Vol. 30, No. 7JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p. 1661-16660095-1137/92/071661-06$02.00/0Copyright ©) 1992, American Society for Microbiology

Detection and Differentiation of Chicken Anemia VirusIsolates by Using the Polymerase Chain ReactionDANIEL TODD,* KAREN A. MAWHINNEY, AND M. STEWART McNULTYVeterinary Sciences Division, Department ofAgriculture for Northern Ireland,

Stormont, Belfast BT4 3SD, United Kingdom

Received 15 November 1991/Accepted 9 April 1992

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of theputative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplificationof CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by usinga 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNAsequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by aguanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involvingphenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany,Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of thePCR-amplified DNAs with the enzymes HaeIII, Hinfl, and HpaII indicated that the 14 CAV isolates can beassigned to seven groups, with isolates from different countries usually exhibiting the greatest number ofrestriction site differences.

Chicken anemia virus (CAV), formerly known as chickenanemia agent, causes a disease in young chickens character-ized by increased mortality, anemia, lymphoid depletion,and hemorrhages throughout the body (17). The agent hasrecently been identified as an icosahedral virus which mea-sures 25 nm in diameter and contains a circular, single-stranded DNA (2,300 bases) genome (4, 13). As such, itcannot be assigned to any known animal virus family (16).Serological evidence indicates that infection of chickens iscommon worldwide (6). Virus isolates originating in differentparts of the world belong to the same serotype and producethe same pathogenic effects in experimentally inoculatedchicks (5).We previously reported the development of a dot blot

hybridization assay for the detection ofCAV DNA in tissuesfrom infected birds (14). As a method of diagnosis, this assayprovided considerable savings in terms of time and laborover conventional procedures involving virus isolation intissue culture or in susceptible 1-day-old chicks. In thisreport, we describe the development of a polymerase chainreaction (PCR)-based enzymatic amplification method whichcan detect CAV DNA with greater sensitivity than can thedot blot hybridization assay. In addition, we report howrestriction endonuclease analysis of the amplified DNAfragments can be used to differentiate virus isolates.

MATERIALS AND METHODSV'iruses, cells, and virus growth. The Cux-1 isolate of CAV

and Marek's disease virus transformed chicken lymphoblas-toid (MDCC-MSB1) cells were obtained from V. von Bulow(Free University, Berlin, Germany). The Japanese isolatesGifu-1 and TK5803 were obtained from N. Yuasa (NationalInstitute of Animal Health, Tokyo, Japan) and M. Goryo(Hokkaido University, Sapporo, Japan), respectively. B.Engstrom (National Veterinary Institute, Uppsala, Sweden)provided the Swedish isolates 1/80 and 1/91. CAVs from theUnited States (isolate EF 88/78/276), the United Kingdom

* Corresponding author.

(isolates 87/10/44 and 87/11/52), and Australia (isolates 89/3711, 89/3713, and IMP704) were isolated in our laboratoryfrom clinical material sent from the countries of origin (3, 8,9). Viruses from Northern Ireland (isolates NI CAV-1 andNI CAV-2) and the Republic of Ireland (isolate EF 90-1)were also isolated in our laboratory. All virus isolates weregrown in MDCC-MSB1 cells as described previously (7).Virus isolates obtained as tissue culture lysates from otherlaboratories were given no more than three passages prior toDNA extraction and subsequent analysis. Viruses isolated inour laboratory were normally subjected to DNA analysisbefore 20 tissue culture passages had been reached.

Experimental infection. One-day-old specific-pathogen-free (SPF) chicks that were free of maternal antibody toCAV, as determined by the indirect immunofluorescencetest (7), were infected by intramuscular inoculation (0.1 ml)of the Cux-1 isolate of CAV (105 to 106 50% tissue cultureinfective doses per ml). Thymuses, which were previouslyshown to be the best source of CAV DNA in infected birds(14), were removed from infected and uninfected SPF chicksat 14 days posthatching.DNA extractions. DNA was extracted from uninfected

MDCC-MSB1 cells and MDCC-MSB1 cells infected witheach of the CAV isolates by using the method describedpreviously (14). DNA was extracted from thymus specimensby three methods. For each method, tissue pieces weighingbetween 0.01 and 0.05 g which were excised from eachthymus were used. With the phenol method, the tissue wasincubated in 100 ,ul of TE (0.001 M EDTA, 0.01 M Tris-HCI[pH 8.0]) containing 1% sodium dodecyl sulfate (SDS) andproteinase K (1 mg/ml; Sigma, Poole, United Kingdom) for 2h or overnight at 370C. Nucleic acid was extracted withphenol, phenol-chloroform-isoamyl alcohol (25:24:1), andchloroform-isoamyl alcohol (24:1). Following ethanol pre-cipitation at -200C, the nucleic acid was collected bycentrifugation at 12,000 x g for 10 min and dissolved in 10 ,ulof TE.The guanidine isothiocyanate method was performed by a

modification of the technique described by Boom et al. (1).This involved vigorous shaking of the thymus tissue with a

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lysis solution containing guanidine isothiocyanate and dia-toms for 2 h at room temperature. Undissolved fibrous tissuewas removed, and the diatoms were collected by centrifu-gation at 12,000 x g for 5 min. Following washing and itsrelease from the diatoms, the nucleic acid was precipitatedwith ethanol at -20°C, and after centrifugation it wasresuspended in 10 ,u1 of TE.The boiling method involved boiling of thymus samples

that were ground with a pestle and mortar in 1 ml of TE for15 min. The supernatant that was collected following cen-trifugation at 12,000 x g for 5 min was used directly for PCR.Thus, samples (1 ml) arising from the boiling method were100-fold more dilute than those (10 ,ul) obtained from eitherthe guanidine isothiocyanate or the phenol methods.

Nucleic acids were extracted from six thymuses fromexperimentally infected chickens by all three methods. Theboiling method was also applied to an additional six thy-muses of similar origin to achieve a fuller evaluation of thisprocedure.PCR conditions. The oligonucleotide primers 5'-GAC TGT

AAG ATG GCA AGA CGA GCT C-3' and 5'-GGC TGAAGG ATC CCT CAT TC-3' were specified by the sequenceof the double-stranded replicative-form DNA of the Cux-1isolate of CAV (9a) (EMBL accession number M81223) andflank a 675-bp fragment which encodes most of the N-termi-nal half of the putative gene for the CAV capsid protein.PCR was carried out in 50-pI reaction volumes, whichincluded 5-pI DNA samples, by using buffer components andTaq polymerase obtained from Promega (Dublin, Ireland).Deoxyribonucleotides (final concentration, 200 pM [each]deoxyribonucleotide) were obtained from Pharmacia, (Mil-ton Keynes, United Kingdom). Following an initial 2-mindenaturation step at 94°C, 50 thermal cycles, with each cyclecomprising 2 min at 94°C, 1 min at 50°C, and 2 min at 72°C,were carried out. A final extension period of 7 min at 72°Cpreceded storage of the reaction product at 4°C. The sensi-tivity of the PCR method was determined by subjectingserial (10-fold) dilutions of cloned CAV DNA to amplifica-tion (14). Prior to dilution, the cloned CAV DNA wasquantified by determination of the A260.Agarose gel electrophoresis. Following PCR amplification,

samples equivalent to one-fifth of each reaction mixtureWere analyzed by electrophoresis in 1% agarose gels asdescribed previously (15). DNA bands were stained withethidium bromide (1 pg/ml) and visualized by using a UVtransilluminator.

Hybridization. Samples of PCR product equivalent to1/100 of the PCR mixture were electrophoresed in 1%agarose gels, and the DNA was blotted by the method ofSouthern (12) onto a nylon membrane as described previ-ously (13). Dot blot hybridization was also performed byusing one-fourth of each PCR mixture as described previ-ously (14). For both hybridizations, a 32P-labeled clonedCAV-specific DNA probe was used (14).

Restriction endonuclease treatment. In those experimentsin which the PCR product was subjected to treatment withrestriction endonucleases, the reaction mixture containingPCR product was diluted by the addition of 50 p.1 of TE andthe amplified DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol(24:1) prior to ethanol precipitation at -20°C. Followingcentrifugation at 12,000 x g for 10 min, pellets of amplifiedDNA were dissolved in 25 or 50 pul of TE, and aliquots (2 to5 ,u1) were treated separately with each of the restrictionendonucleases HaeIII, Hinfl, and HpaII (GIBCO-BRL,Paisley, United Kingdom).

A 1 2 3 B 2 3bp bp

U- 4361

,2322. 2027

'S--564

FIG. 1. (A) Agarose gel electrophoresis of CAV-specific DNAfragment amplified by PCR. Ethidium bromide-stained DNA wasvisualized by UV light. Fragments of bacteriophage X DNA digestedwith HindIlI were included for size reference (lane 1). The productsgenerated by PCR amplification from DNA extracted from unin-fected (lane 2) and CAV (Cux-1 isolate)-infected (lane 3) MDCC-MSB1 cells were analyzed. (B) Southern blot hybridization ofCAV-specific DNA fragments amplified by PCR from extracts ofCAV (Cux-1 isolate)-infected MDCC-MSB1 cells (lane 2) and CAV(Cux-1 isolate)-infected thymus tissue (lane 1). Fragments of bacte-riophage X DNA digested with HindIII were included for sizereference (lane 3). Blots were hybridized with 32P-labeled clonedCAV DNA and X phage DNA probes.

Polyacrylamide gel electrophoresis (PAGE). Restriction di-gests of the PCR-amplified DNA fragment specified by eachof the 14 CAV isolates were analyzed by electrophoresis ingels of 10% polyacrylamide as described previously (13).Fragments generated by digesting the 4X174 (replicativeform) with HaeIII were used as Mr markers. After electro-phoresis at 40 V for 18 h, DNA bands were stained withethidium bromide (1 p.g/ml) or with silver by using theBio-Rad silver staining kit (Bio-Rad Laboratories Ltd.,Hemel Hempstead, United Kingdom).

RESULTS

PCR amplification. Analysis by agarose gel electrophore-sis indicated that a single DNA fragment (Mr 675) wasproduced when DNAs extracted from MDCC-MSB1 cellsinfected with each of the 14 CAV isolates were subjected toPCR enzymatic amplification (Fig. 1A). DNA fragments thatpossessed different electrophoretic mobilities were neverobserved by using these PCR conditions when DNAs ex-tracted from infected or uninfected tissue culture cells ortissue specimens were used for amplification. Southern blothybridization with the cloned CAV DNA probe confirmedthat the 675-bp fragment was CAV specific (Fig. 1B).Experiments designed to assess the sensitivity of the

PCR-based detection method indicated that PCR product(675-bp DNA fragment) amplified from as little as 10 or 100fg of target DNA (cloned CAV DNA) could be detected inethidium bromide-stained agarose gels following electropho-resis. When the dot blot hybridization assay with the 32p_labeled cloned DNA probe was applied, PCR product am-plified from as little as 1 fg and sometimes 0.1 fg of targetDNA could be dctected (data not shown).

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Comparison of DNA extraction methods. Three methods ofDNA extraction were evaluated for their suitability of usewith the PCR-based CAV DNA detection procedure. Theevaluation was performed by using pieces of thymus (0.01 to0.05 g) from CAV-infected and uninfected chicks. Thymusestaken from infected birds were shown to contain CAV-specific DNA by the direct use of the dot blot hybridizationassay and were regarded as positive.By the simple boiling method, none of 12 positive thymus

extracts or dilutions thereof produced an amplified PCRproduct that was detectable by ethidium bromide staining ordot blot hybridization. Both of the more complex methodsthat used phenol and guanidine isothiocyanate were success-ful in extracting CAV DNA suitable for PCR amplification.When half of the total extract was used as sample, DNAextracted by the guanidine isothiocyanate method allowedthe amplification of visually detectable PCR product in all ofthe six positive thymuses tested. This was not the case whenphenol-extracted DNAs were investigated. None of the sixthymus extracts produced visible PCR product when half ofthe total phenol extract was used as the sample. However,following dilution of these samples to 1/20 and 1/200 (asopposed to 1/2) of the total extracts, PCR product wasvisually detectable in two of six and six of six extracts,respectively. Thymus DNA samples that were extracted byeither the guanidine isothiocyanate method or the phenolmethod and diluted to 1/2,000 were capable of producing avisible PCR product. The final dilution of such samples was10-fold greater than that resulting from the boiling method ofextraction. This indicated that the lack of a productive PCRwith boiled samples was not due to the dilution of the extractthat was obtained. These results also supported the view thatthe levels of contaminating molecules were too high topermit enzymatic amplification when the less dilute phenolextracts were used. A visual comparison by using ethidiumbromide-stained agarose gels indicated that 5 to 10 timesmore DNA was extracted from thymus tissue samples by thephenol method than by the guanidine isothiocyanatemethod. Despite this finding, our results indicated that theguanidine isothiocyanate-based method, presumably be-cause of the increased purity that is achieved, is the moreefficient procedure for use with the amplification of CAVDNA by PCR.No PCR product was detected visually in ethidium bro-

mide-stained gels or by dot blot hybridization when DNAextracted from thymus samples obtained from SPF chickswas subjected to amplification. For control purposes, tissuesamples from SPF birds were routinely extracted withbatches of samples from experimentally infected, positivebirds. The absence of amplified DNA in these controlsamples indicated that the detection of PCR product inpositive samples was unlikely to be due to cross-contamina-tion.

Restriction endonuclease analysis. The restriction endonu-cleases HaeIII, Hinfl, and HpaII were selected for analysisafter consideration of the nucleotide sequence of the CAV(Cux-1 isolate) DNA, particular attention having been paidto the number and sizes of the fragments generated and theirease of separation by PAGE. The restriction maps for the675-bp PCR product are shown in Fig. 2. HaeIII, HinfI, andHpaII cleaved the 675-bp Cux-1 DNA fragment at five,three, and three sites, respectively.The PCR product (1 to 5 ,ug) recovered from two 50-,ul

reaction mixtures was usually sufficient for 10 to 20 restric-tion digests. Electrophoresis in gels containing 10% acryl-amide provided effective separation of DNA fragments in the

Hoe III

Hinf I

Hpo II

F1 C 2 A BE4 D S B38 113 238 58 67 161 bp

A D? s C251 60 227 137 bp

12B A C DD170 228 148 129 bp

FIG. 2. Restriction maps of the 675-bp CAV (Cux-1 isolate)DNA fragment amplified by PCR. The positions of the HaelII,Hinfl, and HpaII restriction sites and the fragment sizes (in basepairs) were predicted from the sequence of the CAV (Cux-1 isolate)replicative-form DNA (EMBL accession number M81223). Addi-tional HaeIII (sites 6 and 7 in fragment A) and Hinfl (sites 4 and 5 infragments A and C, respectively) restriction sites were identified forthe DNAs of other CAV isolates.

range of 30 to 300 bp which included most of the fragmentsgenerated by the three enzymes. The single-stranded oligo-nucleotide primers which migrated with electrophoretic mo-bilities equivalent to that of a 50-bp fragment and broadbands present in the top half of each gel, which varied withthe endonuclease used, and which were not stained byethidium bromide were disregarded for restriction analysispurposes.HaeIII proved to be the most useful enzyme, producing

six different patterns with the 14 isolates examined (Fig. 3).The absence of HaeIII restriction sites 1, 3, and 5 in theDNAs of certain isolates resulted in the detection of un-cleaved precursor DNA fragments Z, Y, and X, respectively(Fig. 3). The DNAs of some isolates possess additionalHaeIII restriction sites. For example, in the DNA of isolate

1 2 3 4 5 6 7 8 9 10I ~~~~~~~~bp

~~~ ~~~~~~~3 1 u~~~~1

0i@W -234i_<_ %__t194

6MI1872t#Kv

i;: *1f..

.: eI.0

FIG. 3. Restriction endonuclease (HaeIII) analysis of PCR(675-bp fragment) products amplified from DNAs specified byisolates from Germany (Cux-1; lane 1), Sweden (1/91; lane 2),United Kingdom (87/10/44; lane 3), Northern Ireland (NI CAV-1;lane 4), Republic of Ireland (EF 90-1; lane 5), United States (EF88/78/276; lane 6), Australia (89/3711 [lane 7]; IMP704 [lane 8]), andJapan (Gifu-1; lane 9). Following PAGE, DNA bands were silverstained. Fragments of 4~X174 (replicative form) digested with HaeIIIwere included for size reference (lane 10). The 271- and 281-bp4X174 (replicative form)-HaeIII fragments which are subject toanomalous electrophoretic behavior were left unmarked. Restrictionfragments are indicated by the letters to the right of the lanes, and anarrow indicates the position of the primer oligonucleotides.

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1 2 3 4 5 6 7 8 9 101bp

I ~1

rw;; - B

_M" T

-t-t

FIG. 4. Restriction endonuclease (Hinfl) analysis of PCR(675-bp fragment) products amplified from DNAs specified byisolates from Japan (TK5803 [lane 1]; Gifu-1 [lane 2]), Australia(89/3711; lane 3), United States (EF 88/78/276; lane 4), Republic ofIreland (EF 90-1; lane 5), Northern Ireland (NI CAV-1; lane 6),United Kingdom (87/10/44; lane 7), Sweden (1/91; lane 8), andGermany (Cux-1; lane 9). Following PAGE, DNA bands were silverstained. Fragments of -X174 (replicative form) digested with HaeIIIwere included for size reference (lane 10). See Fig. 3 legend fordescriptions of letters and arrow.

87/10/44 from the United Kingdom, fragment A is cleaved(HaeIII, site 6), yielding fragments W and V; and in theDNAs of the isolates from Northern Ireland and the Repub-lic of Ireland, fragment A is cleaved (HaeIII, site 7) toproduce fragment U, which has the same electrophoreticmobility as fragment X, and a small fragment, which isundetectable in this gel.

Hinfl and HpaII, each of which produce three and twotypes of patterns, respectively, allowed further differentia-

1 2 3 4 5 6 7 8 9

bp

s^^2.i4i310

6_A_-6.d234

~~4~~~-d 194

72

FIG. 5. Restriction endonuclease (HpaII) analysis of PCR prod-uct (675-bp fragment) amplified from DNAs specified by isolatesfrom Germany (Cux-1; lane 1), Sweden (1/91; lane 2), UnitedKingdom (87/10/44; lane 3), Northern Ireland (NI CAV-1; lane 4),Republic of Ireland (EF 90-1; lane 5), United States (EF 88/78/276;lane 6), Australia (89/3711; lane 7), and Japan (Gifu-1; lane 8).Following PAGE, DNA bands were silver stained. Fragments of4X174 (replicative form) digested with HaeIII were included for sizereference (lane 9). See Fig. 3 legend for description of letters andarrow.

tion of the isolates (Fig. 4 and 5, respectively). With Hinfl,the DNAs of the isolates from Northern Ireland and theRepublic of Ireland possess an additional site (Hinfl, site 5)by which fragment C is cleaved to produce fragments T andS. The DNAs of other isolates such as those from Sweden,Australia, the United Kingdom, and Japan (TK5803) appearto have been cleaved by Hinfl within fragment A (site 4) toproduce fragment R, which comigrates with fragment B, anda small fragment that was undetectable in the gel. The DNAsof the isolates from Northern Ireland and the Republic ofIreland also produced distinctive patterns after HpaII diges-tion. The lack of HpaII site 3 in their DNAs resulted in thedetection of fragment Q and the absence of fragments C andD. Examination of the results of HpaII analyses showed thatvariations exist in the electrophoretic mobilities of fragmentA generated from the DNAs of different isolates. Thus,fragment A derived from isolates from the Republic ofIreland and Australia were faster migrating than that derivedfrom the Cux-1 isolate, whereas fragment A derived from theisolate from the United States migrated more slowly. Similarvariation was detected in the electrophoretic mobilities offragment A generated by the endonuclease HaeIII (Fig. 3), afragment that maps in a region which largely overlaps that ofHpaII fragment A (Fig. 2). On the basis of the fact thatHpaII fragment A derived from the DNA of the U.S. isolateis apparently larger than its Cux-1 and Australian 89/3711counterpart, it is unlikely that differences can be attributedto the presence of additional restriction sites. It is thereforepossible that the observed variation in electrophoretic mo-bility is due to differences in the secondary structure of theseDNA fragments. In this connection it is worth noting that, incontrast to all other restriction fragments, the A fragmentsgenerated by HaeIII and HpaII both migrated less rapidlythan expected relative to the Mr standards. Anomalouselectrophoretic migration behavior was also observed withthe (X174 (replicative form)-HaeIII fragments (Fig. 3), forwhich there is uncertainty as to whether the 281- or 271-bpfragment comigrates with the 310-bp fragment.

Epidemiological considerations. Differences in the HaeIII,Hinfl, and HpaII restriction analyses of the PCR-amplified675-bp fragment allowed the 14 isolates to be subdivided intoseven groups (Table 1). CAVs which were isolated fromclinical specimens obtained from the same or closely relatedoutbreaks of disease were identical. This finding applied topairs of isolates from Northern Ireland (NI CAV-1 and NICAV-2), Australia (89/3711 and 89/3713), and the UnitedKingdom (87/10/44 and 87/11/52). Isolates from unrelatedoutbreaks within the same country could sometimes bedifferentiated. For example, the Japanese isolates Gifu-1 andTK5803 could be distinguished by using Hinfl, and theAustralian isolate IMP704 could be distinguished from theother isolates that originated in Australia by using theendonuclease HaeIII. In general, however, although theisolates from Japan, Germany, and Sweden were very sim-ilar, isolates from the other countries-Australia, the UnitedKingdom, the Republic of Ireland, and the United States-could be differentiated, sometimes, as in the case of isolatesfrom Northern Ireland and the Republic of Ireland, by asmany as three of the restriction endonucleases.

DISCUSSION

In this report we described for the first time how enzy-matic amplification by PCR can be used to sensitively detectCAV DNA in tissue extracts from infected birds. We also

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TABLE 1. Restriction endonuclease analysis of PCR-amplified DNAs specified by different CAV isolates

Isolate Country of HaeIII site Hinfl site HpaII siteno.orign Isolate -Groupno. origin 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3

1 Germany Cux-1 + + + + + + + + + + + 12 Japan Gifu-1 + + + + + + + + + + + 13 Japan TK5803 + + + + + + + + + + + + 24 Sweden 1/80 + + + + + + + + + + + + 25 Sweden 1/91 + + + + + + + + + + + + 26 United Kingdom 87/10/44 + + + + + + + + + + + 37 United Kingdom 87/11/52 + + + + + + + + + + + 38 Northern Ireland NI CAV-1 + + + + + + + + + + + 49 Northern Ireland NI CAV-2 + + + + + + + + + + + 410 Republic of Ireland EF 90-1 + + + + + + + + + + + 411 United States EF 88/78/276 + + + + + + + + + 512 Australia 89/3711 + + + + + + + + + + + 613 Australia 89/3713 + + + + + + + + + + + 614 Australia IMP704 + + + + + + + + + 7

showed that CAV isolates can be differentiated by usingrestriction endonuclease analysis of the amplified DNA.The PCR primers that were selected flank a 675-bp frag-

ment which encompasses the first half of the largest openreading frame present in either strand of the cloned 2.3-kbCAV replicative-form DNA (9a). On the basis of the size ofthe protein encoded by this open reading frame (Mr, 52,000)and the presence of a highly basic N-terminal region, whichmay have a DNA-binding function, we believe that this openreading frame codes for the single capsid protein for whichan Mr value of 50,000 has been determined by SDS-PAGE(13). We found that by using the primers described in thisreport, DNA specified by each of the 14 different isolates canbe amplified to produce a DNA fragment of the expected size(675 bp). Given that all the CAV isolates examined to datebelong to the same serotype and presumably possess similarprimary structures in their capsid proteins, this finding isperhaps not unexpected. We also found that, under the PCRconditions used in the study described here, DNA fragmentsof different sizes were never detected when DNAs frominfected or uninfected cells or tissues were subjected toamplification. The specific nature of the PCR product wasconfirmed by hybridization with a cloned CAV DNA probe.Previous work in our laboratory indicated that this probeproduces no hybridization signals with DNAs extracted fromuninfected tissues or from cells infected with a range of avianDNA viruses (14).The PCR-based method for detection of CAV DNA was

sensitive as well as specific. When it was used in conjunctionwith the dot blot hybridization assay, the PCR amplificationmethod allowed as little as 0.1 fg of the target DNA sequence(100 to 200 molecules) to be detected. It is possible that adifferent primer selection and further optimization of thePCR or dot blot hybridization conditions could achieve anadditional 10-fold increase in sensitivity to attain levels (10 to20 molecules) reported for other viruses such as avianpolyomavirus DNA (11). The PCR-based method that incor-porates dot blot hybridization is 103 to 104 times moresensitive than dot blot hybridization used alone (14). Thehigh sensitivity that is possible with this DNA amplificationmethod may be useful for research investigations into thevertical transmission of CAV through the egg or possiblevirus latency.The more simple procedure, in which the PCR product is

detected visually in ethidium bromide-stained gels, is 10 to100 times more sensitive than the dot blot hybridization

assay. In comparison with the existing method of CAVdiagnosis, virus isolation in tissue culture or in SPF chick-ens, the PCR-based method resembles the hybridizationassay in that it saves both time and labor (14). However, forthe amplification procedure to be used diagnostically undercircumstances in which large numbers of samples are exam-ined, additional precautions to avoid contamination must betaken (10). The increasing application of PCR technology inmolecular biological research should ensure that its use indiagnosis will become more widespread.Analyses of the 675-bp PCR products, using three restric-

tion endonucleases, indicated that CAV isolates can bedifferentiated, with the greatest number of restriction sitedifferences occurring between isolates from different coun-tries. These findings may be useful epidemiologically. Forexample, it is conceivable that strains used for vaccinepurposes may be distinguished from isolates circulating inthe field. Since the sequences recognized by the threeenzymes for cleavage make up 44 bp (11 restriction sites onCux-1 isolate DNA), a value which constitutes 7% of the675-bp sequence, it is difficult to assess the sequence diver-sity among CAV isolates over this DNA region. Comparisonof the sequence of a U.S. isolate recently published byClaessens et al. (2) with that of the Cux-1 isolate determinedin our laboratory indicated that of the 56 base changes whichoccurred throughout the genome (2,298 nucleotides), 19were located in the 675-bp region examined in this study(9a). For most diagnostic laboratories, however, sequencedeterminations for epidemiological purposes would not bepractical. Simplified restriction analysis performed withDNA amplified directly from tissue extracts may provide aviable alternative.

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