Veterinary Microbiology, 24 ( 1990 ) 281-295 281 Elsevier Science Publishers B.V., Amsterdam

Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and

latently infected cells

Roger K. Maes, Christopher E. Beisel, Stephen J. Spatz and Brad J. Thacker Department of Microbiology and Public Health and Animal ttealth Diagnostic Laboratory, College q[

Veterinary Medicine, Michigan State University, East Lansing, M148824, U.S.A.

ABSTRACT

Maes, R.K., Beisel, C.E., Spatz, S.J. and Thacker, B.J., 1990. Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells. Vet. Microbiol., 24:281-295.

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency, Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explantation, blot- and in situ hy- bridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences.

Two sets of 20-mer primers were synthesized and used to amplify gX and gIl glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by South- ern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplifica- tion of target sequences by PRC increased their detection limit by a factor of at least 105.

Porcine ganglion samples, in which latency had been demonstrated by in vitro explantation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a por- tion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and conve- nient method for the detection of PRV induced latency.

INTRODUCTION

Pseudorabies or Aujeszky's disease is caused by a herpesvirus belonging to the genus alphaherpesvirinae. Most warmblooded species are either naturally or experimentally susceptible to this virus. Swine are considered to be its nat- ural host and main reservoir. Since 1974, strains of pseudorabies virus (PRV) that are significantly more virulent than those previously encountered, have emerged in the U.S. and spread through the major swine producing areas (Gustafson, 1986 ).

During the acute phase of the disease, PRV replicates in epithelial, lymph-

0378-1135/90/$03.50 © 1 9 9 0 - - Elsevier Science Publishers B.V.

282 R.K. MAES ET AL.

oid and nervous tissues. To diagnose acute infection, the palatine tonsil and trigeminal nerve ganglia are frequently tested for the presence of virus by either the fluorescent antibody technique or cell culture inoculation. Blot hybridi- zation methodology has recently been developed for the detection of viral DNA (McFarlane and Thawley, 1985; Belak et al., 1987; Maes et al., 1988 ).

A characteristic of herpesviruses is that the acute phase of the disease is frequently followed by lifelong latency. The viral genome persists in certain cell types and can be induced to reactivate and replicate infectious virus. The mechanisms of induction, maintenance and reactivation of the latent state are not clearly understood at the present time. Reactivation of the latent state contributes to the perpetuation of PRV in the swine population, although the incidence and true epidemiological significance of latency have not been well defined.

Determining the incidence of latent PRV infections among swine requires a sensitive and convenient assay system. Direct experimental evidence for the existence of latent PRV infections has been obtained by in vitro reactivation of latent virus following tissue explantation (Sabo and Rajcani, 1976; Beran et al., 1980; Van Oirschot and Gielkens, 1984), in vivo reactivation following repeated administration of large doses of corticosteroids (Crandell et al., 1979), blot hybridization (Gutekunst, 1979; Ben-Porat et al., 1984; McFarlane et al., 1986; Rziha et al., 1986) and in situ hybridization (Rziha et al., 1984; Rock et al., 1988).

Blot hybridization would be a convenient method for screening of large numbers of samples. We have recently determined that a probe consisting of a cloned PRV DNA fragment labeled with biotin or digoxygenin could detect l -5 pg ofPRV DNA deposited on nitrocellulose by slot blotting (Maes et al., 1988). Radiolabeled probes of the highest specific activity combined with longer term exposures could further reduce this detection limit. While this level of sensitivity is sufficient to consistently detect acute PRV infections, it is often not sufficient to detect latent PRV DNA in tissues shown to be posi- tive by in vitro explantation.

The polymerase chain reaction (PCR) is a novel technique which makes possible the amplification of rare DNA sequences by a factor of 105 to 10 6. The method consists of cyclic denaturation of the DNA template, annealing of oligonucleotide primers to the single stranded DNA and extension of the primers by a thermostable DNA polymerase. PCR has proven to be very use- ful for the detection of low levels of genomic sequences of several different viruses including human immunodeficiency virus (Kwok et al., 1987; Laure et al., 1988; Murakawa et al., 1988; Ou et al., 1988), human T cell leukemia virus (Duggan et al., 1988; Kwok et al., 1988; Bhagavati et al., 1988 ), human B lymphotropic virus (Buchbinder et al., 1988 ), human papillomavirus (Shi- bata et al., 1988a,b), human rhinovirus (Gama et al., 1988) and hepatitis B virus (Larzul et al., 1988).

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA 283

This report describes the successful adaptation of PCR to the amplification of DNA sequences from two different areas of the PRV genome. In addition, we show the usefulness of this method in the detection of latent PRV DNA.

MATERIALS AND METHODS

Virus strains and propagation The P-2208 strain of PRV was isolated in 1975 from the tonsils of three 16-

day-old piglets. Death losses amongst infected pigs of all ages continued for more than 2 months. The Rice strain of PRV was obtained from Dr. D.P. Gustafson of Purdue University. Both of these strains are considered highly virulent.

Both strains of PRV were grown in a mycoplasma-free Crandell-Rees feline kidney cell line (CRFK). The growth medium consisted of Eagle's min imum essential med ium (EMEM), supplemented with 10% fetal bovine serum, penicillin ( 100 U / m l ) , streptomycin ( 100 pg /ml ) and glutamine (2 mM) . The CRFK monolayers were inoculated at a multiplicity of infection (MOI) of 0.01. The infected cultures were processed when the viral cytopathic effect was generalized. The virus containing medium was clarified by low speed cen- trifugation. Cell-free virus was titrated by the microtiter method.

Infection of experimental pigs Pigs were purchased at 6-8 weeks of age from a pseudorabies-free swine

operation. They were divided into experimental groups and housed in sepa- rate isolation units at least 1 week prior to the start of the experiments. Care of the experimental pigs was provided according to the guidelines for the use and care of laboratory animals provided by the National Institutes of Health.

The ganglion samples tested for the presence of latent PRV originated from ( 1 ) Four pigs from a pseudorabies-free herd. Three of these were exposed to 5 × 105TCIDso of the P-2208 strain of PRV at 120 days of age, the fourth to 1 × 105 TCIDso of the same strain at 50 days of age. (2) Two pigs from a pseudorabies-free herd vaccinated at 3 days of age with approximately 104s TCIDs0 of a tk - , gIII-PRV vaccine and challenged at 4 months of age with 5 × 105 TCIDso of the P-2208 strain. (3) One pig with passive immuni ty to PRV, exposed to 105 TCIDso of the P-2208 strain at 28 days of age. (4) One control pig from a pseudorabies free herd. Clinical signs and temperatures were recorded daily. Nasal swabs were collected at 2-day intervals and blood samples were taken weekly.

Surviving pigs were kept in isolation for at least 60 days post-inoculation. Virus isolation was at tempted from nasal swabs at weekly intervals during this period to ensure the absence of acute infections. The pigs were euthan- ized by i.v. injection of an overdose of T-61. Nervous tissues, including the

284 R.K. MAES ETAL.

trigeminal nerve ganglia, were collected aseptically, kept on ice briefly, fur- ther processed immediately or frozen at - 7 5 ° C.

Latency control To assess the presence of latent PRV in the experimental pigs, one of the

trigeminal nerve ganglia was collected under aseptic conditions and placed on ice in 4 ml of EMEM, containing 10% fetal bovine serum, 100 U / m l of penicillin, 100/~g/ml of streptomycin and 50 ~g/ml of fungizone (EMEM- B 10-psf ). Within 30 min after collection, the ganglia were washed in EMEM- B 10-psf and minced into fragments of approximately 1 m m 3. The fragments were then explanted in vitro according to four different methods. A detailed description of these methods will be published elsewhere (Maes and Thacker, in preparation). Briefly, ganglion fragments were placed at the bot tom of 16 × 125 m m plastic tissue culture tubes, deposited on pieces of sterile 0.45 /lm nitrocellulose paper or coated with chicken embryo extract and allowed to adhere to the inner wall of tubes, precoated with avian plasma. The media used were DMEM or EMEM, supplemented with 10% lamb serum or 10% FBS. Medium samples were collected at 2 or 4 day intervals and assayed on CRFK cells.

Preparation qf DNA Total DNA was extracted from infected cell monolayers or from frozen

ganglia essentially as described by Hermann and Frischauf ( 1987 ). PRV viral DNA was prepared from infected CRFK monolayers using methods de- scribed by Rota et al. (1986). Briefly, infected cells were washed in PBS, re- suspended in TE ( 10 mM Tris-HC1, pH 7.5, 1 mM EDTA) and adjusted to 0.5% NP-40 (Sigma, St. Louis, MO). Nuclei were pelleted by centrifugation at 2000 × g for 7.5 min. The supernatant containing the viral nucleocapsids was adjusted to 1% SDS, 25 mM EDTA, 1 m g / m l predigested Pronase and incubated overnight at 37 ° C. The viral DNA was banded by equilibrium cen- trifugation in sodium iodide with ethidium bromide. Viral DNA bands were harvested, extracted with isoamyl alcohol and dialyzed against TE.

DNA amplification Oligonucleotides were synthesized by Research Genetics (Huntsville, AL).

Primer sequences were derived from the sequences of the PRV gX gene (Rea et al., 1985) and gII gene (Robbins et al., 1987). Primer designations and sequences were: gX-P2 (dATGTAGATCGAGGTGCGGAT) , gX-P3 (dATCTTCCGCTCGGAGTCTGA) . gII-P1 (dTGTACGTGCAGAACT CCATG ) and gII-P2 (dGACATGTACCGGATCATGTC) .

Varying amounts of DNA were used in each amplification (see Fig. leg- ends). In some cases, the DNA was denatured in H20 at 100°C for 7.5 min before being added to the reaction. All reactions were in 100 ¢tl and contained

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA 285

10 mM Tris-HCl, pH 8.3, 50 mM KCI, 1.5 mM MgC12, 0.01% gelatin, 200 /~M of each dNTP, 1.0/~M of each primer and 2.5 units of Taq polymerase (Perkin Elmer-Cetus, Norwalk, CT). The reactions were overlayed with 45 /tl of mineral oil to prevent evaporation.

Thirty cycles of amplification were performed in a Perkin Elmer-Cetus DNA Thermal Cycler. Prior to cycling, the samples were heated to 95 °C for 1 min. For each cycle, samples were denatured at 95 °C for 1 min, annealed at 55 °C for 1 min and extended at 72°C for 2 rain. After cycling, the final extension was continued for 8 rain.

Analysis of amplified DNA All reactions were extracted once with chloroform: isoamyl alcohol (24:1 ).

Samples of each reaction were electrophoresed on agarose gels or transferred directly to nitrocellulose using a slot blotting apparatus. The DNA was dena- tured in 0.3 M NaOH and neutralized prior to slot blotting. Restriction di- gests were performed prior to electrophoresis under conditions specified by the enzyme supplier. Electrophoresed DNA was transferred to nitrocellulose membranes using standard techniques (Maniatis et al., 1982).

Hybridization probes were prepared from specific restriction fragments purified from low-melting temperature agarose. Fragments were labeled with 32p-dCTP by nick-translation using a commercial kit (BRL, Gaithersburg, MD). The probe for gX consisted of the BamHI 7 fragment of the P-2208 strain of PRV isolated from a pBR322 clone (Ruiz et al., 1982). A probe specific for the amplified gII fragment but lacking the primer sequences was prepared by cloning a subfragment of the amplified region. The 778 bp am- plified gII fragment was digested with NciI to produce a 727 bp internal frag- ment (see Fig. 1 ). The recessed 3' ends were filled in with Klenow (Maniatis et al., 1982 ) and the fragment blunt-end ligated into Sinai cut, phosphatase- treated pBluescript KS- (Stratagne Cloning Systems, La Jolla, CA). Clones containing a 0.7 kb insert were digested with SalI, SstI, and XhoI to confirm the presence of restriction sites predicted by the gII sequence.

Blots were prehybridized for at least 6 h at 42 °C in 10% formamide, 0.6 M NaC1, 0.06 M Na-citrate, 0.01 M EDTA, 0.1% SDS, 5XDenhardt 's and 100 pg/ml denatured salmon sperm DNA. Probes were denatured at 95°C and added to a final concentration of 100 ng/ml in a hybridization solution con- sisting of 50% formamide, 7.0% dextran sulfate, 1 X Denhardt's, 4 X SSC, 0.1 M EDTA, 0.1% SDS and 50 #g/ml denatured salmon sperm DNA. Blots were hybridized at 42 ° C for 48 h and washed under stringent conditions (Maniatis et al., 1982).

RESULTS

Specificity of amplification Two regions of the PRV genome were selected for amplification (Fig. 1 ).

Since the polymerase chain reaction often produces non-specific amplifica-

286 R,K. MAES ET 4L.

5 I 2 9 II 4

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N c i I S a l I Sst T Xh0I Sst][ N c i I I I I I ' - -

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Fig. 1. Regions of the PRV genome amplified by PCR. The BamHl map of the Rice strain is shown (Rea et al., 1985). The locations of the glI and gX genes are as reported by Robbins et al. (1987) and Rea et al. (1985), respectively. Restriction sites within the amplified regions were derived from the reported sequences. Primers are represented as filled rectangles at the ends of each amplified fragment.

tion products (Saiki et al., 1988 ), it was necessary to confirm the specificity of the amplification products by restriction digestion, Southern hybridization or both.

Amplification with primers gX-P2 and gX-P3 should produce a 647 bp fragment with two BstEII sites (Fig. 1 ). Digestion of the reaction product with BstEII produced the predicted 0.32 and 0.26 kb fragments (Fig. 2 ). On Southern blots, 3Zp-labeled purified PRV BamHI 7 hybridized specifically to the 0.65 kb amplified band (Fig. 2).

Amplification with primers gII-P1 and gII-P2 should produce a 778 bp fragment with two SstI sites and one XhoI site (Fig. 1 ). Digestion with SstI produced the expected 0.45, 0.21 and 0.12 kb fragments; digestion with XhoI produced the expected 0.47 and 0.31 kb fragments (Fig. 3).

Optimization of reaction conditions For several of the PCR reaction conditions, slight deviations from the op-

timal values can result in reduced or undetectable amplification (Saiki et al., 1988 ). We found the magnesium concentrat ion and the denaturat ion temper- ature to be critical. Significant reduction in amplification of both gX and gII was observed at MgC12 concentrations above 1.5 m M (Fig. 4). For gII ampli-

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA 287

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Fig. 2. Specificity of amplification of gX sequences. Approximately 1/zg of DNA from PRV- infected CRFK cells was amplified and 20% of the products analyzed on agarose gels. (A) Am- plified product from Rice (lane 1 ) and P-2208 (lane 2) strains. Amplified product from lambda control DNA (2), a BamHI digest of P-2208 viral DNA (V) and size markers (M) are in- cluded. (B) Southern blot of gel from (A) hybridized with PRV BamHI fragment 7. (C) BstEII digests of amplified product from Rice (lane 1 ) and P-2208 (lane 2) strains. Corresponding undigested product is in lanes 3 and 4.

fication, a denaturation temperature of 95 °C was required: denaturation at the standard 94 °C produced no detectable amplification product (data not shown).

Quantitation of amplification Serial dilutions of purified PRV viral DNA were amplified with gII primers

in order to determine the least amount of starting material that can be de- tected following PCR. On ethidium bromide stained gels, the amplified prod- uct from 2 X 10-1 z g of viral DNA can be detected visually (data not shown ). Following blotting and hybridization, the amplified fragment from 2 X 10- ~ 6g of viral DNA is easily detected (Fig. 5 ).

Amplification of latent DNA The four nonimmunized pigs exposed to virulent PRV had elevated tem-

peratures between the second and seventh day post-inoculation. Other clini- cal signs included depression and purulent nasal discharge. Two of these pigs

2 8 8 R.K. MAES ET AL.

1 2 3

Fig. 3. Specificity of amplification of gII sequences. Approximately l /zg of DNA from PRV- infected CRFK cells was amplified and 20% of the product analyzed on an agarose gel, Lane 1, undigested amplification reaction; lane 2, Sstl digest; lane 3, XhoI digest.

A t3 M J

Fig. 4. Effect of Mg 2+ concentration on amplification efficiency. Approximately 1 /~g of DNA from PRV ( Rice )-infected CRFK cells was amplified in the presence of different concentra- tions of MgCI2 and 20% of the product run on agarose gels. Millimolarity of MgClz is indicated above the lanes. (A) Amplification with gX primers. The second lane in each pair is an unin- fected CRFK control. The bands in the control lanes are non-specific and do not hybridize with the BamHI-7 probe (data not shown ). (B) Amplification with glI primers.

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA

1 2 3 4 5 6 7

289

j 0.78

Fig. 5. Sensitivity of detection of amplified PRV DNA. Dilutions of purified PRV (P-2208) viral DNA were amplified with glI primers and 20% of the product electrophoresed on an aga- rose gel. The gel was blotted to nitrocellulose and probed with the sub-cloned gII probe. Amounts of PRV viral DNA per lane: 1, 2 X 10- 9g, 2, 2 × 10- ~ ~g; 3, 2 X 10- ~ 2; 4, 2 × 10- ~ 3g; 5, 2 × 10-14g; 6, 2X 10-15g; 7, 2X 10-16g.

developed nervous signs. The two pigs vaccinated with the t k - , glII-PRV vac- cine prior to challenge with the virulent P-2208 strain showed a 1 to 2-day temperature elevation. Other clinical signs were not observed except for a mucoid nasal discharge in one pig on 2 separate days. The passively immune pig exposed to virulent PRV in the presence of passive immunity did not show any clinical signs. The control pig was also clinically normal throughout the experiment.

The P-2208 challenge virus was consistently isolated from the nasal swabs of all the nonimmunized pigs between days 2 and 6 post-infection. Nasal swabs from one of the previously vaccinated pigs yielded virulent virus on both the 2nd and 4th day post-challenge. Consecutive nasal swabs collected from the other pig were positive only on the 4th day post-infection. The P-2208 chal- lenge virus was isolated between the 2nd and 8th day post-infection from the passively immune pig. Virus isolation at tempts from the control pigs were consistently negative.

Virus neutralizing antibodies were detected 7-14 days post-infection in the serum of the non immunized pigs that were exposed to the virulent P-2208 strain. Virus neutralizing ant ibody responses as a result of vaccination were fully developed at the t ime of challenge. A marked anamnestic response was apparent 7 days after exposure to the virulent virus. The virus neutralizing ant ibody titer also increased in the passively immune group following expo-

2 9 0 R.K. MAES ET AL.

Lotents:

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Fig. 6. Amplification of PRV sequences in latent ganglion DNA. gll primers were used to am- plify 5/tg of ganglion DNA or 1/zg of PRV (Rice)-infected CRFK DNA (positive control ). 40% of the product was slot blotted and probed with the gII subclone probe. Lanes 1 and 2 are repli- cate amplifications, lane 3 is unamplified DNA. Negative control is ganglion DNA from an uninfected pig.

sure to virulent PRV, indicating the development of an active immune re- sponse. The control pig remained serologically negative for PRV throughout the experiment.

Ganglion DNA from pigs latently infected with PRV (P-2208) and from uninfected control pigs was amplified with glI primers. Amplification prod- ucts were transferred to nitrocellulose by slot blotting and probed with the subcloned glI probe. Slot blotting was selected because it is faster and more convenient than gel electrophoresis and Southern blotting, and therefore will be more suitable to the eventual screening of large numbers of latency sam- ples. Two separate amplifications of the same samples are included on the blot in Fig. 6. By combining the results of both sets, seven of eight amplified latency samples showed hybridization signals above background.

DISCUSSION

A common feature of herpesvirus infections is that recovery from clinical disease is frequently followed by lifelong persistence of the virus in nervous and possibly lymphoid tissues (van Oirschot and Gielkens, 1984) of its host in a latent form. Reactivation of the latent genome and the associated viral shedding is a mechanism in the perpetuation of herpesviruses. In order to better assess the epidemiological significance of latency, it is essential to have both a sensitive and convenient detection method. Our goal is to use a hybrid- ization based probe-assay system to detect latent PRV in swine tissues.

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA 291

We have previously shown that 1-5 pg amounts of slot-blotted PRV target sequences can be detected by hybridization with both radioactive and non- radioactive probes (Maes et al., 1988). This level of sensitivity was still not sufficient to consistently detect PRV DNA sequences in latently infected tis- sues. The purpose of the experiments described in this paper was to show that target DNA amplification by PCR can significantly reduce this detection limit.

We designed primers for the amplification of regions of both the gX and gII genes of PRV. In initial amplifications of gX, the reaction product was char- acterized by size and by hybridization with the labeled BamHI 7 fragment. However, these methods were decided to be insufficient, as PCR often pro- duces non-specific amplification products that may cross-hybridize with the desired product or may coincidentally be of the same size as the expected fragment (Saiki et al., 1988; Abbott et al., 1988 ). Therefore, restriction diges- tion was used to verify the specificity of the amplified fragments from both the gX and gIl genes.

Both strains of PRV examined contained PRV gX and gII sequences that were amplified. It is unclear how well the amplified regions are conserved among PRV isolates. At the protein level there are good indications that PRV gX sequences are conserved (Molitor and Thawley. 1988 ). The PRV gII gene is homologous to the HSV gB gene, which is generally conserved amongst the herpesviruses (Hampl et al., 1984; Pellett et al., 1985; Snowden et al., 1985; Gong et al., 1987; Conraths et al., 1988). More strains will have to be exam- ined, however, to establish the general usefulness of these two particular primer sets.

The PCR technique requires a certain amount of optimization. It was found that the reaction became consistent only after opt ima for several of its param- eters were defined individually. Complete denaturation of the template ap- peared to be very important. Perhaps due to the high GC content of the am- plified region (67.4%), it was necessary to increase the recommended denaturation temperature of 94 ° to 95 ° C. This is the max imum temperature allowable for the enzyme to retain sufficient activity throughout 30 amplifi- cation cycles.

The Mg 2+ concentration of the PCR buffer has a fairly narrow opt imum. The results of the titration done for optimization of amplification of both the gX and gII gene fragments indicated that the op t imum Mg 2+ concentration was close to 1.5 mM. Highly purified DNAs of both viral and tissue origin were used in these experiments. At the point where the technique is simplified to incorporate the use of crude lysates Mg 2+ concentration may have to be optimized again.

Another at tempt to optimize the reaction was the use of an automated tem- perature cycling device. Direct comparisons between yields obtained in the manual and automated modes were not made as part of this study. Others have found that the success rate of amplification was significantly higher and

292 R.K. MAES ET AL.

more consistent when the reaction was automated (Kazazian and Dowling, 1988).

One of the potential applications of PCR is to detect latent PRV DNA in porcine tissues. We were able to detect the amplified product from 0.2 X 10-15g or less of purified viral DNA. This is an improvement in sensitivity by a fac- tor of at least 105 over unamplified DNA, where the detection limit with a 9.4 kb cloned PRV DNA probe was 1-10-~2g of homologous target sequences or 1.5 X 10-~lg of genomic PRV DNA. The theoretical relationship between the improved detection limit and the amount of viral DNA in latent tissues can be determined. In situ hybridization has been used to demonstrate that 1 in 400 neurons, implying 1 in 4000 ganglion cells, contains latent PRV. Given a molecular weight for PRV DNA of 90 X 106 daltons (Rubenstein and Kaplan, 1975) and a porcine cellular DNA content of approximately 5 X 10-12 g, it can be calculated that there should be 7.5 X 10-~Sg of latent PRV DNA per/tg of cellular DNA, well within our experimentally determined detection limit. This represents a m in imum estimate, assuming the presence of only a single copy of the PRV genome per latently infected cell. These calculations, com- bined with the experimentally determined detection limits, show the theoret- ical potential for detecting PRV DNA in latent tissues in quantities as low as 1 PRV genome in 106 cells.

As a practical test, a set of eight ganglion DNA samples, shown to contain latent PRV by in vitro explanation, were examined by PCR and compared to positive and negative controls. Although we have previously been able to de- tect latent PRV DNA in some ganglion DNAs, none of the unamplified sam- ples in this set yielded a detectable signal. A possible explanation is that only 2/~g of ganglion DNA was blotted, instead of the 10-20/tg used previously.

The calculated amount of amplified PRV DNA in the 2/xg samples assayed, assuming an amplification factor of 105, was approximately 8 pg. This is well within the detection limits of blot hybridization that we have previously de- termined. There was considerable variation in hybridization intensity be- tween samples. This variability could be the result of differences in sample quality effecting amplification efficiency, or of variations in the amount of latent PRV DNA between samples. Experiments currently in progress are ad- dressing these possibilities.

Under the amplification conditions used, six of eight samples were positive in each of two duplicate runs, and seven of eight when both runs are taken together. This inconsistency of amplification in some DNA samples has been noted by others (Kazazian and Dowling, 1988 ). The reasons for this are un- clear, but could be the result of unoptimized reaction conditions. We are in the process of adjusting our reactions to maximize amplification efficiency.

In vitro explantation, in situ hybridization and slot blot hybridization are the laboratory methods available to examine the presence of latent PRV in- fections. Optimized in vitro explantation methodology is capable of detecting

PCR AMPLIFICATION OF PSEUDORABIES VIRUS DNA 293

latent infections in most or all pigs experimentally inoculated with virulent PRV. Significant drawbacks of the explantation method are the requirement for sterile, viable tissue and the time interval between explantation and de- monstrable reactivation. In situ hybridization is an excellent method for ob- taining data on the cellular and sub-cellular location of latent PRV infections (Rock et al., 1988 ). Providing proper controls are included, sensitivity and specificity are high. This method is, however, labor intensive, technically de- manding and time consuming: exposure times following hybridization range from 1 to several weeks.

The drawbacks mentioned make neither in vitro explantation nor in situ hybridization ideally suitable for simultaneous screening of large numbers of samples. Blot hybridizations of tissue extracts would be faster and more con- venient for this purpose, but the amount of latent PRV DNA present in the 10-20/tg of total DNA typically used for these hybridizations is often too low to be consistently detected by conventional methods. PCR provides a sub- stantial increase in blot hybridization sensitivity and is faster than either in situ hybridization or in vitro explantation. By optimizing the reliability of PCR, adapting it to allow latent PRV amplification from crude tissue extracts and by incorporating the use of nonradioactive probes, it would appear to be the method of choice for the analysis of large numbers of samples.

ACKNOWLEDGEMENTS

We would like to than Dr. James Tiedje for the use of his thermocycler, Dan Sullivan for excellent technical assistance and Tony Randall for expert typing of the manuscript.

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