Quantification of Feline Herpesvirus 1 DNA in Ocular Fluid Samples of Clinically Diseased Cats by Real-Time TaqMan PCR

Abstract

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R² = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

The most important microorganisms causing diseases of the upper respiratory tract in cats include feline herpesvirus 1 (FeHV-1), feline caliciviruses, and Chlamydia spp. (4, 8, 10, 22, 31, 32, 34). FeHV-1 is a member of the family Alphaherpesvirinae (18, 27). It has no known extrafeline reservoir or alternative host. To survive in nature, it relies, as other herpesviruses, on the establishment of latency, with intermittent episodes of reactivation and virus shedding. Infection with this virus has been associated with abortion, neonatal diseases, classical rhinotracheitis, chronic conjunctivitis and keratitis, recurrent rhinitis, chronic sinusitis, and central nervous system disease (19, 24). One dreaded complication associated with reactivation of FeHV-1 is herpetic stromal keratitis (HSK) (19, 24), which is supposed to rely on an immunopathological basis (1, 21, 24, 28, 33, 36). However, a chronic course of primary infection may also lead to HSK.

Upon primary infection, the virus replicates in the mucous membranes of the nose, larynx, trachea, and genital tract but also in the conjunctivae. A vigorous immune response is able to terminate the clinical illness, usually within 10 to 20 days. However, it is estimated that 80% of infected cats will establish latency in the trigeminal ganglia (9, 20, 23). Upon reactivation from latency, recurrent episodes of rhinitis, conjunctivitis, and keratitis are common.

Both fluorescent antibody tests and virus isolation procedures are considered to be reliable for diagnosing FeHV-1 during the acute primary infection. Unfortunately, during chronic and recurrent infections both tests often yield negative results (8). Therefore, DNA detection techniques, e.g., PCR, have become extremely useful in the diagnosis of FeHV-1 (3, 12, 26, 29, 35).

However, with conventional PCR, quantitative aspects are difficult and cumbersome to resolve. Furthermore, conventional PCR is prone to contamination, which increases the danger of false-positive results. Therefore, fluorogenic real-time PCR was expected to allow the development of reproducible, sensitive, and specific quantification of FeHV-1 genomes (11, 15, 16). The objective of this study was, therefore, to establish a quantitative test for the detection of FeHV-1 DNA in ocular fluid samples of cats with clinical signs of HSK or conjunctivitis.

Here, an excellent correlation between virus isolation and quantitative fluorogenic PCR was demonstrated. When clinical samples were analyzed, the combination of virus titration and quantitative PCR revealed insight into the stage of the infection.

MATERIALS AND METHODS

Cell cultures and virus strains.

Dulbecco's modified essential medium (GIBCO BRL, Life Technologies) containing 10% fetal bovine serum and bicarbonate buffer was used. Crandell Reese feline kidney (CRFK) cells were grown at 37°C in the presence of 5% CO₂. The following virus strains were used: FeHV-1 (isolate UT 88), bovine herpesvirus 1 (BHV-1) (strain Jura) (25), BHV-5 (strain N569) (6), pseudorabies virus (PRV) (vaccine strain Begonia; Intervet) and equine herpesvirus 1 (EHV-1) (strain 502) (5).

Spiked samples.

Tenfold dilutions of FeHV-1, starting from 10^7.5 50% tissue culture infective doses (TCID₅₀) per ml, were made in Dulbecco's modified essential medium. Aliquots (200 μl) of each dilution were used for DNA extraction and PCR as described below. Aliquots of 800 μl were used for virus isolation and titration in 96-well microtiter plates (Costar, Fisher Scientific AG). For that purpose, 20,000 CRFK cells were seeded per well before being inoculated with 100 μl of diluted virus stock.

Experimental infection.

Two specific-pathogen (FeHV-1)-free cats (Harlan, Sprague-Dawley, Inc., Madison, Wis.) were infected with 10^7.5 TCID₅₀ of FeHV-1 per m. For experimental inoculation, 25 μl of virus suspension per location was dropped into each eye as well as into the mouth and nose.

Clinical samples.

Standard methods were used to collect eye fluid samples (Schirmer tear test strips) (14) and Cytobrush smears (2) from 10 specific-FeHV-1-free cats (Harlan) as well as from 20 cats that showed clinical signs of FeHV-1-associated disease, including keratitis or conjunctivitis. Cytobrushes (Cytobrush), brush-like devices for collecting exfoliative cytology specimens from cornea and conjunctiva, were obtained from Medscan (Malmö, Sweden).

Virus isolation.

The contents of the Cytobrushes were eluted in 1 ml of sterile phosphate-buffered saline. This suspension was used to inoculate subconfluent CRFK cell cultures grown in 25-cm² flasks (Corning, O. Kleiner AG). The flasks were incubated at 37°C. The cultures were examined daily for a cytopathic effect indicative of herpesviruses.

Extraction of DNA.

Viral DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. AE buffer (Qiagen) containing 50 μg of carrier DNA (salmon sperm DNA from Gibco, BRL Life Technologies) per ml was used to elute DNA for the 10-fold dilution series of FeHV-1.

DNA amplification by TaqMan technology.

The DNA amplification reaction was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems, Rotkreuz, Switzerland). Primers and probe were designed with Primer Express software (version 1.0; Applied Biosystems, Foster City, Calif.) to amplify a conserved 81-bp sequence within the open reading frame of the glycoprotein B (gB) gene of FeHV-1 (GenBank accession no S66371 [Table 1] Primers and probe were synthesized by Applied Biosystems, Weiterstadt, Germany. PCR amplification was carried out in 25-μl reaction mixtures containing (final concentrations) 12.5 μl of Mastermix (Applied Biosystems, Rotkreuz, Switzerland), 0.5 μl (400 nM) of each primer, 0.2 μl (80 nM) of probe, 1.3 μl of sterile water, and 10 μl of the extracted DNA. The conditions were set as follows: 2 min at 50°C and 10 min at 95°C followed by 40 cycles consisting of denaturation at 95°C for 15 s and annealing-elongation at 60°C for 1 min. The data were analyzed with the sequence detector software (version 1.6). Signals were regarded as positive if the fluorescence intensity exceeded 10 times the standard deviation of the baseline fluorescence (threshold cycle [Ct]).

TABLE 1.

Primers and probe for the FeHV-1 specific DNA amplification

Method and oligonucleotide	Sequence (5"-3")a	Amplifica-tion product size (bp)
Taqman PCR
Forward primer	AGA GGC TAA CGG ACC ATC GA
Reverse primer	GCC CGT GGT GGC TCT AAA C	81
Probe	FAM-TAT ATG TGT CCA CCA CCT TCA GGA TCT ACT GTC GT-TAMRA
Conventional PCR
Forward primer	GCA CAC GAC CGG CTA ATA CAG G
Reverse primer	CAG CTT TCG AGA GGC ACA TAC CC	737

^aAnd fluorogenic labels where applicable.

DNA amplification by conventional PCR and sequencing.

Primers detecting a more extended part of the FeHV-1 gB gene (737 bp) were designed with the Primer Select program from DNA Star (Lasergene, Madison, Wis.) (Table 1). Oligonucleotide primers were synthesized by Microsynth GmbH. The PCR mix, with a total volume of 30 μl, contained 1 μl of extracted DNA, 3 μl of 10× Pfu polymerase buffer, 2 μl (0.66 μM) of each primer, 1 μl of deoxynucleoside triphosphate (10 mM) solution, 1 μl (2.5 U) of Pfu polymerase, and 20 μl of sterile water. A hot-start PCR was performed, starting with 30 min at 95°C. The temperature profile consisted of two different cycles. The first cycle, which was repeated three times, was composed of denaturation at 95°C for 4 min followed by 1 min at 55°C and 1 min at 74°C. The second cycle was repeated 27 times and consisted of denaturation at 95°C for 2 min followed by 1 min at 59°C and 1 min at 74°C. A final elongation was performed at 74°C for 10 min. The PCR products were cleaned using 300 S Microspin columns (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). Cycle sequencing reactions were performed by Microsynth GmbH, using the same primers used for conventional PCR.

RESULTS

Analytical specificity and sensitivity of FeHV-1 DNA detection by fluorogenic PCR.

Sequences of the primers and the probe for the detection of FeHV-1 DNA by fluorogenic PCR were chosen from the open reading frame of the gB gene of FeHV-1 (Table 1). Initially, the conditions for amplification were established using extracted FeHV-1 DNA. To determine the analytical specificity of the reaction, DNA preparations of various herpesviruses, including BHV-1, BHV-5, PRV, and EHV-1, or sterile water were used as controls. With all those controls no positive signal was detectable, while DNA of FeHV-1 gave a dose-dependent positive signal (data not shown). Finally, eye fluid samples and Cytobrush smears were taken from the eyes of 10 healthy, specific-FeHV-1-free cats and subjected to either virus isolation (Cytobrush smears) or fluorogenic PCR for FeHV-1 DNA (eye fluid samples). As expected, none of those samples reacted positively (data not shown).

In order to compare the analytical sensitivity of the TaqMan PCR with virus isolation in cell culture, 10-fold dilutions of viable FeHV-1 stocks were made in cell culture medium, starting with a concentration of 10^7.5 TCID₅₀/ml. Each dilution was processed for testing, either by inoculation of cell cultures or by fluorogenic PCR. The results (Table 2) indicated that both methods had a similar overall sensitivity, with slight advantages for PCR. The detection limits of both tests, virus isolation, and PCR, ranged around 0.06 TCID₅₀, while the 100% detection rate was 60 TCID₅₀ for virus isolation and 6 TCID₅₀ for fluorogenic PCR.

TABLE 2.

Comparison of virus isolation and quantitative fluorogenic PCR using spiked samples with predetermined amounts of virus

TCID50 of input virusa	Isolationb (+/−)	% Positive	PCRc(+/−)	% Positive	Ct ave (s)d
6 × 105	16/0	100	16/0	100	15.0 (0.00)
6 × 104	16/0	100	16/0	100	18.0 (0.00)
6 × 103	16/0	100	16/0	100	20.6 (0.72)
6 × 102	16/0	100	16/0	100	24.9 (0.25)
6 × 101	16/0	100	16/0	100	28.1 (0.57)
6 × 100	13/3	81	16/0	100	33.2 (0.83)
6 × 10−1	3/13	19	11/5	69	38.9 (4.36)
6 × 10−2	1/15	6	4/12	25	42.7 (3.77)
6 × 10−3	0/16	0	0/16	0	45.0 (0.00)
6 × 10−4	0/16	0	0/16	0	45.0 (0.00)

^aTenfold dilutions of virus stock were used to compare the analytical sensitivities of either virus isolation or fluorogenic PCR.

^bOne portion was used to inoculate cell cultures for virus detection. The ratios of successful (+) or unsuccessful (−) virus isolations are presented.

^cFrom the second portion, DNA was extracted and tested for presence of FeHV-1 DNA by fluorogenic PCR. The ratios of positive (+) or negative (−) results are presented.

^dAverage (ave) and standard deviations (s) of values were calculated from 16 experiments.

The Ct values obtained in the TaqMan PCR correlated well with the infectious dose of virus in the sample. Notably, average Ct values of 28 (with a standard deviation of 0.57) correlated to approximately 60 TCID₅₀ of FeHV-1, an amount of virus which is usually easily isolated in cell culture. With less virus in the sample, the Ct values increased, and the frequency of successful virus isolation decreased simultaneously in a linear fashion. Over a range of at least 4 logs (6 to 6,000 TCID₅₀), a linear regression curve was calculated, using the following formula: log (TCID₅₀) = −0.2611 + 9.4373 (R² = 0.9858). From these experiments, it was expected that virus can be isolated from samples with Ct values of 28 and less. On the other hand, the linearity of correlation between virus titer and Ct value was expected to be less stringent at either very high or very low virus concentrations.

Analysis of samples from cats experimentally infected with FeHV-1.

In order to test the above prediction under in vivo conditions, two cats were experimentally inoculated with FeHV-1 and were sampled at intervals for virus shedding by either virus isolation and titration or by the newly developed fluorogenic PCR. One specific-FeHV-1-free cat was used as a negative control. The results are summarized in Fig. 1. Virus isolation was successful from day 1 through day 24, while positive PCR signals could be detected from day 1 to day 80. Early in the infection, an excellent correlation between virus titer and the quantitative PCR signal was observed. However, between day 19 and day 24, the close correlation between virus titer and PCR signal was lost. Virus was not or only barely isolated from samples with Ct values corresponding to high titers of FeHV-1. On day 24 the last positive virus isolation was made, while even at day 80 one out of four Schirmer test samples was still weakly positive by PCR. It was concluded from this experiment that, also under in vivo conditions, virus isolation and quantitative PCR correlated well, although only early in infection. A quantitatively elevated PCR signal combined with negative or low virus titer was observed in a second stage. The final stage was characterized by a decreasing PCR signal accompanied by negative virus isolation.

FIG.1.

Comparison of TaqMan PCR and virus titration in the course of experimental FeHV infection. Schirmer tear test strips taken from the left (samples 1 and 3) or right (samples 2 and 4) eyes of FeHV-1 inoculated cats or a control cat (Control) were analyzed by TaqMan PCR. The Ct values are given on the y axis at left. Simultaneously, Cytobrush smears were analyzed by virus isolation and titration. Virus titers are indicated on the y axis at right, and the highest titer observed per analysis is indicated by the gray area on the figure. The three stages of infection, which are indicated on the figure, were determined as explained in the text.

Analysis of eye fluid samples and Cytobrush smears from diseased cats.

A comparison of virus isolation and quantitative PCR for FeHV-1 DNA was done using eye fluid samples and Cytobrush smears collected from 20 cats that were suspected to suffer from FeHV-1 infection. The results, including clinical and anamnestic data, are shown in Table 3. A total of 10 cats were found to be negative for FeHV-1 both by virus isolation and by fluorogenic PCR. Ten other cats showed a positive PCR signal in the diseased eye, but FeHV-1 was isolated from only four of those 10 cats. The isolates were identified as FeHV-1 by conventional PCR followed by cycle sequencing of the PCR product. Furthermore, typical herpes virus particles were identified in the supernatants of the positive cultures by electron microscopy (not shown). However, without quantitative analysis of the PCR results, the discrepancies between virus isolation and PCR were difficult to explain.

TABLE 3.

Comparative analysis of eye fluid samples and Cytobrush smears from cats with clinically suspected FeHV-1 infection

Agea	Symptom(s)b	Ct valuec	Titer		Interpre-tationf
			Expectedd	Observede
11	K, C	>40	Negative	Negative	Not FeHV
3	C	>40	Negative	Negative	Not FeHV
3	Sneezing, coughing	>40	Negative	Negative	Not FeHV
4 mo	C	>40	Negative	Negative	Not FeHV
4	Ocular discharge	>40	Negative	Negative	Not FeHV
4	K, C	>40	Negative	Negative	Not FeHV
5	K, C	>40	Negative	Negative	Not FeHV
2	K, C	>40	Negative	Negative	Not FeHV
11	K	>40	Negative	Negative	Not FeHV
1	C	>40	Negative	Negative	Not FeHV
4	K	14	5.7	6.8	Stage 1
13	K	20	4.2	6.8	Stage 1
14	K	18	4.7	4.3	Stage 1
2	K, C	19	4.5	3.3	Stage 1
1	C	26	2.6	Negative	Stage 2
1.5 mo	C	23	3.4	Negative	Stage 2
6	C	24	3.1	Negative	Stage 2
11	K	25	2.9	Negative	Stage 2
5	C	35	0.5	Negative	Stage 3
6 mo		38	−0.4	Negative	Stage 3

^aAge in years, except where indicated.

^bAbbreviations: K, keratitis; C, conjunctivitis.

^csamples for PCR taken from the same eye as sample for virus isolation. Ct values correspond to a volume of 200 μl. Values above 40 are considered negative.

^dLog values of expected virus titers were calculated by using the following formula: log (TCID₅₀) = −0.2611 + 9.4373 (R² = 0.9858).

^eLog values of actually measured virus titer per 200 μl in same eye as in the column with the heading “Ct value.”

^fLaboratory data were interpreted as follows: disease not associated to FeHV-1 infection, Ct > 40 and virus isolation negative; stage 1, Ct value <28; virus titer corresponding to Ct value; stage 2, Ct value <28; virus isolation negative or titer not corresponding to Ct value; stage 3, Ct value >28-40; accompanied by negative virus isolation.

For quantitative comparisons, a standard curve was generated using serial dilutions of virus stocks in both fluorogenic PCR and virus isolation. Then, the Ct values of the PCR-positive samples were used to calculate the expected titers of infectious virus using the previously established formula [log (TCID₅₀) = −0.2611 + 9.4373 (R² = 0.9858)]. The results obtained with the standard samples matched very closely with the calculated values (not shown). The results from clinically diseased animals are shown in Table 3. Samples positive by virus isolation revealed Ct values ranging between 14 and 20 (determined in a volume of 200 μl), which correspond to an infectious dose between 10⁴ and 10⁵ TCID₅₀/ml. The FeHV-1 titers determined in the virus-positive Cytobrush samples were 10^7.5 (two samples), 10⁵, and 10⁴ TCID₅₀/ml. The previous results from experimentally infected cats suggested that those two cats with FeHV-1 titers of 10^7.5 TCID₅₀/ml had been sampled during the first stage of FeHV-1 infection. Four samples with Ct values ranging from 23 to 26 remained negative by virus isolation, indicating that the sampling from those cats had been during the second stage. Finally, no virus could be isolated from two samples with Ct values of 35 and 38. It was concluded that those cats had been in the final phase (third stage) of infection.

DISCUSSION

Upper respiratory diseases, also referred to as feline rhinotracheitis, are very frequent in cats. The corresponding most important etiologic agents include FeHV-1, feline caliciviruses, and Chlamydia (7, 8, 10, 31, 32, 34). In order to better understand the pathogenesis of individual infections and corresponding diseases, it is essential to track the particular agents in their original host on a qualitative as well as on a quantitative basis.

A fluorogenic PCR for the detection and quantification of FeHV-1 DNA in ocular fluid samples from diseased cats was therefore established. Based on the known conservation of the targeted gB sequence of FeHV-1 (13), the new assay was able to amplify DNA from geographically (European and American) and biologically (vaccine strains as well as wild-type isolates) different virus strains (data not shown). Using a variety of animal herpesviruses as well as samples from specific-FeHV-1-free cats, the analytical specificity of the new assay was demonstrated.

Then, using spiked samples with known quantities of virus, the analytical sensitivity of the new quantitative PCR assay was determined to be threefold higher than that of the nested PCR assay for FeHV-1 DNA described by Hara and coworkers (12).

Under in vitro conditions, an excellent correlation between virus isolation and quantitative PCR was demonstrated. However, when samples from experimentally infected cats were tested, it turned out that in vivo this correlation was dependent on the stage of the infection. At early times postinfection, the correlation between the two tests was high. This is in agreement with the observation that virus isolation as well as the detection of viral antigens by fluorescent antibody staining is highly reliable for diagnosing FeHV-1, but only during the acute, primary infection (17). In contrast, during chronic and recurrent infections antigen is often not detected (19). Yet, recent studies using PCR have suggested that shedding of FeHV-1 may be more frequent than previously determined by virus isolation studies (29, 35).

Throughout our study, a quantitatively elevated PCR signal combined with a negative or low virus titer was typical for a second stage of the infection. The final stage was characterized by a decreasing PCR signal accompanied by negative virus isolation. Essentially the same observation was made when samples from either experimentally infected cats or cats clinically suspected of FeHV-1 infection were tested. Notably, cats of all ages, very young or very old, were found among the PCR-positive, virus isolation-negative group.

Thus, our study indicated that a positive PCR result did not merely replace virus isolation with a more-sensitive biochemical method as suggested by others (7, 29, 30, 35). Indeed, testing patients by both methods, virus isolation and quantitative PCR, yielded a valuable insight into the stage of the infection. In the daily routine, the expenditures of combined testing may be reduced by the analysis of consecutive samples. Indeed, the greatest advantage of the new quantitative fluorogenic PCR over both classic PCR and virus isolation and titration comes with the analysis of consecutive samples. This allows the tracking of the course of the infection even without the need for costly time- and labor-intensive virus isolation and virus titration procedures.

In contrast, a single PCR-positive result will not discriminate between the different stages of the infection, while nonquantitative consecutive results will not reveal the course of the viral load. Furthermore, classic PCR is much more prone to inadvertent contamination of the sample and, therefore, to the occurrence of false-positive results than the new method presented here. Gaining more information with regard to the course of the infection yields an important clue to help form an educated prognosis for the disease.