Real-Time Reverse Transcription PCR for Detection and Quantitative Analysis of Equine Influenza Virus

Equine influenza is a cause of epizootic respiratory diseaseof the equine. The detection of equine influenza virus usingreal-time Light Cycler reverse transcription (RT)-PCR technologywas evaluated over two influenza seasons with the analysis of171 samples submitted for viral respiratory disease. Increasedsensitivity was found in overall viral detection with this systemcompared to Directigen Flu A and virus isolation, which were40% and 23%, respectively, that of the RT-PCR. The assay wasalso evaluated as a viable replacement for the more traditionalmethods of quantifying equine influenza virus, 50% egg infectiousdose and 50% tissue culture infectious dose. There was a significantpositive correlation (P < 0.05) between the quantitativeRT-PCR and both of these assays.

INTRODUCTION

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Introduction

Equine influenza is considered the most economically importantrespiratory disease of the equine in countries with substantialbreeding and racing industries. Equine influenza virus belongsto the Orthomyxoviridae family and there are two distinct subtypes,H7N7 (11) and H3N8 (13). It is generally accepted that the formeris no longer in circulation as the last confirmed outbreak causedby this virus was in 1978 (14), however, outbreaks caused byH3N8 viruses occur annually. A study conducted in Colorado in1998 showed that the pathogen was responsible for two-thirdsof equine viral respiratory infections (7).

It is imperative to have specific and sensitive virus detectionsystems available for rapid diagnosis of equine influenza. Traditionally,the gold standard has been virus isolation from nasopharyngealswabs using embryonated hen eggs. Other diagnostic tests employedinclude virus culture in Madin Darby canine kidney cells ormeasurement of a rise in antibody titer in paired sera by hemagglutinationinhibition assay (9). These established techniques are timeconsuming and laborious. Furthermore, the sensitivity of virusisolation is dependant on the presence of infectious particlesand some virus strains are difficult to isolate (8). Serologyprovides only retrospective data which do not facilitate promptintervention and appropriate case management. In recent yearsenzyme-linked immunosorbent assays such as Directigen FluA (BectonDickson) and reverse transcription (RT)-PCR have been introducedin some laboratories to provide more timely results althoughthe sensitivity of the former has been shown to be quite variable(6, 10, 12).

The Light Cycler PCR system from Roche Molecular Biochemicals,allows for high-speed thermal cycling and quantitative RT-PCR(qRT-PCR) using SYBR Green I dye which binds specifically todouble-stranded DNA. The aim of this study was to transfer ourmost sensitive tool for equine influenza virus detection (10)to a more rapid, real-time RT-PCR system suitable for routinediagnostics using the Light Cycler technology. The advantagesof this system include faster result turnaround time, a closedsystem less prone to contamination and the elimination of gelelectrophoresis. We also sought to examine real-time RT-PCRas a viable replacement for the more traditional methods ofquantifying equine influenza virus, i.e., the determinationof 50% egg infectious dose (EID₅₀) or tissue culture infectiousdose (TCID₅₀).

MATERIALS AND METHODS

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Materials and Methods

Samples. A total of 171 nasopharyngeal swabs collected from suspect casesof viral respiratory disease during a 2-year period were testedfor the presence of equine influenza virus. Some of these diagnosticsamples were accompanied by sera for hemagglutination inhibitiontesting. The second set of samples used to validate the qRT-PCRsystem were previously described (10). Briefly, nasopharyngealswabs from four seronegative foals exposed to an aerosol ofinfluenza strain A/Equi/Kildare/89 at 10⁶ EID₅₀/ml were collectedfor 10 consecutive days postchallenge.

Virus isolation, Directigen Flu A, and hemagglutination inhibition. Virus isolation in embryonated eggs was carried out as previouslydescribed (10). The immunoassay Directigen Flu A for the detectionof nucleoprotein was carried out in accordance with the manufacturer'sinstructions (10). The hemagglutination inhibition assay forthe measurement of antibodies to type 1 and type 2 equine influenzaviruses was adapted from the Office International des Epizooties(OIE) Manual of Standards for Diagnostic Tests and Vaccines(9). Seroconversion was defined as a fourfold or greater risein antibody titer.

PCR primers. Primers M52C (5'-CTTCTAACCGAGGTCGAAACG-3') and M253R (5'-AGGGCATTTTGGACAAAGCGTCTA-3')designed by Fouchier et al. (4) were used to amplify a 244-bpamplicon from nucleotides 32 to 276 of the matrix gene. A secondprimer set was designed to confirm the samples positive onlyby RT-PCR. A 373-bp region of the hemagglutinin gene (nucleotides566 to 938) was amplified using primer HA3F (5'-GAATGTGACAATGCCTAAC-3')and primer HA3R (5'-GATGCTTCCATTTGGTGTA-3').

RT-PCR. One-step RT-PCR was performed using the Light Cycler RNA amplificationkit, SYBR Green I (Roche). RNA was extracted and purified from100 µl of nasal secretions using the RNAgents Total RNAisolation system (Promega Corporation, Madison, Wisconsin) accordingto the manufacturer's recommendations. A 10 µl RT-PCRmix consisted of 6.0 mM MgCl₂, 2.0 µl of 5X reaction mix,0.2 µl enzyme mix, and 1.5 µl of RNA. Primers wereused at a final concentration of 0.5 µM. Reverse transcriptionwas carried out at 55°C for 10 min, followed by an initialdenaturation step at 95°C for 30 seconds. cDNA was thenamplified with 40 to 50 cycles of 95°C held for 0 seconds,65°C for 5 seconds (secondary target temperature 55°C),and 72°C for 10 seconds. Fluorescence data were acquiredat the end of each cycle in a single step. Once the plateauphase of the PCR had been reached, amplification was stoppedand a standard melting curve analysis was performed (95°Cheld for 0 seconds, 65°C for 10 seconds, and a 0.1°C/secondrise to 95°C) with continual fluorescence measurement. Asecond independent RT-PCR protocol was carried out with hemagglutinin(HA) gene primers for validation purposes. RT-PCR conditionsused with the second primer set were the same as those usedwith the Matrix primers except the 65°C annealing temperaturewas held for 17 seconds.

qRT-PCR. For quantification of virus shedding, purified viral genomicRNA was used as a standard. The concentration of the standardnucleic acid was determined by measuring the absorbance at 260nm. Serial log dilutions of the RNA were used to generate astandard curve ranging from 5 x 10⁷ to 500 genome copies/µlof viral RNA. The standard curve is the linear regression linethrough the data points on a plot of C_t (threshold cycle) versuslogarithm of standard sample concentration. The crossing pointsof unknown samples were determined from the regression lineand the corresponding concentrations calculated. The genomecopy number was then calculated.

Statistical analysis. Microsoft SPSS 12.0.1 for Windows was used for formal statisticaltesting, and the significance level was set at 0.05. The bivariatecorrelations function was used to compute Pearson's correlationcoefficient for comparing the quantitative tests.

RESULTS AND DISCUSSION

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Results and Discussion

One hundred and seventy-one nasopharyngeal swabs submitted overa two year period from cases of suspected viral respiratorydisease were tested for the presence of equine influenza virus(Table 1). Virus was isolated from eight horses and antigenwas detected by Directigen Flu A in 14 swabs. Acute and convalescent-phasesera were only available for seven horses of which six on fivedifferent premises seroconverted to H3N8 equine influenza. Thirteenof the 23 unpaired serum samples submitted to the laboratoryand the acute sample from the horse that did not seroconverthad high antibody titers to H3N8 equine influenza virus butno detectable antibody titers against H7N7. As all of the vaccinesavailable during the study period contained both H3N8 and H7N7viruses, these results were considered to suggest exposure tovirus by natural infection. However, while such serologicalresults may corroborate a diagnosis of influenza, they are notconfirmatory.

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TABLE 1. Results for clinical samples

The RT-PCR proved to be the most sensitive of the assays used.Equine influenza virus nucleic acid was detected in the nasalswabs from 35 horses on 17 different premises, i.e., the overalldetection rate was 20%, compared to 8% and 5% for DirectigenFlu A and virus isolation, respectively. Of the 35 RT-PCR-positiveswabs 27 were positive by one or more of the other diagnostictests employed. All samples positive only by RT-PCR were alsotested with the second primer set against the HA gene (datanot shown) and there were no discrepancies.

This study demonstrates that RT-PCR is effective in the detectionof virus shedding from seropositive horses. Virus was isolatedfrom only 15% of seropositive horses that tested positive byRT-PCR. The detection of virus shedding in subclinically infectedvaccinated horses is extremely important as the introductionof such animals into an immunologically naïve populationcan lead to explosive virus spread and high morbidity.

Vaccination remains the most effective control measure for equineinfluenza. Vaccines need to be safe and efficacious. Vaccineefficacy is measured by the reduction in clinical signs andthe decrease in virus shedding. We examined whether qRT-PCRcould viably replace the traditional methods of quantifyingequine influenza virus shedding, i.e., EID₅₀ and TCID₅₀ analysis.Figure 1 shows the mean amount of virus shed by the four horsesper day postchallenge by qRT-PCR analysis. We found a significantpositive correlation (P < 0.05) between the traditional methodsof viral quantification and the qRT-PCR results. RT-PCR wasnot only less labor intensive than the other two methods butwas also far faster and 17% and 29% more sensitive than virusisolation in eggs and tissue culture, respectively.

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FIG. 1. Mean and standard error of the mean of viral RNA copies/ml (log₁₀) in nasal secretions of four foals from days 2 to 7 postchallenge with equine influenza virus.

In conclusion, the Light Cycler RT-PCR protocol described herefor the detection of equine influenza virus has been shown tobe a more sensitive assay in the clinical diagnosis of influenzavirus infection than standard techniques. The RT-PCR can alsobe reliably used for the quantification of equine influenzavirus in vaccine efficacy studies Furthermore, the primers employedwere designed to amplify a region of the matrix gene which ishighly conserved across different subtypes of influenza A viruses(4). Thus, this RT-PCR detects influenza virus from multiplespecies. Given the propensity of influenza A viruses to crossthe species (1, 15), 18 and their zoonotic potential (2, 3,5) this assay can be used with relative ease and speed to aidvirus surveillance that is advantageous not only to the equineindustry but also to public health.

ACKNOWLEDGMENTS

The initial investigations of outbreaks of respiratory diseasewere sponsored by Interchem. All other laboratory work was fundedby the Department of Agriculture, Food and Rural Development,under the National Development Plan (Irish Equine Centre NDP\9\R\2).Michelle Quinlivan is a Ph.D. student funded by the Irish ResearchCouncil for Science, Engineering and Technology and Eugene Dempseyis a Ph.D. student aided by the Research Support Unit, DublinInstitute of Technology.

FOOTNOTES

* Corresponding author. Mailing address: Virology Unit, The Irish Equine Centre, Johnstown, Naas, Co. Kildare, Ireland. Phone: 353 45 866266. Fax: 353 45 866273. E-mail: acullinane{at}equine-centre.ie.

REFERENCES

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