Introduction

Equid herpesvirus (EHV) 1 and -4 are highly contagious and ubiquitous pathogens that commonly cause fever and respiratory disease in horses. EHV-1 can achieve systemic distribution and in severe cases, results in abortion or neurological disease known as equine herpesviral myeloencephalopathy (EHM), while EHV-4 infection and symptoms are usually limited to the upper respiratory tract1,2. Generally, EHV-1 causes more severe disease than EHV-4, although the prevalence of EHV-4 is higher3,4,5. In recent PCR-based surveillance studies of horses presenting with fever and/or respiratory signs, EHV-1 was detected in 0.7 to 3.0% of the cases, contrasting with EHV-4 which was detected in 6.6 to 10.8% of cases3,4,5. Retrospective studies of infectious abortions in horses revealed that EHV-1 caused 3–6% of infectious abortions in Australia, United Kingdom, and United States, and up to 26% in Poland6,7,8,9, whereas EHV-4 was associated with only 0.3% of the abortions in the United Kingdom8. Outbreaks of encephalomyelitis and/or abortion in horses due to EHV-1 infection have been reported in the United States, Europe, Ethiopia, New Zealand, and China10,11,12,13,14,15,16,17. A few outbreaks have been attributed to EHV-4; those that have been reported describe respiratory disease of horses and donkeys in Denmark, Germany, and Romania18,19,20.

EHV-1 and EHV-4 are members of the subfamily Alphaherpesvirinae and genus Varicellovirus, along with EHV-3, EHV-6, EHV-8, and EHV-9 according to the International Committee on Taxonomy of Viruses21. EHV-3, also known as equine coital exanthema, is distinct from EHV-1 and EHV-4 in that it causes mucocutaneous disease and is spread through venereal transmission rather than causing respiratory or neurological symptoms or abortion22. EHV-6, EHV-8, and EHV-9 were not initially isolated from horses21. EHV-6 was initially named asinine herpes virus (AHV) 1 because it was the first herpesvirus isolated from a donkey23,24. EHV-8 (previously referred to as AHV-3) was initially isolated from a donkey presenting respiratory symptoms; additional cases of respiratory disease, viral encephalitis, or abortion have been reported in donkeys and horses23,24,25,26,27,28. Notably, two EHV-8 abortion cases in horses were misdiagnosed as EHV-1 due to PCR cross-reactivity28. EHV-9 was first isolated from a captive gazelle and hence named gazelle herpesvirus 1, subsequently it has been shown to be pathogenic in horses29,30.

EHV-1 infection of non-equid hosts including polar bears, black bears, Thomson’s gazelles, and guinea pigs in zoo settings manifested as neurological disease and often resulted in death of affected animals31. Occasionally equid herpesvirus species recombine with each other or with “zebra EHV-1”, which can lead to disease and death in unexpected species32,33.

Control of EHV-1 and EHV-4 is difficult in part because foals can be infected by two weeks of age, regardless of vaccination protocols34. Also, EHV-1 and EHV-4 establish latency after infection, consistent with other alphaherpesviruses, creating a reservoir of virus that can reactivate from latency and transmit to other susceptible animals35,36,37. Notably, a study of 70 routine necropsies following musculoskeletal injuries in horses identified latent EHV-1 infection in 25.7% of horses and latent EHV-4 in 82.8% 41.

Clinical symptoms are shared among respiratory infections caused by equine viral and bacterial pathogens, necessitating specific and sensitive diagnostic assays4. Rapid diagnosis of EHV-1 and EHV-4 is imperative considering their highly infectious nature and potential for severe disease outbreaks. Real-time PCR-based diagnostics are the recommended method for direct detection of EHV-1 infection when respiratory symptoms are present and to manage quarantines and biosecurity during outbreaks10,39,40. Likewise, the World Organization for Animal Health (WOAH)) identified PCR as the standard method of EHV-1 and EHV-4 detection followed by virus isolation41.

Here we describe a multiplex real-time PCR assay that was developed and validated to simultaneously test for EHV-1 and EHV-4. The built-in equid MC1R assay provides an endogenous extraction control to detect equid DNA in samples and verify sample and species integrity. An exogeneous extraction control - the bacteriophage MS2 - is also included as an additional quality control check to monitor assay performance.

Materials and methods

Real-time PCR assay design

De novo real-time PCR assays targeting the EHV-1 glycoprotein B (gB) gene ORF33, EHV-4 gB ORF33, and the equid MC1R gene (Table 1) were designed with Primer3 web service42, https://bioinfo.ut.ee/primer3/ and Primer3 2.3.7 in Geneious Prime 2023.1.2 to enable specificity testing (Biomatters, Inc., Boston, MA). A total of 135 EHV-1 and 50 EHV-4 sequences available in the NCBI nucleotide database on June 6, 2022, as well as 12 EHV-1 sequences determined at the Virology Laboratory at the Cornell Animal Health Diagnostic Center (AHDC), were used to build sequence alignments and identify regions with high sequence conservation. Representative gB sequences from EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, EHV-6, EHV-8, and EHV-9 (NC_001491, HQ247739, NC_024771, NC_001844, AF050671, MT012704, MF431611 and MW822570, and NC_011644, respectively) were analyzed to confirm optimal specificity.

Table 1 Equid herpesvirus 1 and 4 multiplex real-time PCR assay primer and probe sequences.
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MC1R sequences were obtained from the NCBI nucleotide database on June 3, 2022 from the following equids and aligned to identify conserved regions: horse (Equus caballus, n = 21), Przewalski’s horse (Equus przewalskii, n = 2), donkey (Equus asinus, n = 6), kiang (Equus kiang, n = 2), onager (Equus hemionus, n = 3), and zebra (Equus burchelli, Equus grevyi, Equus zebra, n = 5). The forward and reverse primers and the probe are identical to these 39 sequences except for one nucleotide mismatch in the probe sequence compared to the 2 Equus grevyi sequences at position 7 out of 24 from the 5’ end (Supplementary Figure S1). The equid MC1R real-time PCR assay primers and probe are within a single exon of the MC1R gene which enables detection of either genomic DNA or mRNA. In addition to the MC1R assay, an exogenous extraction control real-time PCR assay targeting the Escherichia coli bacteriophage MS2 was also included our multiplex assay43. Primers were synthesized by Thermo Fisher Scientific, Inc., Waltham, MA. Hydrolysis probe dyes were selected to facilitate multiplexing (synthesized by Sigma-Aldrich, St. Louis, MO, or Thermo Fisher Scientific).

Viral and bacterial isolates, positive amplification controls, and clinical samples

Well-characterized EHV-1 and EHV-4 isolates that are used in routine diagnostic testing in the Animal Health Diagnostic Center (AHDC) Virology laboratory and have been confirmed by whole genome sequencing were used to characterize the assay performance. The EHV-1 strain Ab4 (99.94% identical to GenBank accession AY665713.1) and the EHV-4 strain NS80567 (99.9% identical to GenBank accession AF030027.1) were used. A panel of characterized viral (n = 16) and bacterial (n = 12) equine respiratory isolates were used to evaluate assay specificity (Supplementary Table S1).

Synthetic positive amplification controls (PACs) were designed for each assay containing the target sequence region within a 395–400 base pairs DNA fragment (gBlocks, Integrated DNA Technologies, Coralville, IA). Synthetic fragments of EHV-8 and EHV-9 gB regions were also obtained, based on GenBank NC_075566 and NC_011644 sequences, respectively (Supplementary Table S2).

A plasmid containing 200 bases of the MS2 TM2 region from GenBank nucleotide sequence V00642 was synthesized and cloned into pUC57 by GenScript USA, Inc (Piscataway NJ) to serve as an exogenous control.

Residual diagnostic samples submitted for testing at the AHDC Molecular Diagnostic Laboratory at Cornell University between January 18, 2019 and April 4, 2023 and archived at −80 °C were used to evaluate the diagnostic sensitivity and specificity of the EHV1-4MP assay. A panel of 60 samples was selected to include 30 for which EHV-1 or EHV-4 was detected (15 EHV-1 positive plus 15 EHV-4 positive) and another 30 for which neither EHV-1 nor EHV-4 were detected (Supplementary Table S3). An equine respiratory pathogen other than EHV-1 or EHV-4 was detected in 25 of the latter 30 samples, including EHV-2, EHV-5, equine adenovirus 1 (EAdV1), equine rhinitis A virus (ERAV), equine rhinitis B virus (ERBV), equine influenza virus (EIV), Streptococcus equi sbsp equi (S. equi equi), Streptococcus equi sbsp zooepidemicus, or Mycoplasma felis. The panel included 8 different specimens (nasal exudate, nasal/pharyngeal swab, whole blood, nasal/pharyngeal wash, tracheal wash, placenta, brain, and lung) collected from 21 horse breeds plus a mule and a miniature donkey, with ages spanning from an 8-month aborted fetus to 22 year-old adult horse. Samples originated from 24 states (AR, CT, DE, FL, GA, ID, IL, MA, MD, MN, ND, NH, NJ, NV, NY, OH, OR, PA, SC, TX, VA, VT, WA, WI). To further evaluate diagnostic specificity, 16 donkey and mule samples that previously tested positive for EHV-1 using the assay from Elia and co-workers44 or a pan-herpesviral species PCR45 were tested. Thirty samples used to characterize the equid MC1R assay specificity are listed in Supplementary Table S4.

Nucleic acid extraction and real-time PCR

Nucleic acid extraction was performed with the MagMAX CORE Nucleic Acid Purification Kit following the simple workflow described by the manufacturer and using the KingFisher Flex (Thermo Fisher Scientific) automated magnetic extractor. Nucleic acid was eluted in 100 µL nuclease-free water. The MS2 plasmid was added to the extraction lysis/binding solution to serve as an exogenous DNA extraction control. For viral isolates, nucleic acid was extracted from the supernatant of infected cells after three rounds of freeze/thawing.

Real-time PCR was optimized and validated using the SensiFAST™ Probe No-ROX Kit (Meridian Bioscience, Memphis, TN) and 7 µL of nucleic acid in a total reaction volume of 25 µL. Real-time PCR was performed on ABI 7500 Fast instruments with cycling conditions of 95 °C for 5 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 50 s, as recommended by the PCR kit manufacturer. The results of the multiplex real-time PCR run were analyzed with 7500 software v2.3 (Life Technologies Corp, Thermo Scientific).

Analytical assay performance evaluation

Analytical sensitivity was evaluated with ten-fold serial dilutions of EHV-1 and EHV-4 viral isolates and synthetic PACs (Integrated DNA Technologies, Coralville, IA) for each assay target so that the limit of detection could be quantified using digital PCR and expressed in copy numbers.

Digital PCR was used to quantify EHV-1 and EHV-4 genome copy numbers in the isolate and PACs serial dilutions using the singleplex EHV1g1775, EHV1gB444, and EHV4gB assays. Absolute Q™ Universal DNA Digital PCR Master Mix (Thermo Fisher Scientific) was combined with 900 nM primers and 250 nM probe (Table 1) and 3.5 µL nucleic acid in a total volume of 9 µL. Cycling conditions were 96 °C for 10 min followed by 40 cycles of 96 °C for 5 s and 60 °C for 15 s on a QuantStudio Absolute Q digital PCR system with software version 6.3.5 (Thermo Fisher Scientific). EHV-1 and EHV-4 assays were tested individually because the Absolute Q cannot analyze an assay containing five different probes. The combined PACs were tested in triplicate digital PCR wells, providing data for at least 61,387 microchambers per assay.

The multiplex assay performance was evaluated with competition experiments performed with 4 replicates each: (1) an EHV-1 isolate was serially diluted in the presence of an EHV-4 isolate at a high concentration, (2) an EHV-4 isolate was serially diluted in the presence of an EHV-1 isolate at a high concentration, and (3) combined EHV-1 and EHV-4 isolates serially diluted. Dilutions were performed prior to nucleic acid extraction and the same nucleic acid elutions were tested side-by-side with the singleplex and multiplex versions of the assays. The EHV-1 and EHV-4 genome copy number in these serial dilution series was quantified using the digital PCR as described above. Analytical specificity of each assay was evaluated in silico with PrimerTree to perform Primer-BLAST, on November 1, 202446.

Diagnostic assay performance evaluation

Results of the EHV1-4MP assay were compared to results of previously published singleplex EHV-1 and EHV-4 real-time PCR assays44,47 which are currently used at the Molecular Diagnostics Laboratory at the AHDC to evaluate diagnostic sensitivity and specificity.

Historical diagnostic testing results

Results of EHV-1, EHV-4, and equine respiratory pathogen panel testing at the Molecular Diagnostics Laboratory at the AHDC from 2011 to December 31, 2024 were downloaded from the laboratory information management system (LIMS) VetView (version 4.0.9.9; University of Georgia, Athens, GA) on January 7, 2025 using Tableau Software Server Version: 2022.3.6 (20223.23.0507.0956) (Salesforce Inc, Seattle, WA). The equine respiratory pathogen panel includes real-time PCR tests for EAdV1, equine adenovirus 2 (EAdV2), ERAV, ERBV, EIV, equine arteritis virus (EAV), and S. equi equi in addition to EHV-1 and EHV-4. Submissions identified for research purposes were excluded from this analysis. This allowed us to identify a panel of samples known to be positive for other common equine pathogens to determine the diagnostic specificity of our newly developed EHV1-4MP assay.

Statistics

GraphPad Prism 10.0.0.153 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com was used for analysis of standard curve data by linear regression, measuring the difference between slopes of serial dilutions by analysis of covariance, for 2 × 2 test agreement, and to generate plots.

Results

Equid herpesvirus target selection. The glycoprotein B (gB) was selected as an equid herpesvirus real-time PCR assay target because it allows efficient differentiation between EHV-1, EHV-4 and other equid herpesviruses48,49. Alignment of the full-length gB gene sequences of EHV-1 revealed 97.42 to 100% nucleotide identity, with 114 single nucleotide polymorphisms (SNPs) among the 2,943 nucleotide coding sequence. Alignment of 28 EHV-4 full-length gB gene sequences available in GenBank revealed 99.80 to 100% nucleotide identity, with only 12 SNPs among the 2,928 nucleotide coding sequence. Alignment of gB sequences from EHV-1 (NC_001491.2) and EHV-4 (NC_001844.1) reference genomes yielded 82.9% nucleotide identity with 500 SNPs.

The EHV-1 gB assay (EHV1gB444) was designed such that the 3’ end of both primers and probe were anchored on nucleotide positions that differed from EHV-8 and EHV-9. However, this assay still detected EHV-8 and EHV-9 synthetic fragments. Despite additional attempts, we were not able to design a real-time PCR assay in the gB gene that only detected EHV-1 because of the high sequence identity between EHV-1, EHV-8, and EHV-9 (93.2% identical sites, 95.4% pairwise identity). We then used the entire EHV-1 genome sequence, performed an alignment and identified regions that could potentially enable exclusion of EHV-8 and EHV-9. Analysis and comparison of the all EHV-1, EHV-8 and EHV-9 sequences available on public databases led to the design of an assay targeting the EHV-1 genome just upstream the ORF1 coding sequence (EHV1g1775 real-time PCR assay).

Real-time PCR assay in silico specificity assessment.

The EHV1gB444 forward and reverse primer pair were evaluated for specificity in silico using NCBI nucleotide database. The EHV-1 gB primers were 100% identical to 143 Equid alphaherpesvirus 1 sequences (Supplementary Table S5) and were predicted to generate an 184 bp amplicon, as expected. Predicted off-target hits included 8 species of Schistosoma and one Marasmarcha lunaedactyla sequence (Supplementary Table S5). All hits used the forward primer at both ends of the amplicon with 2–3 mismatches and generated amplicons ranging from 354 to 3,965 base pairs. To verify assay specificity, another query was performed using the forward primer and probe using PrimerTree. The EHV-1 gB forward primer and probe pair were 100% identical to the same 143 Equid alphaherpesvirus 1 sequences and were predicted to generate a 64 bp amplicon, as expected; no Schistosoma or Marasmarcha hits were identified with this search. The EHV1g1775 forward and reverse primer pair was evaluated for specificity in silico using NCBI nucleotide database. All 162 hits (Supplementary Table S5) were EHV-1 sequences and 160/162 generated the expected amplicon size. No off-target hits were identified.

EHV-4 gB primer specificity was assessed in silico using the NCBI nucleotide database. The EHV-4 gB forward and reverse primers were 100% identical to 30 Equid alphaherpesvirus 4 sequences (Supplementary Table S5) and were predicted to generate an 82 bp amplicon, as expected. No off-target hits were identified.

Equid MC1R assay primer specificity was assessed in silico using the NCBI nucleotide database. The equid MC1R assay forward and reverse primers match identical sequences from the following 8 equid species and are predicted to generate a 73 bp amplicon: Equus asinus, Equus caballus, Equus grevyi, Equus hemionus, Equus kiang, Equus przewalskii, Equus quagga burchellii, and Equus zebra (Supplementary Table S5). Sequences from several other species were identified as possible hits but all have 3 to 7 mismatches with the equid MC1R assay primers and/or probe and the position of many of these mismatches are likely to hinder amplification. These species include tapir (3 mismatches), rhinoceros (5 mismatches), beaver (5 mismatches), dolphin (5 mismatches), squirrel (6 mismatches), lynx (6 mismatches), and pig (7 mismatches) (Supplementary Table S5). Tapirs, rhinoceroses, and horses all belong to the taxonomic order Perissodactyla.

Analytical assay performance

The performance of each singleplex assay was characterized by testing four replicate serial dilutions of a well-characterized viral isolate or equine control on each of three days. All assays demonstrated acceptable performance based on slope, amplification efficiency, and linearity (Table 2). Intra- and inter-assay variation levels were also evaluated and found to be acceptable.

Table 2 Singleplex assay performance on replicate serial dilutions.
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The multiplex EHV1-4MP assay performance was compared to singleplex assay performance with 3 experiments: (1) serial dilution of individual EHV-1 or EHV-4 isolate, (2) serial dilution of the target EHV isolate in the presence of the competing EHV isolate maintained at a high concentration, and (3) serial dilution of combined EHV-1 and EHV-4 isolates. After four replicate serial dilutions were generated and extracted, multiplex and singleplex real-time PCR were performed. Assay performance and sensitivity were comparable between singleplex and multiplex formats when the EHV-1 or EHV-4 isolate was diluted in the absence or presence of a strong competitor (Fig. 1) and when EHV-1 and EHV-4 isolates were combined before dilution. The slopes generated from amplification of dilution series were analyzed with simple linear regression and compared using analysis of covariance. Slopes did not differ between singleplex and multiplex assays for EHV-1g1775 (p = 0.6956), EHV-1 gB (p = 0.5922), or EHV-4 gB (p = 0.8747).

Fig. 1
figure 1

Performance of EHV1-4MP real-time PCR assay in presence of strong competitor. Copy number of isolate shown on x-axis and Ct value shown on y-axis. A) Performance of the EHV1-4MP assay on a serial dilution of EHV-1 isolate in presence of high concentration EHV-4 isolate competitor, B) Performance of the EHV1-4MP assay on a serial dilution of EHV-4 isolate in presence of high concentration EHV-1 isolate competitor, C through E) comparison of Ct values determined by multiplex assay (filled symbols) and singleplex assay (open symbols). Genome copy numbers of EHV-1 and EHV-4 in the serial dilution series were determined by digital PCR.

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Limit of detection

The limit of detection (LoD) of each assay was initially evaluated with four replicates of 10-fold serial dilutions of the synthetic PACs combined in equal copy numbers, ranging from 106 to 0.1 copies of each PAC per reaction. This identified the preliminary LoD to be 10 copies per reaction, based on the lowest copy number for which all replicates were detected. The final LoD was defined by detection of ≥ 19 of 20 replicates (95%) of the combined PACs. The equid MC1R assay detected 20/20 replicates of 10 copies per reaction. Digital PCR was then used to precisely quantify the EHV1g1775, EHV1gB444, and EHV4gB targets present in the serial dilution series of the PACs. The final limit of detection for the EHV1g1775 assay was approximately 2 copies, the EHV1gB444 assay detected ~ 5 copies, and the EHV-4 assay detected ~ 4 copies (Table 3).

Table 3 Limit of detection for EHV1-4MP real-time PCR assay.
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Analytical specificity

Analytical specificity of the EHV1-4MP assay was assessed by performing real-time PCR on a panel of characterized equine viral (n = 16) and bacterial (n = 12) respiratory isolates. Because EHV-8 and EHV-9 isolates were not available, synthetic DNA fragments were used to evaluate specificity in vitro. All EHV-1 isolates were detected by EHV-1 assays, and all EHV-4 isolates were detected by both EHV-4 assays (Table 4). No off-target amplification was detected by the EHV1g1775 (CY5) assay in the EHV1-4MP. The published EHV-1 assay44 and the EHV1gB444 assay (FAM) in the EHV1-4MP detected EHV-8 and EHV-9 templates, although the EHV1gB444 Ct values were 5–6 cycles later. The previously published EHV-4 assay47 detected the EHV-3 isolate.

Table 4 Analytical specificity of EHV1-4MP assay.
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Diagnostic sensitivity and specificity

Diagnostic sensitivity and specificity of the EHV1-4MP assay was evaluated on a panel of 60 residual samples (30 EHV-1 or EHV-4 positive and 30 EHV-1 or EHV-4 negative samples) submitted for diagnostic testing at the AHDC. The tested samples differed regarding specimen type, animal characteristics such as breed, age, and sex, geographic location, and the presence of other equine respiratory pathogen(s) (Supplementary Table S3). All 30 EHV-1 and EHV-4 positive samples were detected by the EHV1-4MP and respective singleplex published real-time PCR assays44,47 and the 30 negative samples were not detected, yielding estimates of 100% diagnostic sensitivity and 100% diagnostic specificity (Figs. 2A, B). Twenty-five of the samples that did not harbor EHV-1 or EHV-4 were positive for one or more of the following equine pathogens: EHV-2, EHV-5, EAdV1, ERAV, ERBV, EIV, S. equi equi, S. zooepidemicus, or Mycoplasma felis. The EHV1-4MP assay also provided equid MC1R Ct values from 16.34 to 35.42 (Fig. 2C). The exogenous extraction control MS2 DNA was detected in 56 of 60 samples with Ct values 32.75 to 38.77; the other 4 samples had weak or no MS2 amplification but detected the equid MC1R control (Ct values ≤ 22.6) (Fig. 2C).

Fig. 2
figure 2

Diagnostic sensitivity and specificity of the EHV1-4MP real-time PCR assay. EHV1-4MP assay results for 60 clinical samples were compared to results of the published singleplex EHV-1 and EHV-4 assays. (A) EHV1g1775 Ct values are plotted on the left y-axis and represented by teal circles. EHV1gB444 Ct values are plotted on the right y-axis and represented by black circles. The previously published EHV-1 assay44 Ct values are plotted on the x-axis. (B) EHV-4 Ct values determined by the EHV1-4MP assay are plotted on the y-axis and are represented by pink circles. The previously published EHV-4 assay47 Ct values are plotted on the x-axis. Not detected results were assigned Ct value of zero. The gray line shows the line of identity. (C) EHV1-4MP control assay Ct values are plotted on the y-axis with equid MC1R Ct values represented by purple circles and MS2 DNA exogenous extraction control Ct values represented by light blue circles. The 60 samples are plotted on the x-axis.

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EHV1-4MP specificity on herpesviruses carried by donkeys

To further characterize the specificity of the EHV1-4MP assay, 16 donkey and mule samples that previously tested positive for EHV-1 using the assay from Elia and co-workers44 or a pan-herpesviral species PCR45 were tested (Table 5). Three of the four specimens detected by the published EHV-1 real-time PCR were also detected by the EHV1gB444 assay but not by EHV1g1775, suggesting that these samples may harbor EHV-8 or EHV-9. Sequencing of the herpesvirus polymerase gene fragment amplified by the pan-herpesviral species PCR revealed EHV-8 was present in these samples (Table 5). No other herpesviruses carried by these donkeys and mule were detected by the EHV1-4MP assay.

Table 5 EHV1-4MP specificity assessment on Donkey and mule specimens containing a herpesvirus.
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EqMC1R real-time PCR assay specificity

A panel of 30 samples representing a variety of species and specimen types were selected to characterize the equid MC1R real-time PCR assay specificity (Supplementary Table S4). Inclusive equid samples encompassed 10 horses, 2 donkeys, a mule, and 3 zebras. All equid specimens were detected with this assay, including nasal/pharyngeal swabs, whole blood, serum, plasma, cerebrospinal fluid, uterine/vaginal swabs, brain, lung, liver, and formalin-fixed paraffin-embedded liver. For exclusivity testing, non-equid species included cows, goats, sheep, white-tailed deer, reindeer, alpaca, dog, cats, beaver, and dolphin. No non-equid samples were detected by this assay. Exogenous extraction controls were used to confirm successful nucleic acid extraction for all these specimens.

Historical EHV-1 and EHV-4 detection trends

We investigated the detection rates of EHV-1 and EHV-4 by real-time PCR at the AHDC between 2013 and 2024. EHV-1 was detected in 1.0 to 7.3% of EHV-1 tested samples on an annual basis since 2013 (Fig. 3A). EHV-4 detection rates ranged between 2.5 and 7.7% of EHV-4 samples tested since the real-time PCR assay became available at the AHDC in 2015 (Fig. 3A). When considered in the context of equine respiratory panel real-time PCR test requests since 2016, EHV-1 was detected in 0.3 to 1.9% of tests annually whereas EHV-4 was detected in 3.9 to 9.4% of tests (Fig. 3B). Other equine respiratory pathogens accounted for 16.0 to 30.4% of positive tests, including EAdV1, EAV, ERAV, ERBV, EIV, S. equi equi, S. zooepidemicus, and M. felis, while approximately 64 to 73% of panel tests did not identify a pathogen (Fig. 3B).

Fig. 3
figure 3

EHV-1 and EHV-4 detected by real-time PCR at the Animal Health Diagnostic Center. A) Percent of EHV-1 and EHV-4 tests with detected result shown on y-axis and year shown on x-axis. EHV-1 detected results are presented in black squares, and EHV-4 results are presented in pink circles. B) Frequency of EHV-1 and EHV-4 detection from equine respiratory pathogen real-time PCR panel test requests. EHV-1 detected results shown in black (0.3 to 1.9%), EHV-4 detected results shown in pink (3.9 to 9.4%), other equine respiratory pathogens detected shown in teal (16.0 to 30.4%), and no pathogen detected shown in purple (63.8 to 73.7%).

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This data was further analyzed to assess specimen types that harbor EHV-1 and EHV-4 DNA. EHV-1 was detected in nasal exudate most frequently, followed by blood and respiratory/vaginal swabs; these matrices constitute 85.3% of detected results (Fig. 4A). EHV-4 was also detected in nasal exudate specimens predominantly, followed by respiratory swabs; together these two matrices comprise 79.9% of detected results (Fig. 4B). These results underscore the importance and utility of the EHV1-4 MP assay developed in the present study.

Fig. 4
figure 4

Specimen types of EHV-1 and EHV-4 real-time PCR detected results at the Cornell University Animal Health Diagnostic Center. A) EHV-1 was detected by real-time PCR from 13 different specimen types since 2011. B) EHV-4 was detected by real-time PCR from 8 different specimen types since 2015.

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Discussion

Our goal was to develop a specific and efficient real-time PCR diagnostic assay to enable rapid and simultaneous detection of EHV-1 and EHV-4 infections, in order to support disease control and transmission mitigation efforts. Without prompt and accurate detection, EHV-1 or EHV-4 infections can lead to substantial economic losses due to abortions/neonatal mortality, death following severe disease including EHM, lost training time, and movement restrictions during outbreaks1,2,40. We verified that the multiplex assay format did not affect assay performance and provided sensitive detection of these pathogens. To ensure that this assay provided valid results, both endogenous and exogenous assay controls were included. The endogenous equid control (MC1R) verifies that relevant equid nucleic acid is present in the sample, which may be horse, donkey, mule, hinny, and zebras. The exogenous control provides a measure to assess the presence of PCR inhibitors in the test sample. The importance of monitoring assay performance is illustrated by a real-time PCR study of nasal swab samples in which 27.5% of samples failed quality control50.

A key feature of the EHV1-4MP assay is its improved specificity over existing assays for EHV-1 detection. The de novo EHV1g1775 assay only detects EHV-1, and although the EHV1gB444 assay was designed to exclude amplification of EHV-8 and EHV-9, if high viral loads of these agents are present in the sample, this assay will still cross react and amplify these pathogens. Both EHV-1 assays (EHV1g1775 and EHV1gB444) were kept in the final multiplex assay to increase sensitivity for EHV-1 detection as well as to monitor for EHV-8 or EHV-9, as the prevalence of the latter may be underestimated. Existing EHV-1 gB assays either share 100% identity or differ by 2–3 nucleotides from EHV-8 and EHV-9 gB sequence44,47. EHV-1 and EHV-8 sequence similarity contributed to the misdiagnosis of two equine abortions caused by EHV-8 that were initially attributed to EHV-1 31, and there could be more unrecognized misdiagnoses. Unfortunately, neither EHV-8 nor EHV-9 isolates or characterized positive samples were available for in vitro analytical specificity assessment, and so synthetic fragments were utilized for analytical testing. Considering this, and to better characterize the EHV1-4MP assay, we also tested 16 donkey and mule samples that were detected by an EHV-1 real-time PCR44 or a pan-herpesviral species PCR assay45. The EHV1g1775 assay did not detect any samples, indicating that EHV-1 was not present. However, the EHV1gB444 assay detected three samples with later Cts than the Elia et al. assay44, suggesting that EHV-8 could be present in these samples. Sequences obtained from these three samples confirmed the presence of EHV-8. None of the other donkey samples which were confirmed by sequencing to be positive for EHV-7 or other equid herpesviruses were detected by the EHV1-4MP assay, confirming that it does not amplify these herpesviruses.

A previously described EHV-4 real-time PCR assay47 amplified EHV-3 DNA in this study, despite only 81% nucleotide identity over the assay target region. Although the differences between EHV-4 and EHV-3 in transmission, tissue distribution, and pathogenesis make misdiagnosis unlikely, it is important to know the inclusivity and exclusivity of assays being used. The de novo EHV-4 gB real-time PCR assay validated herein did not detect any other equine pathogens.

Surveillance studies of equine respiratory pathogens by real-time PCR of nasal secretions from thousands of equids with acute fever and/or respiratory symptoms performed by others detected EHV-1 in 0.7–3.0% of submissions and EHV-4 was detected in 6.6–10.8%3-5. These rates are consistent with those detected by equine respiratory panel real-time PCR tests at the AHDC since 2016, where EHV-1 was detected in 0.3–1.9% of cases and EHV-4 was detected in 3.9–9.4% (Fig. 3B). This pattern of higher EHV-4 prevalence than EHV-1 is supported by higher rate of latent EHV-4 detection (83 vs. 26%)38. Other viral and bacterial equine respiratory pathogens including EIV, ERAV, ERBV, EAdV1, EAdV2, EAV, S. equi, and M. felis were also tested for by real-time PCR at the AHDC or by these other studies, accounting for approximately 20–30% of the respiratory cases. Notably, these pathogens were not detected in the remaining ~ 70% of cases.

In conclusion, we developed a multiplex real-time PCR assay that provides simultaneous, sensitive, and rapid detection of EHV-1 and EHV-4 DNA with improved specificity compared to previously described assays. The EHV1-4MP assay also verifies sample integrity and assay performance at the same time. The multiplex format enables testing for two pathogens and two assay controls within a single well, which may decrease costs and increase workflow efficiency.