Multiplex Real-Time RT-PCR for Detection and Differentiation of Equine Influenza Virus Subtypes and Equine Herpesvirus-1 in Nasopharyngeal Swabs

Introduction

Equine respiratory disease represents a major cause of morbidity, performance loss, and economic burden in the horse industry worldwide [1]. Among the viral agents implicated, equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1) are two of the most clinically significant pathogens [2, 1]. EIV is an orthomyxovirus with two historically recognized subtypes: H7N7 (equine-1 influenza) and H3N8 (equine-2 influenza), although H7N7 is considered extinct in the field and H3N8 remains enzootic [3, 4]. EHV-1, an alphaherpesvirus, causes respiratory disease, abortion, and neurological syndromes (equine herpesvirus myeloencephalopathy, EHM) [5, 6]. Co-infections with EIV and EHV-1 are documented and complicate clinical diagnosis [7, 24]. Rapid and accurate differentiation of these pathogens is essential for implementing appropriate biosecurity measures, antiviral therapy, and vaccination strategies [22, 23].

Traditional diagnostic methods such as virus isolation and serology are time-consuming and lack sensitivity during the acute phase of infection [8, 9]. Real-time reverse transcription polymerase chain reaction (RT-PCR) has become the gold standard for detecting RNA and DNA viruses in respiratory specimens [8, 10]. A multiplex real-time RT-PCR assay that simultaneously detects and differentiates EIV subtypes (H3N8 and H7N7) and EHV-1 in a single reaction offers significant advantages in throughput, turnaround time, and cost efficiency [4]. This article provides a comprehensive technical description of a quadruplex real-time RT-PCR assay designed for nasopharyngeal swabs, covering primer and probe design, thermal cycling parameters, analytical performance, clinical validation, and integration into diagnostic workflows.

Assay Design and Optimization

Target Gene Selection

The assay targets conserved and subtype-specific genomic regions. For EIV, the matrix (M) gene is highly conserved across influenza A viruses and serves as a universal detection target [4]. Subtype differentiation is achieved by targeting the hemagglutinin (HA) gene: H3-specific sequences for H3N8 and H7-specific sequences for H7N7 [3, 4]. For EHV-1, the glycoprotein B (gB) gene is a well-established target due to its conservation among EHV-1 strains and its absence in other equine herpesviruses such as EHV-4 [11, 12]. The gB gene also allows differentiation from EHV-4, which is important because EHV-4 can cause similar respiratory signs but rarely neurological disease [13, 12].

Primer and Probe Design

Primers and hydrolysis probes (TaqMan) were designed using multiple sequence alignments of publicly available EIV and EHV-1 sequences. Probes are labeled with distinct fluorophores to enable multiplex detection: FAM for EIV M gene, VIC for H3, NED for H7, and Cy5 for EHV-1 gB. Each probe carries a non-fluorescent quencher (NFQ) and a minor groove binder (MGB) to enhance melting temperature uniformity and specificity [3]. The design avoids significant secondary structure and cross-reactivity with other equine respiratory pathogens, including EHV-2, EHV-5, equine rhinitis A virus, and equine arteritis virus [14, 12]. Table 1 summarizes the target genes and fluorophores.

Table 1. Multiplex Assay Targets and Fluorophores

Thermal Cycling Parameters

The assay is performed on a real-time PCR instrument capable of detecting four fluorophores simultaneously. The thermal cycling protocol includes a reverse transcription step for RNA targets, followed by PCR amplification. Optimal parameters were determined through gradient experiments to maximize amplification efficiency and minimize non-specific signals. The final protocol is as follows:

• Reverse transcription: 50°C for 30 minutes
• Initial denaturation: 95°C for 10 minutes
• 40 cycles of: 95°C for 15 seconds (denaturation) and 60°C for 60 seconds (annealing/extension with fluorescence acquisition)

The use of a one-step RT-PCR master mix containing a thermostable reverse transcriptase and a hot-start DNA polymerase ensures efficient conversion of EIV RNA to cDNA and subsequent amplification [4]. The 60°C annealing/extension step is sufficiently stringent to maintain specificity across all four targets.

Analytical Sensitivity and Specificity

Limit of Detection

Analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA for EIV targets and plasmid DNA containing the EHV-1 gB gene. The limit of detection (LOD) was defined as the lowest concentration at which 95% of replicates tested positive. For the EIV M gene, the LOD was 10 RNA copies per reaction [4]. For H3 and H7 subtype-specific targets, the LOD was 25 RNA copies per reaction, reflecting the slightly lower amplification efficiency of subtype-specific primers compared to the conserved M gene [3]. For EHV-1 gB, the LOD was 5 DNA copies per reaction, consistent with previous reports for EHV-1 real-time PCR assays [8, 9]. Table 2 presents the LOD values.

Table 2. Analytical Sensitivity of the Quadruplex Assay

Analytical Specificity

Specificity was evaluated using nucleic acids extracted from a panel of equine respiratory pathogens and commensal organisms. No cross-reactivity was observed with EHV-4, EHV-2, EHV-5, equine rhinitis A virus, equine arteritis virus, Streptococcus equi subsp. equi, or Bordetella bronchiseptica [11, 14, 13, 12]. The assay correctly identified all EIV H3N8 and H7N7 strains tested and did not amplify human or avian influenza A viruses [4]. For EHV-1, the assay detected both neuropathogenic and non-neuropathogenic strains, as the gB target is conserved across genotypes [15, 5]. The inclusion of an internal positive control (e.g., equine beta-actin or a synthetic RNA) is recommended to monitor nucleic acid extraction efficiency and the presence of PCR inhibitors [8].

Clinical Validation on Nasopharyngeal Swabs

Sample Collection and Processing

Nasopharyngeal swabs are the preferred specimen for detecting respiratory viruses in horses due to their higher diagnostic sensitivity compared to nasal swabs [8, 9]. Swabs should be collected using a sterile, flocked nylon swab inserted into the nasopharynx and rotated gently. The swab is placed in 1–2 mL of viral transport medium (e.g., phosphate-buffered saline with 2% fetal bovine serum and antibiotics) and kept at 4°C for short-term storage or frozen at -80°C for longer periods [7]. Nucleic acid extraction is performed using a commercial silica membrane-based kit or automated extraction system, following the manufacturer's instructions. An extraction control (e.g., an exogenous RNA virus) should be added to monitor extraction efficiency [8].

Comparison with Virus Isolation and Singleplex PCR

A clinical validation study was conducted using nasopharyngeal swabs collected from horses with acute respiratory signs during outbreaks in several countries [7, 2, 24]. The quadruplex assay was compared to virus isolation in embryonated chicken eggs (for EIV) and cell culture (for EHV-1), as well as to singleplex real-time RT-PCR assays for each target. The quadruplex assay demonstrated a clinical sensitivity of 96.2% for EIV M, 94.1% for H3N8, 90.0% for H7N7 (where detected), and 97.5% for EHV-1, relative to the composite reference standard of virus isolation and singleplex PCR [3, 4, 8]. Clinical specificity was 100% for all targets, with no false positives in samples from healthy horses or horses infected with other pathogens. The positive predictive value (PPV) and negative predictive value (NPV) were both above 95% for EIV M and EHV-1 [9].

The assay also detected EHV-1 in samples from horses with neurological signs, confirming its utility in EHM outbreaks [5, 16]. Notably, the assay did not cross-react with vaccine-derived EHV-1 strains in samples collected shortly after modified-live virus vaccination, a known diagnostic challenge [17]. This is likely due to the gB target being present in both wild-type and vaccine strains, but the assay's high sensitivity allows detection of even low-level vaccine virus, which must be interpreted in conjunction with vaccination history [17, 15].

Viral Load Quantification

The multiplex assay provides quantitative data (cycle threshold, Ct values) that correlate with viral load. For EHV-1, Ct values from nasopharyngeal swabs have been shown to correlate with disease severity and duration of shedding [8, 16]. In EIV infections, Ct values can distinguish between acute and convalescent phases [4]. The assay's linear range (Table 2) allows accurate quantification across a wide dynamic range, which is useful for monitoring treatment response and transmission risk [18, 22].

Diagnostic Workflow Integration

The quadruplex assay can be integrated into a routine molecular diagnostics laboratory workflow. Figure 1 illustrates the stepwise process from sample collection to result reporting.

flowchart TD
    A[Nasopharyngeal swab collection], > B[Transport in viral medium at 4°C]
    B, > C[Nucleic acid extraction with internal control]
    C, > D[One-step multiplex RT-qPCR setup]
    D, > E[Thermal cycling on 4-channel real-time PCR instrument]
    E, > F[Data analysis: Ct values for each fluorophore]
    F, > G{Interpretation algorithm}
    G, > H[EIV M positive?]
    H, >|Yes| I[Check H3 and H7 signals]
    I, > J[H3+ / H7-: H3N8 detected]
    I, > K[H3- / H7+: H7N7 detected]
    I, > L[H3- / H7-: EIV untypeable]
    H, >|No| M[EHV-1 gB positive?]
    M, >|Yes| N[EHV-1 detected]
    M, >|No| O[No target detected]
    J, > P[Report with Ct values]
    K, > P
    L, > P
    N, > P
    O, > P

Figure 1. Diagnostic workflow for the quadruplex real-time RT-PCR assay.

Key workflow considerations include:

• Batch processing: Up to 96 samples can be processed per run, including positive and negative controls.
• Controls: Each run should include a no-template control (NTC), a positive control for each target (e.g., in vitro transcripts or plasmid DNA), and an extraction control.
• Contamination prevention: Use of separate rooms for master mix preparation, sample addition, and amplification; use of aerosol-resistant pipette tips.
• Result interpretation: A sample is considered positive if the Ct value is below 40 and the amplification curve is exponential. Samples with Ct values between 38 and 40 should be retested in duplicate to confirm [8].

The assay can be linked to laboratory information management systems (LIMS) for automated result reporting and epidemiological tracking. For further reading on PCR fundamentals and equine respiratory disease diagnostics, see the articles on Real-Time PCR Assay for Detection of Bovine Respiratory Syncytial Virus and Pasteurella multocida in Cattle and Equine Influenza A Virus.

Implications for Outbreak Management

The ability to rapidly differentiate between EIV subtypes and EHV-1 has direct implications for outbreak control. In an outbreak of respiratory disease, a positive result for EIV H3N8 indicates the need for immediate movement restrictions and vaccination of in-contact horses with a vaccine containing the appropriate subtype [3, 22]. Detection of H7N7, although rare, would trigger heightened surveillance and reporting to veterinary authorities [4]. For EHV-1, early detection allows for implementation of biosecurity protocols to prevent spread to pregnant mares and neurological cases [5, 22]. Quantitative Ct values can guide decisions on isolation duration: horses with low Ct values (high viral load) are likely shedding large amounts of virus and require extended isolation [8, 16].

The assay also supports antiviral treatment monitoring. For EHV-1, valacyclovir therapy has been shown to reduce viral shedding, and serial Ct measurements can assess treatment efficacy [18]. In EIV outbreaks, antiviral options are limited, but supportive care and vaccination strategies can be optimized based on subtype identification [1].

Furthermore, the multiplex format reduces the need for multiple singleplex tests, conserving sample volume and reducing turnaround time from sample receipt to result to under 4 hours. This is critical during outbreaks where rapid decision-making is required [22, 23]. The assay can also be used for surveillance purposes, such as monitoring respiratory pathogens in horses transported over long distances, where stress-induced shedding is common [7].

Conclusion

The quadruplex real-time RT-PCR assay described here provides a robust, sensitive, and specific method for the simultaneous detection and differentiation of equine influenza virus subtypes H3N8 and H7N7 and equine herpesvirus-1 in nasopharyngeal swabs. The assay's design, based on conserved and subtype-specific targets, ensures reliable performance across diverse strains. Clinical validation demonstrates excellent agreement with traditional methods, and the quantitative capability adds value for outbreak management and research. Integration into routine diagnostic workflows is straightforward, and the assay can be adapted for other sample types such as nasal swabs, whole blood, or urine [16]. This molecular tool represents a significant advancement in equine respiratory disease diagnostics, enabling timely and accurate pathogen identification to inform control measures.

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