Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products

Journal of Virological Methods, 43 (1993) 233-246 ‘ix) 1993 Elsevier Science Publishers B.V. All rights

reserved

VIRMET

01510

/ 0166-0934/93/$06.00

Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products Jung-Chung

Lin, Barun Kumar

De and Seh-Ching

Molecular Biology Section, Hematologic Diseases Branch, Division of HIV/AIDS, Centers for Disease Control, Atlanta. GA (USA) (Accepted

27 January

Lin NCID, MS-DOZ.

1993)

Summary Two distinct wild-type Epstein-Barr virus (EBV) strains (A and B) that have significantly diverged at the two small RNA-encoding region (EBER) have been identified (Arrand et al., 1989). In order to test whether single-strand conformation polymorphism analysis (SSCP) would correlate with these sequence variations, we designed primer pairs specific for EBER-encoding regions for amplification of divergent sequences by polymerase chain reaction (PCR). The PCR-amplified products from six EBV-positive cell lines were analyzed by SSCP method, and the results were compared with the prototype strains B958 (type A) and AG876 (type B). Type-specific point mutations were detected as demonstrated by shifts in mobility due to conformational changes of DNA sequences. The locations of point mutations were identified by direct sequencing of the PCR amplified DNA. Of the three primer pairs designed, the pair that amplified a 190 bp fragment spanning six type-specific point mutations gave the best resolution in SSCP analysis. This pair is now preferred for initial genotyping of EBV-infected tumor biopsies. Thus, SSCP is a simple, fast and efficient technique for genotyping of EBV-associated diseases. Epstein-Barr virus; Genotyping; Polymerase chain reaction; Single-strand conformation polymorphism analysis

Correspondence to: HIV/AIDS,

NCID,

J.-C. Lin, Molecular Biology Section, Hematologic Diseases Branch, MS-DO2, Centers for Disease Control, Atlanta, GA 30333, USA.

Division

of

234

Introduction Epstein-Barr virus (EBV), the etiologic agent of infectious mononucleosis (Henle et al., 1979) has a clearly established association with endemic Burkitt’s lymphoma (de-The, 1980), nasopharyngeal carcinoma (Henle et al., 1985) secondary B-cell proliferation in immunosuppressed individuals (Purtilo et al., 1981) and a wide range of other clinical conditions. Based on the organization of the BamHI WYH gene region that encodes for EBV nuclear antigen 2 (EBNA-2), two distinct types of EBV (type A and B) have been identified (Adldinger et al., 1985; Dambaugh et al., 1984; King et al., 1982). The distinction between type A and type B EBV isolates by the immunoblotting method has extended beyond the EBNA-2 gene to the EBNA-3 family of proteins (EBNA-3a, 3b, and 3c) (Rowe et al., 1989) which was further confirmed by DNA sequence analysis (Sample et al., 1990). Analysis by restriction fragment-length polymorphism (RFLP) in the two small RNAencoding regions (EBERs) of EBV has also demonstrated type-specific cleavage patterns between type A and B (Arrand et al., 1989). The specificity of RFLP analysis was conlirmed by DNA sequencing that detected single-base changes (Arrand et al., 1989). Biologic differences between type A and B strains have been noted (Rickinson et al., 1987). Cell lines containing type B virus display a lower growth rate and a significantly lower saturation density compared to cell lines containing type A virus (Rickinson et al., 1987). These differences have been attributed to the divergent forms of the EBNA-2 gene which accounts for the reduced transformation capacity of the type B viruses, making the establishment of B-type EBV cell lines difficult. The type A virus has a world-wide distribution and is regarded as the overwhelmingly predominant strain in Western communities. In contrast, type B virus has been found mainly in Central Africa and New Guinea (Young et al., 1987; Zimber et al., 1986). However, recent studies of throat washings from both normal and HIV-infected persons by polymerase chain reaction (PCR) have found that type B virus infection in a Western population may be more prevalent than previously believed (Sixbey et al., 1989). As part of our studies to assess the role of EBV and its subtypes in EBVassociated hematologic disorders including Hodgkin’s lymphoma (HL), we have designed primer pairs specific to EBER-encoding regions for single-strand conformation polymorphism analysis (SSCP) (Orita et al., 1989). The EBER regions were specifically selected for SSCP analysis because within the chosen regions the polymerase chain reaction (PCR) amplified products encompass strain-specific point mutations (Arrand et al., 1989) but do not contain strainspecific deletions. The purpose of this study was to establish the feasibility of employing PCR-based SSCP analysis to determine if genetic variations among EBV isolates can be detected by this technique. We have adapted and optimized this technique for initial quick genotyping using a panel of cell lines infected by known EBV strains. Type-specific point mutations in the EBER

235

region were identified and confirmed by direct sequencing of the PCRamplified products. The results of EBER genotyping by the PCR-SSCP method correlated with EBNA-2 typing data.

Materials and Methods Cell cultures

The lymphoblastoid cell lines, B95-8, AG876, BJAB, P3HR-l(LS), Raji, Jijoye, and BJAB/GC, were propagated as described (Lin et al., 1982). Burkitt hybrid cells, D98/HR- 1 (a gift from Dr. Ronald Glaser, Ohio State University), were grown in Eagle’s minimal essential medium with 10% fetal calf serum. Isolation of DNA DNA was isolated by incubation for 3 h at 50°C in a digestion buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, 0.5% Sarkosyl, and proteinase K (0.1 mg/ml)) followed by phenol-chloroform extraction and RNase A (100 pg/ml) treatment. Selection

qf sequences for primers and probe

The nucleotide sequences in the EBER region were examined, and the oligonucleotide sequences for primers and probe were selected (Fig. 1). The 20base primers were designated Pl : 5’-GTGGTCCGCATGTTTTGATC-3’ (nucleotide positions 6780-6800) P2: 5’-GCAACGGCTGTCCTGTTTGA-3’ (6969-6950) P3: 5’-GTGTCTACCTGAACTAAGAC-3’ (7269-7251) and P4: 5’-CCTAGTGGTTTCGGACACAC-3’ (6970-6989); probe: 5’-AACGGGGCTTAACGTTGCAT-3’ (6850-6869). F or EBNA-2 typing, we selected a primer pair spanning a region that has 14 type-specific point mutations and a 16 bp deletion in B95-8 strain. The primers were designated E5: 5’AGGCTGCCCACCCTGAGGAT-3’ (48 162-48 18 1); E3: 5’-GCCACCTGGCAGCCCTAAAG-3’ (48330-483 11). A 19-mer internal probe (5’-GTTGCCGCCAGGTGGCAGC-3’; 48253-4827 1) with a common sequence between AG876 and B95-8 strains was selected. PCR ampllyication Aliquots of 0.1 pug of genomic DNA were amplified manufacturer’s protocol (Perkin-Elmer Cetus). The mixtures 35 cycles of amplification (30 s at 94°C 30 s at 57°C 1 min cycling, the samples were denatured at 94°C for 5 min. After polymerization step was extended by 10 min.

according to were subjected at 72°C). Prior the last cycle,

the to to the

236

Southern blot PCR amplified products were analyzed by 2% agarose gel electrophoresis and Southern blotted onto a Hybond-N paper (Amersham). The sheets were hybridized with an internal 5’-end labeled oligo probe. PCR-SSCP

analysis (Orita et al., 1989)

The primers (20 pmol) were 5’-end labeled with [y-“P]dATP (3000 Ci/mmol, NEN) and polynucleotide kinase in 5 ~1 of 50 mM Tris-HCl, pH 8.3, 10 mM MgC12, and 5 mM DTT at 37°C for 30 min. Aliquots of 0.1 pg of genomic DNA was amplified in a total volume of 5 ~1 containing 0.25 pmol each of the labeled primers, 2 nmol each of the four deoxynucleotides, and 0.25 units of Taq polymerase. The amplification conditions were the same as above. After completion of the reaction, 45 ~1 of formamide loading buffer (95% formamide, 10 mM EDTA, 0.05% each of bromophenol blue and xylene cyanol) was added to the reaction mixture. The mixture was heated at 80°C and applied (2 @ane) to 5% polyacrylamide gel containing 45 mM Trisborate, pH 8.3, 1 mM EDTA, and 5% glycerol. Electrophoresis was performed at 40 W for 5 h with a cooling fan. The gel was dried and exposed to X-ray film. Direct DNA sequencing of PCR products The PCR products of expected fragments were purified and sequenced directly by the dideoxy-termination method @anger et al., 1977) using the Sequenase Version II kit (US Biochemicals, OH).

Results Determination

of spectficity qf PCR products

In order to amplify the DNA sequences in the EBER region, primers complementary to regions containing restriction endonuclease polymorphisms were designed for PCR (Fig. 1). With primer pair Pl/P3, a 490 bp fragment was amplified from genomic DNA of B95-8 (Fig. 2, panel A, lane 2) and AG876 (lane 3); a band of identical size was also amplified from a DNA sample of Hodgkin’s lymphoma (HL) (lane 1). Similarly, with primer pair Pl/P2, a 190 bp fragment was detected in all cases (lane 4, HL; lane 5, B95-8; lane 6, AG876). No similar bands were amplified from DNA of peripheral blood of a healthy donor (lane 7). Some other bands in the higher molecular weight region were also detected in the HL sample. To determine the specificity of these bands, Southern blot hybridization was performed using an internal 5’-end labeled probe. In all cases, this probe detected only the 490 bp fragment amplified by Pl/P3 and the 190 bp fragment amplified by Pl/P2 (Fig. 2, panel

237

B95-8 AG676

T A PIPI -190

GTAG AGGA

G A

A G

490bp -pa bp e

~2 -

Fig. I. Summary strategy for PCR sites of sequence 7123, 7198,

CA AG

300bp-

~3

of the sites of sequence variation in the EBER region of two prototype amplification. The hatched boxes are the coding sequences for the two variation and the positions are labeled as follows: 6808, 6884, 6886, and 7231. This chart was adapted from a previous report (Arrand et

EBV strains and small RNAs. The 691 I, 6927, 6944, al., 19893).

B). The other bands were not hybridized by this probe, and therefore were nonspecific. Hybridization thus confirmed that the 490 bp and 190 bp fragments visualized by ethidium bromide staining represent the authentic PCR products of the primer pairs. With primer pair P4/P3, a 300 bp fragment was amplified, and the specificity of this band was also confirmed by Southern blot hybridization (data not shown).

Fig. 2. Determination of specificity of amplified products by Southern-blot hybridization. Genomic DNA samples were amplifed using two primer pairs (Pl/P2, Pl/P3), and the amplified products were analyzed by ethidium bromide staining (panel A) and by Southern-blot hybridization (panel B). Lane I and 4, HL sample; lane 2 and 5, AG876; lane 3 and 6, B95-8; lane 7, BJAB. Samples in lane I, 2, and 3 were the products amplified by primer pair Pl/P3, and in lane 4, 5, and 6 were from the products generated by PI/ P2. Mixture of PI/P2 and Pl/P3 was included in the control DNA in lane 7. An internal probe with common sequence for both 190 bp and 490 bp fragment was 5’-end labeled and hybridized to the Southern blot (panel B) as detailed in the text. M, molecular weight marker (HaeIII-digested x174 DNA).

238

Gemtyping

by PC’R-SSCP

As shown in Fig. 2, the agarose gel electrophoretic analysis could not differentiate the PCR products of the same size amplified from B95-8 (type A) and AG876 (type B) DNAs. To rapidly detect type-specific genetic polymorphisms of EBV from multiple samples without DNA sequencing, we applied PCR-SSCP analysis. Each primer set (Pl/P2, Pl/P3, and P4/P3) was labeled at the S-end, and was included in the PCR mixture. The products were denatured and applied to a non-denaturing polyacrylamide gel. Fig. 3 shows two distinct patterns of shift in mobility due to conformational changes of DNA sequences. DNA from Raji (lane 3) and P3HR-l(LS) cells (lane 6) exhibited a pattern of mobility similar to DNA from B95-8 cells (lane 1, EBNA-2 type A), whereas D98/HR- 1 (lane 4) and Jijoye DNA (lane 5) produced similar patterns but with a subtle difference in mobility to AG876 DNA (lane 2, EBNA-2 type B). The difference in mobility were also observed in 300 bp- and 400 bp-fragments. Of the thee primer pairs designed, the pair that amplified a 190 bp fragment (Pl/ P2) gave the best resolution in SSCP analysis (Fig. 3, panel A). Thus, this pair is now preferred in our molecular epidemiologic studies of EBV-associated diseases. Specificity

of single-strand

DNA

To facilitate the identification of each band that represents single-strand DNA prior to electrophoresis, PCR products amplified with both labeled

123456

123456

C

123456

190 bp Fig. 3. PCR-SSCP analysis of point mutations of EBER gene region with various primer sets. Three primer pairs (Pl/P2, Pl/P3, and P4/P3) were 5’-end labeled and included in each of the PCR reaction mixture. The amplified products were denatured and subjected to SSCP analysis. Panels A, B, and C were amplified products of 190 bp, 300 bp, and 490 bp using primer pair Pl/P2, P4,‘P3, and Pl/P3, respectively. Lane I, 895-8; lane 2, AG876; lane 3. Raji; lane 4, D98/HR-I; lane 5, Jijoye; lane 6, P3HR-l(LS).

239

1234

A

_'a

1234

B

1234 ," _/

c

1234

D

Fig. 4. Determination of specificity of single-strand DNA. Primer pair PljP2 was used to amplify 190 bp fragment for SSCP analysis. In panel A, both primers were 5’-end labeled and the PCR amplified products were heat denatured in formamide buffer prior to electrophoresis. Panels B and C were the amplified products of primer pair PI/P2 using either labeled PI (panel B) or labeled P2 (panel C) in the PCR reaction mixture. Panel D, the same products derived from both primers labeled as in panel A except the samples were not heat denatured in formamide buffer. Lane 1, B95-8; lane 2, AG876; lane 3, D98/HR-1; lane 4, Jijoye. ds and ss stand for double strand and single strand.

primers (Pl/P2) were either heat-denatured in formamide buffer or not denatured by placing in loading buffer minus formamide. As expected, nondenatured double-stranded DNA migrated as a single band with faster mobility than single-stranded DNA (Fig. 4D). In contrast, the denatured DNA resolved into two bands (Fig. 4A; lane 1, B95-8; lane 2, AG876; lane 3, D9S/ HR-1; and lane 4, Jijoye). Again, the subtle differences in mobility among type B viruses (lanes 2, 3, and 4) were detectable. These subtle differences in mobility reflected minor base changes as confirmed by DNA sequencing (Table 1). To determine whether the faster moving band derives from the sense or antisense strand of DNA, we labeled either sense or antisense primer for PCR amplification. When sense primer complementary to the antisense strand was labeled, only the faster moving band was detected (Fig. 4B). In contrast, the slow-moving band was detected with labeled antisense primer (Fig. 4C). These results, taken together, indicate that each band represents single-stranded DNA, and the sense strand moves faster than the antisense strand. Type-spec$c characteristic

differences in the EBNA-2 gene between B95-8 and AG876 are of other type A and B isolates

We determined whether the sequences selected for the primer pairs were common to other type A and B strains, and whether the results of EBNA-2

240

TABLE

1

Sequence analysis of EBER Cell line

region

of DNA

Substitution

B95-8 P3HR-l(LS) Raji D98/HRl Jijoye AC876

from

EBV-infetected

cell lines.

at positions*

6808

6884

6886

6911

6927

6944

T(J) T(f) T(f) A(2) A(2) A(2)

G(f) G(f) G(f) A(2) A(2) A(2)

T(t) T(1) T(f) G(2) G(2) G(2)

A(f) A(1) A(1) G(2) G(2) G(2)

G(f) G(f) G(f) G(J) G(1) A(2)

G(f) G(f) G(f) A(2) A(2) A(2)

*PCR amplified 190-bp fragment was directly suquenced as detailed **EBNA-2-encoding region is deleted in P3HR-I DNA. however,

P3HR-I.

Consensus

EBNA-2

type

type

1 1 1

A

2

2 2 2

(BB)** B

in the text.

Jijoye, the parental

strain of

is a type B virus (Adldinger et al., 1985).

genotyping agreed with those generated by PCR-SSCP specific primers generated the predicted amplified products

1

6

analysis. EBNA-2of 184 bp for type B

23456709

8, I.

-184 bp +168 bp

Fig. 5. EBNA-2 genotyping of EBV DNA from different cell lines. The primers chosen for this study generated a fragment of 168 bp from cell lines containing type A virus and 184 bp fragment from cell lines with type B strain. Panel A, ethidium bromide staining: lane 1, BJAB; lane 2, D98/HR-1; lane 3, HL; lane 4, Raji; lane 5. P3HR-l(LS); lane 6, Jijoye; lane 7, AG876; lane 8, B95-8. Panel B, Southern-blot hybridization: lane I, B95-8; lane 2, D98/HR-1; lane 3, Raji; lane 4, P3HR-I(LS); lane 5, AG876; lane 6, Jijoye; lane 7, BJAB/GC; lane 8, HL sample; lane 9, BJAB. Note that D98/HR-1 has a deletion in EBNA-2 gene.

241

and 168 bp for type A, which were easily detectable by ethidium bromide staining of the gel (Fig. 5A). In a separate experiment, hybridization of a Southern blot to an internal probe common to both type A and B revealed a single band in each lane of either 184 bp for each type B strain or of 168 bp for each type A virus (Fig. 5B). These results indicated that the amplified DNAs had the expected type-specific deleted sequences in type A viruses. The absence of a signal in D98/HR-1 (Fig. 4B, lane 2) confirmed that this hybrid cell line originally derived from fusion of a variant of HeLa cells with P3HR-1 cells, an EBV-infected cell line known to have a deletion in the EBNA-2 gene (Bornkamm et al., 1982; Hayward et al., 1982; King et al., 1982; Rabson et al., 1982). P3HR- 1(LS) cells derived from cloning of P3HR- 1 cells in low-serum

TYPE

A

TYPE

B

GATC

6886 6884

Fig. 6. Examples of direct DNA sequencmg of PCR products of EBER gene region showing sites of base transition and transversion between a type A virus (B95-8) and a type B virus (AG876). PCR amplified products were puritied and sequenced directly by dideoxy-termination method using the Sequenase as described in the text. Arrows indicate the base changes, and the nt positions are labeled on the left.

242

conditions lane 1).

(Lin et al., 1986) had type A sequence in the EBNA-2 gene (Fig. 5B,

Direct DNA sequencing of’ PCR products To assess whether the type-specific mobility shifts observed in the SSCP analyses resulted from type-specific point mutations within the DNA fragment, we directly sequenced the 190 bp fragment. A comparison of the sequences of the type A virus (B95-8) and type B virus (AG876) revealed six point mutations at nucleotide positions 6808, 6884, 6886, 6911, 6927, and 6944 (Fig. 1). A representative DNA sequencing result is shown in Fig. 6 (only five point mutations are shown). Among these six point mutations, three positions (nt 6884, 6927, and 6944) had G in type A virus substituted by A in type B virus. In type A virus, the bases at 6911 and 6886 were both changed to G from A and T, respectively, whereas at 6808, from T to A. The results of DNA sequencing of six EBV-positive cell lines are summarized in Table 1. B95-8 and AG876 cells exhibited two distinct patterns of nucleotides at six positions. From these nucleotide patterns the EBV DNA in the EBER region (EcoRI-J fragment) could be assigned to the B95-8 (type 1) or AG876 (type 2) family. Any pattern variations from analysis of other EBV-infected cell lines and clinical samples were compared with these two prototypes as a standard. It is evident (Table 1) that the two sequence families within the 190-bp fragment correlated well with the results obtained by PCR-SSCP analysis (Fig. 3 and 4) and by EBNA-2 typing (Fig. 5). There was a small variation at nt position 6927; both D98/HR-1 and Jijoye had an A to G transition at this nt position. However, this minor variation (1 out of 6) did not appear to affect arriving at the ‘consensus type’.

Discussion Distinguishing type A and B virus isolates at the DNA or at the protein level requires EBNA-2 type-specific probes (Zimber et al.. 1986) or antiserum capable of recognizing structurally distinct EBNA-2 proteins (Rowe et al., 1985; Sauter et al., 1987). Restriction fragment length polymorphisms (RFLP) have been used as epidemiological markers for genotyping of natural EBV isolates associated with various diseases (Katz et al., 1986; Katz et al., 1988; Lung et al., 1988; Lung et al., 199 1). This method requires restriction endonuclease digestion of DNA followed by Southern blot hybridization, a tedious and time-consuming procedure. The identification and analysis of DNA polymorphisms has been greatly facilitated by the development of PCR (Saiki et al., 1988), a method that rapidly and exponentially amplifies the specific target sequences located between two primers. The PCR-based SSCP method applied in this study for genotyping has proven to be not only simple and fast, but also highly sensitive for multiple sample analysis. Under the assay conditions, the single strands assume a secondary structure by folding back on

243

themselves in a sequence-dependent manner. A single-base change can alter this secondary structure and, consequently, the electrophoretic mobility. By using this method we were able to detect the differences between type A and B virus in the EBER encoding region using the primers spanning type-specific point mutation sites (Fig. 1). In PCR-SSCP analysis, these base substitutions were visualized as shifts in mobility due to conformational changes of DNA sequences (Figs. 3 and 4). However, the ability of each single-base change to alter the sequence-dependent conformation of the single strands is unknown. The technique, although powerful and sensitive, has its inherent limitations. For instance, a particular point mutation might not be detected if it occurred in a loop or in a long stable stem of the secondary structure. Thus, single-base changes that do not alter the conformation of the single strands would remain undetectable by SSCP. Heteroduplex analysis (White et al., 1992), an alternative method to detect single-base substitutions, does not rely on secondary structure formation. Another limitation is that the base substitutions in a fragment as long as 490 bp would be difficult to resolve electrophoretically, as illustrated in Fig. 3. This problem can be resolved by digesting the PCR products with the appropriate restriction enzyme and analyzing the fragments. The primer pair (PIP2) that amplified a 190 bp fragment encompassed six point mutations clustered in the ‘spacer’ region between the termination site of EBER-I and the initiation point of EBER-2 (Fig. 1). In PCR-SSCP analysis, this fragment gave the most striking characteristics of type-specific mobility shifts (Figs. 3A and 4). DNA sequencing analysis of 190 bp fragment has revealed that the pattern of six single-base changes (nt positions: 6808, 6884, 6886, 6911, 6927, and 6944) within this fragment is specific for each viral genotype (Table l), which can be assigned to either the B95-8 (type 1) or AGE76 (type 2) family. Thus, this fragment is a suitable genetic marker for EBV genotyping. In this connection, the optimal length of fragment for high resolution in this assay is approximately 200 bp containing at least one singlebase change. The sequences from P3HR- 1(LS) and Raji cells were identical to that of B958, whereas those of D98/HR-1 and Jijoye cells were similar to the sequences of AG876, except at nt position 6927 where A was substituted by G in both cases. It is not known whether the occurrence of the A to G transition at nt position 6927 found in the somatic hybrid cell line (D98/HR-1) was inherited from the parental line Jijoye, nor it is clear whether this change has any biological significance. The present results indicate that the sequence variations in the EBER region were clearly nonrandom since they either belong to B95-8 or AG876. It should be noted that P3HR-l(LS) cells were derived from P3HR-1 by lowserum cloning (our unpublished data). The heterogeneous EBNA-1 staining patterns observed previously (Freese et al., 1977) indicate that P3HR-1 may contain a mixture of viruses. Indeed, under our assay conditions a residual type A sequence in EBNA-2 gene was detectable (Lin et al., unpublished data). It is

244

likely that under the low-serum conditions (0.3% FCS) the type A virus might have been selected as a result of growth advantage of this virus (Rickinson et al., 1987). In SSCP analysis, the variants within type 2 (AG876, D98/HR-1, and Jijoye) that differ only in one base were distinguishable based on the banding patterns and mobility shifts (Fig. 3, compare lanes 2, 4, and 5 in panel A; these subtle differences were more discernible in gels with shorter exposure). By DNA sequencing analysis, six single-base changes were identified (Table 1). Based on the patterns of these single-base changes detected in the 190 bp fragment, we arrived at the ‘consensus type’ and grouped the strains into two sequence families (type 1 and 2). Our results are in agreement with that of previous report (Arrand et al., 1989) indicating that the base changes in EBER region are type-specific. In order to determine whether the two sequence families within the 190 bp fragment correlated with EBNA-2 type, the primer pairs for EBNA-2 amplification were so chosen that the resultant amplified products of type A and B could be readily distinguished based on the size without hybridization. In addition to the size difference in PCR products, the hybridization can be carried out under the same conditions for both type DNAs, which is impossible with the primers reported by Sixbey et al. (1989). Their primers encompassed sequences highly divergent between two types. Distinguishing of EBV types of PCR products by hybridization had to be done individually since the optimal conditions of hybridization were different for each type-specific probe (Sixbey et al., 1989). The EBNA-2 typing (type A or B) of the strains correlated well with the EBER typing. Our results were in agreement with that of a previous report (Arrand et al., 1989) indicating that EBNA-2 type A DNA regularly contained EBER type 1, while EBNA-2 type B DNA generally carried EBER type 2. However, some exceptions were found in cell lines, which carried a type 2 EBER region and a type A EBNA-2 gene, whereas other cell lines contained a type 1 EBER region and a type B EBNA-2 gene (Arrand et al., 1989). These ‘mixed’ viral sequences were interpreted as the possible recombination between a type A and a type B virus since dual EBV infections do occur naturally (Bornkamm et al., 1982; Katz et al., 1986; Lung et al., 1988; 1991; Sixbey et al., 1989). This study has established the feasibility of PCR-SSCP analysis in genotyping of EBV. Because of the rapidity, simplicity and sensitivity of the method we envision a large number of applications. The method has several advantages over other methods, which should make it applicable to molecular epidemiologic studies, diagnostic virology, and various other experiments in which it is desirable to screen multiple samples for EBV DNA.

References Adldinger,

H.K.,

Delius,

H.,

Freese,

U.K.,

Clarke,

J. and

Bornkamm,

G.W.

(1985)

A putative

245 transforming gene Jijoye virus differs from that of Epstein-Barr virus prototypes. Virology 14, 221-234. Arrand, J.R., Young, L. and Tugwood, J.D. (1989) Two families of sequences in the small RNAencoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B. J. Viral. 63, 9833986. Bornkamm, G.W., Hudewentz, J., Freese, U.K. and Zimber, U. (1982) Deletion of the nontransforming Epstein-Barr virus strain P3HR-1 causes fusion of the large internal repeat to the DSI region. J. Viral. 43, 9522968. Bornkamm, G.W., Knebel-Doeberitz, N.V. and Lenoir, G.M. (1984) No evidence for differences in the Epstein-Barr virus genome carried in Burkitt lymphoma cells and nonmalignant lymphoblastoid cells from the same patients. Proc. Natl. Acad. Sci. USA 81, 4930-4934. Dambaugh, T., Hennessy, K., Chamnankit, L. and Kieff, E. (1984) U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2. Proc. Natl. Acad Sci. USA 81, 7632-~7636. de-The, G. (1980) Role of Epstein-Barr virus in human diseases: infectious mononucleosis, Burkitt’s lymphoma and nasopharyngeal carcinoma. p 7699797. In Klein, G. (ed.) Viral oncology. Raven Press, New York. Freese, U.K., Merkt, B., Bornkamm, G.W. and zur Hausen, H. (1977) Heterogeneity of EpsteinBarr virus originating from P3HR-1 cells. I. Studies on EBNA induction. Int. J. Cancer 19, 317323. Hayward, S.D., Lazarowitz, S.G. and Hayward, G.S. (1982) Organization of the Epstein-Barr virus DNA molecule. II. Fine mapping of the boundaries of the internal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-I deletion. J. Viral. 43, 201~212. Henle, G. and Henle, W. (1979) The virus as the etiologic agent of infectious mononucleosis. p 257320. In M.A. Epstein and B.C. Achong (ed.), Epstein-Barr virus. Springer-Verlag K.G. Berlin. Henle, W. and Henle, G. (1985) Epstein-Barr virus and human malignancies. pp. 2Oll238. In: G. Klein (ed.), Advances in Viral Oncology. Raven Press, New York. Katz, B.Z., Andiman, W.A., Eastman, R., Martin, K. and Miller, G. (1986) Infection with two genotypes of Epstein-Barr virus in an infant with AIDS and lymphoma of the central nervous system. J. Inf. Dis. 153, 6Oll604. Katz, B.Z., Niederman, J.C., Olson, B.A. and Miller, G. (1988) Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts. J. Inf. Dis. 157, 2999308. King, W., Dambaugh, T., Heller, M., Dowling, J. and Kieff E. (1982) Epstein-Barr virus DNA. XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-I deletion. J. Virol. 43, 9799986. Lin, J.C., Smith, M.C and Pagan0 J.S. (1982) Effect of l2-O-tetradecanoyl-phorbol-l3-acetate on cell proliferation and Epstein-Barr virus DNA replication. Virology 117, 186-194. Lin, J.C., Nelson, D.J., Lambe, C.U. and Choi E.I. (1986) Metabolic activation of 9([2-hydroxy-l(hydroxymethyl)ethoxy]methyl)guanine in human lymphoblastoid cell lines infected with Epstein-Barr virus. J. Virol. 60, 5699573. Lung, M.L., Chang, R.S. and Jones J.H. (1988) Genetic polymorphism of natural Epstein-Barr virus isolates from infectious mononucleosis patients and healthy carriers. J. Viral. 62, 3862-3866. Lung, M.L., Lam, W.P., Sham, J., Choy, D., Zong, Y.S., Gou H.Y., and Ng M.H. (1991) Detection and prevalence of the ‘f variant of Epstein-Barr virus in Southern China. Virology 185, 67-71. Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874-879. Purtilo, D.T., Sakamoto, K., Saemundsen, A.K. (1981) Documentation of Epstein-Barr virus infection in immunodelicient patients with life threatening lymphoproliferative disease by clinical, virological and immunopathological studies. Cancer Res. 41, 42264235. Rabson, M., Gradoville, L., Heston, L. and Miller, G. (1982) Non-immortalizing P3J-HR-I Epstein-Barr virus: a deletion mutant of its transforming parent Jijoye. J. Viral. 44, 834-844. Rickinson, A.B., Young, L.S. and Rowe, M. (1987) Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells. J. Virol. 61, I3 IO-1 3 17.

246 Rowe, D., Heston, L., Metlay, J. and Miller, G. (1985) Identification and expression of a nuclear antigen from the genomic region of the Jijoye strain of Epstein-Barr virus that is missing in its non-immortalizing deletion mutant, P3HR-I. Proc. Natl. Acad. Sci. USA 82, 7429-7433. Rowe, M., Young, L.S., Cadwallader, K., Petti, L., Kieff, E. and Rickinson, A.B. (1989) Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 28) isolates extends to the EBNA 3 family of nuclear proteins. J. Virol. 63, 1031~~1039. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487488. Sample, J., Young, L.S., Martin, B., Chatman, T., Kieff, E., Rickinson, A.B. and Kieff, E. (1990) Epstein-Barr virus type I and 2 differ in their EBNA-3A, EBNA-3B and EBNA-3C genes. J. Virol. 64, 40484092. Sanger, F., Nicklen, S. and Coulson A. (1977) DNA sequencing with chain terminating inhibitors. Proc. Nat]. Acad. Sci. USA 74, 546335467. Sauter, M. and Mueller-Lantzsch, N. (1987) Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera. Virus Res. 8, 141-152. Sixbey, J.W., Shirley, P., Chesney, P.J., Buntin, D.M. and Resnick, L. (1989) Detection of a second widespread strain of Epstein-Barr virus. Lancet 2, 761-765. White, M.B., Carvalho, M.. Derse, D., O’Brien, S.J. and Dean. M. (1992) Detecting single base substitutions as heteroduplex polymorphisms, Genomics 12, 301-306. Young, L.S., Yao, Q.Y., Rooney, C.M., Sculley, T.B.. Moss, D.J., Rupani, H., Laux, G., Bornkamm, G.W. and Rickinson, A.B. (1987) New type B isolates of Epstein-Barr virus from Burkitt’s lymphoma and from normal individuals in endemic areas. J. Gen. Virol 68, 285332862. Zimber, U., Adldinger, H.K., Lenoir, G.M., Vuillaume, M., Knebel-Doeberitz, M.V., Laux, G., Desgranges. C., Wittmann, P., Freese, U.K., Schneider, U. and Bornkamm, G.W. (1986) Geographical prevalence of two Epstein-Barr virus types. Virol. 154, 5666.