How to Use and Interpret Quantitative Polymerase Chain Reaction for the Laboratory Diagnosis of Equine Herpesvirus-1 Infection

1. Introduction

Rapid laboratory tests for detection of equine herpesvirus-1 (EHV-1) are most useful in potential epidemic situations because immediate identification of the causative agent is often critical for guiding management strategies. Real-time polymerase chain reaction (qPCR) has become the diagnostic test of choice due to its quick turn-around-time and high analytical sensitivity and specificity. The qPCR is an enzymatic exponential DNA amplification technique that, under optimal conditions, is capable of detecting a small number of target viral genes. The PCR detection of EHV-1 is routinely performed in respiratory secretions from a nasal or nasopharyngeal swab and uncoagulated blood samples collected into Ethylenediaminetetraacetic acid (EDTA) tubes. Many conventional PCR protocols (single or nested PCR) targeting specific genes of EHV-1 have been published for the molecular detection of EHV-1.^1–6

The increasing application of PCR for the molecular detection of EHV-1 in practice settings has presented new dilemmas with regard to how test results are interpreted and used by both equine practitioners and regulatory veterinarians since routine PCR assays are unable to differentiate between replicating (lytic), nonreplicating, or latent virus. Advances in technology and the use of novel PCR platforms, such as qPCR, allow calculation of viral loads for equine herpesviruses.^7–13 Although considerable progress has been made in developing PCR assays, the lack of protocol standardization between laboratories and the variability in the use of contamination and quality assurance controls remain an ongoing challenge. 

Viral load testing, although not routinely offered by many veterinary laboratories, represents a major improvement in the interpretation of PCR results in the EHV-1 field, allowing better characterization of disease stage, better assessment of risk of exposure to other horses, and better monitoring of response to treatment. The objective of this paper is to discuss the use and interpretation of quantitative PCR for the laboratory diagnosis of EHV-1 infection and to illustrate viral kinetics using field cases.

2. Materials and Methods

Quantitative PCR for EHV-1

Biological samples (whole blood and nasal secretions) were collected from symptomatic and asymptomatic horses naturally infected with EHV-1. The horses either presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, School of Veterinary Medicine at Davis or were part of recent outbreaks of EHV-1. Blood samples and nasal swabs were processed for nucleic acid purification using an automated nucleic acid extraction system,^a according to the manufacturer’s recommendations. All samples were assayed for the presence of the equine glyceraldehyde-3-phosphate dehydrogenase (eGAPDH) and the glycoprotein B (gB) gene of EHV-1 using previously reported TaqMan PCR assays.¹¹ Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and eGAPDH and expressed as EHV-1 gene copies per million cells.¹¹

Molecular Surrogate for Lytic EHV-1

The lytic cycle of infection for EHV-1 results in the release of new virus particles from the infected cells. During lytic infection, viral gene transcription and regulation is sequentially regulated into three distinct phases: immediate early, early, and late phase. The detection of transcripts for late genes has been successfully used as a surrogate for lytic infection of equine gammaherpes viruses.^11,14 One hundred and thirty dual blood and nasal secretion samples from naturally infected horses were used to correlate the presence or absence of late gene transcripts, by measure of messenger RNA (mRNA) of the gB gene of EHV-1, with absolute quantitation of EHV-1 target genes. Total RNA purification and transcription of RNA to complementary DNA (cDNA) was performed according to previously reported protocols.¹¹

Characterization of Disease Stage

To determine viral load signature profiles of horses in different stages of EHV-1 infection, blood and nasal secretions were processed and quantitation was performed as mentioned earlier. The study population was comprised of 27 horses with neurological signs (EHM group), 28 horses with fever and no neurological signs (EHV-1 group), and 52 exposed but asymptomatic horses (asymptomatic group). The statistical difference (P < 0.05) in EHV-1 viral loads between the groups was determined using the Mann-Whitney Test.

Monitoring of Response to Treatment

Twelve horses presenting to the William R. Pritchard Veterinary Medical Teaching Hospital at Davis from 2006 to 2013 with acute onset of neurological deficits and laboratory confirmation of EHV-1 infection were sampled daily during their hospitalization period to determine the viral kinetics of EHV-1 in blood and nasal secretions. All horses received similar medical treatment, which included the administration of dexamethasone (0.05 mg/kg, 0.1 mg/kg IV q 24 h for 2–3 days), valacyclovir (30 mg/kg PO q 8 h for 6 treatments followed by 20 mg/kg PO q 12 h for up to a total of 10 days), flunixin meglumine (0.5–1.1 mg/kg IV q 12 h), α-tocopherol (10,000 IU PO q 24 h), di-methyl sulfoxide (1 g/kg IV diluted in fluids q 24 h for 3 days) and pentoxifylline (10 mg/kg PO q 8 h). Duration of EHV-1 detection in blood and nasal secretions was determined for each horse.

Indicator for Survival

Absolute quantitation for EHV-1 was performed on admission for 22 EHM horses presenting to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California at Davis. The horses were grouped into survivors¹⁵ and non-survivors.⁴ Non-survivors were horses euthanized because of progression of disease despite intensive treatment and did not include horses euthanized because of financial reasons. Statistical difference (P < 0.05) in EHV-1 viral loads between the two groups was determined using the Mann-Whitney Test.

3. Results

The presence or absence of mRNA transcripts for the gB gene directly related to the absolute quantitation of this gene at the genomic DNA level (Fig. 1). Nasal secretions with the presence of lytic EHV-1 showed an average of 170 times higher viral load than nasal secretions with no lytic EHV-1 (P = 0.0001). 
 

[...]