of 356 (0.8%) healthy horses tested qPCR-positive for ERBV. Median age for ERBV qPCR-positive horses was 3 years of age, and fever, coughing and nasal discharge were the most common signs reported. Further, co-infections with other respiratory pathogens were reported in 73 (21.9%) of ERBV qPCR-positive samples. CONCLUSION: ERBV is a commonly detected respiratory virus from nasal secretions of young horses presenting with fever, nasal discharge and coughing.

Equine rhinitis B viruses in horse fecal samples from the Middle East.

BACKGROUND: Among all known picornaviruses, only two species, equine rhinitis A virus and equine rhinitis B virus (ERBV) are known to infect horses, causing respiratory infections. No reports have described the detection of ERBV in fecal samples of horses and no complete genome sequences of ERBV3 are available. METHODS: We performed a molecular epidemiology study to detect ERBVs in horses from Dubai and Hong Kong. Complete genome sequencing of the ERBVs as well as viral loads and genome, phylogenetic and evolutionary analysis were performed on the positive samples. RESULTS: ERBV was detected in four (13.8 %) of the 29 fecal samples in horses from Dubai, with viral loads 8.28 × 10(3) to 5.83 × 10(4) copies per ml, but none of the 47 fecal samples in horses from Hong Kong by RT-PCR. Complete genome sequencing and phylogenetic analysis showed that three of the four strains were ERBV3 and one was ERBV2. The major difference between the genomes of ERBV3 and those of ERBV1 and ERBV2 lied in the amino acid sequences of their VP1 proteins. The Ka/Ks ratios of all the coding regions in the ERBV3 genomes were all <0.1, suggesting that ERBV3 were stably evolving in horses. Using the uncorrelated lognormal distributed relaxed clock model on VP1 gene, the date of the most recent common ancestor (MRCA) of ERBV3 was estimated to be 1785 (HPDs, 1176 to 1937) and the MRCA dates of ERBV1 and ERBV2 were estimated to be 1848 (HPDs, 1466 to 1949) respectively. CONCLUSIONS: Both acid stable (ERBV3) and acid labile (ERBV2) ERBVs could be found in fecal samples of horses. Detection of ERBVs in fecal samples would have implications for their transmission and potential role in gastrointestinal diseases as well as fecal sampling as an alternative method of identifying infected horses.

Seroprevalence study of Equine rhinitis B virus (ERBV) in Australian weanling horses using serotype-specific ERBV enzyme-linked immunosorbent assays.

Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses.

BACKGROUND: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. RESULTS: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). CONCLUSION: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.

Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.

REASONS FOR PERFORMING STUDY: 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. OBJECTIVES: 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. POTENTIAL RELEVANCE: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.

Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens.

Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duplex PCR was developed for the detection and differentiation of both ERAV and ERBV. The method was evaluated for its ability to detect viral RNA in cell culture supernatants and nasal swabs, and lung and urine spiked with known quantities of virus. The assay demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation (CV%) ranging from 1% to 7.4% and from 1.2% to 12% for intra- and inter-assay variability respectively. The assay detected ERBV in 14 of 86 nasal swabs collected from horses with respiratory disease. The real-time duplex PCR is a useful new diagnostic method for the rapid detection and differentiation of ERAV and ERBV.

Denatured virion protein 1 of equine rhinitis B virus 1 contains authentic B-cell epitopes recognised in an enzyme-linked immunosorbent assay--short communication.

Equine rhinitis B virus 1 (ERBV1), genus Erbovirus, family Picornaviridae, is a pathogen of horses which causes clinical and subclinical infection of the upper respiratory tract in horses. The virus is widespread in European horse populations and the current standard method for the detection of antibody against ERBV1 is by virus neutralisation (VN). VN tests, however, are labour-intensive and time-consuming, require tissue culture facilities, and generally do not provide same-day results. In this study, a protocol for the high-level expression and purification of recombinant virion protein 1 (rVP1) was established using metal-chelate affinity chromatography under denaturing condition. When used as a coating antigen in a prototype enzyme-linked immunosorbent assay (ELISA), denatured rVP1 was recognised by ERBV1 antibody present in horse serum. This finding suggests that denatured rVP1 is a promising candidate for the development of an ELISA to be used in the routine laboratory diagnosis of ERBV1 infection in horses.

Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serology.

OBJECTIVE: To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. METHODS: Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in Australia. A type specific ELISA was used to measure equine herpesvirus (EHV1 and EHV4) antibody, and serum neutralisation assays were used to measure equine rhinitis A virus (ERAV) and equine rhinitis B virus 1 and 2 (ERBV1 and ERBV2) antibody titres in serum samples. RESULTS: Virus was isolated from 4 of 20 nasal swab samples. There were three isolations of EHV4 and one of ERBV2. By PCR, virus was identified in the nasal swab samples of 12 of the 20 horses. Of the 12 horses [corrected] that were positive, 17 viruses were detected as follows: there was [corrected] one triple positive (EHV4, EHV2, and EHV5), three double positives (EHV4, ERBV and EHV5, ERBV (2 horses)) and 8 [corrected] single positives (EHV4 (2 horses), EHV5 (3 horses) and ERBV (3 [corrected] horses). CONCLUSION: By virus isolation and PCR, 17 viruses were identified in nasal swab samples from 12 of 20 horses that had acute febrile respiratory disease consistent with a diagnosis of virus infection. Initial PCR identification and subsequent virus isolation led to the isolation of ERBV2 for the first time in Australia and the second time anywhere of ERBV2.

Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus.

Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae occurs as two serotypes, ERBV1 and ERBV2. An ERBV-specific nested reverse transcriptase-polymerase chain reaction (RT-PCR) that amplified a product within the 3D(pol) and 3' non-translated region of the viral genome was developed. The RT-PCR detected all 24 available ERBV1 isolates and one available ERBV2 isolate. The limit of detection for the prototype strain ERBV1.1436/71 was 0.1 50% tissue culture infectious doses. The RT-PCR was used to detect viral RNA in six of 17 nasopharyngeal swab samples from horses that had clinical signs of acute febrile respiratory disease but from which ERBV was not initially isolated in cell culture. The sequences of these six ERBV RT-PCR positive samples had 93-96% nucleotide identity with six other partially sequenced ERBV1 isolates and one ERBV2. ERBV was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of 20 mg/mL MgCl(2) to the cell culture medium enhanced the growth of the virus. This isolated virus was antigenically similar to ERBV2.313/75. Determination of the nucleotide sequence of the P1 region of the genome also indicated that the isolate was ERBV2, and it was therefore designated ERBV2.1576/99. This is the first reported isolation of ERBV in Australia. The study highlights the utility of PCR for the identification of viruses in clinical samples that may initially be considered negative by conventional cell culture isolation.