From www.bloodjournal.org by guest on December 29, 2014. For personal use only. Common Epstein-Barr Virus (EBV) Type-l Variant Strains in Both Malignant and Benign EBV-Associated Disorders By Volker Schuster, German Ott, Silvia Seidenspinner, and Hans Wolfgang Kreth In the present study, Epstein-Barr virus (EBV) isolates from l 8 malignant tumors (angioimmunoblastic lymphadenopathy [AILD], n = 4; Hodgkin‘s disease [HDI,n = 3; pleomorphic T-cell non-Hodgkin‘s lymphoma [l-NHLI, n = 1; &cell nonHodgkin‘s lymphoma [B-NHL], n = 8; gastric carcinoma, n = 2) as well as from 10 tonsils of EBV-seropositive children and from peripheral blood mononuclear cells of 12 children with uncomplicated infectious mononucleosis (IM) and of a boy with severe chronic active EBV infection ware genotyped in the EBV nuclear antigen-2 (EBNA-2) gene. A total of 40 of 41 isolates harbored EBV type 1; in 1 specimen (tonsil), only EBV type 2 was found. Further molecular chartp e l wild-type isolates in the EBNA-2 acterization of EBV y gene and in the 40-kb distant EBV-encoded small RNAs (EBER) region showed that different groups of stable EBV y tp e variant l strains exist in vivo both in benign and malignant lymphatic tissue. Group 1 is composed of EBV type-l isolates (B-NHL, n = 3;T-NHL, n = 1; HD, n = 1; IM, n = 4) that showed a 695-8-Iike DNAsequence pattern in both viral genes. Group 2 isolates (HD, n = 1; AILD, n = 1; B-NHL, n = 1; tonsils of EBV-seropositive children, n= 9; IM, n = 2) showed a nucleotide change at position 49095 in the EBNA2 gene, leading t o an amino acid substitution (Pro -,Ser), and EBV type9 sequences in the EBER region. EBV type-l isolates that fall into group 3 (AILD, n = 3; HD, n = 1; B-NHL, n = 4; gastric carcinoma, n = 2; IM, n = 6; severe chronic active EBV infection, n = 1) were characterized by typical nucleotidechanges and a 3-bp insertion (CTC;extra Leu residue) in the EBNA-2 gene and anEBV type-2-specific sequence pattern in the EBER region. These EBV type-l variant strains may represent the most prevalent circulating EBV type-l strains in the exposed population and seem not t o be restricted t o a certain EBV-associated disease or tumor type. However, analysis of more EBV isolates from benign and malignant lesions must show whether more EBV type1 substrains exist in vivo. 0 1996 b y The American Society of Hematology. E a common phenomenon of EBV type-l wild-type isolates derived from patients with different malignant lymphomas and from children with benign clinical conditions. We extended this analysis to another locus, the 40-kb distant EBER region, using single-strand conformation polymorphism (SSCP). PSTEIN-BARR VIRUS (EBV), the causative agent of infectious mononucleosis (M), has been associated with endemic Burkitt’s lymphoma (BL), with nasopharyngeal carcinoma, with certain B- and T-cell lymphomas, with approximately 50% of Hodgkin’s disease (m), with different types of gastric carcinomas, and with B-cell lymphoproliferative disorders in immunosuppressed individuals.”’ According to their DNA sequence (and protein) divergence within the EBV nuclear antigens (EBNA-2, -3a, -3b, -3c) and the 40-kb distant EBV-encoded small RNAs (EBER) region, two types of EBV (EBV type 1, EBV type 2) have been identified.”” EBNA-2 is of particular interest because this protein plays an essential role in the transformation process of B lymphocytes by EBV”*” and seems to be a critical determinant for EBV-induced lymphoma tumor growth in immunodeficient severe combined immunodeficiency disease mice.I3 The biological differences between EBV type 1 and EBV type 2 appear to be mainly caused by the divergence within the EBNA-2 protein, because EBV type 2, when compared with EBV type 1, has a lower transforming efficiency, a poorer initial outgrowth, and a higher cell-density dependence for cell viability in ~ i t r 0 . lBoth ~ EBV types occur worldwide, with different geographical distributions. In general, EBV type 1 is the predominant type in lymphatic cells of EBV-infected individuals. Immunodeficient hosts, especially those with acquired immunodeficiency syndrome, show increased infection rates with EBV type 2.15However, little is known of whether both strains really differ in their biological activities in vivo. Recently, Aitken et a l l 6 found EBNA-2 sequence heterogeneity in EBV type-l isolates from four African and New Guinea endemic BL cases, suggesting that variant EBV type-l substrains may be associated with these tumors. Different point mutations in the EBNA-2 gene have been described in some EBV type-l isolates from human immunodeficiency virus-infected patients but not from human immunodeficiency virus-negative subjects.” The present study was undertaken to evaluate if stable and constant nucleotide changes in the EBNA-2 gene are Blood, Vol 87, No 4 (February 15). 1996: pp 1579-1585 MATERIALS AND METHODS Clinical samples. Biopsy specimens of 16 malignant tumors (centroblastic B-cell non-Hodgkin’s lymphoma [B-NHL], n = 7; HD, n = 2; angioimmunoblastic lymphadenopathy [AILD], n = 4; pleomorphic T-cell NHL [T-NHL], n = 1; and gastric carcinoma, n = 2) were selected from the files of the lymph node registry at the Department of Pathology in Wiirzburg, Germany. In addition, 2 biopsy samples (Hodgkin’s disease [HD-21 and small-cell lymphocytic B-NHL with plasmacytoid features [B-NHL-4], respectively), were derived from 2 brothers with X-linked lymphoproliferative disease, an inherited immunodeficiency with a high vulnerability to EBV infection^.'^,'^ Furthermore, peripheral blood mononuclear cells (PBMCs) from 12 children with uncomplicated acute IM and from a 6-year-old boy with severe chronic active EBV infection (SCAEBV) as well as tonsils from 10 tonsillectomized EBV-seropositive children were examined for the presence of EBV type-l variant strains. Cell lines. The B95-8 line was used as a source of EBV type1 prototype.” This EBV strain was originally established from an From the Children’s Hospital and Institute of Pathology, University of Wiirzburg, Wiirzburg, Germany. Submitted June 5, 1995; accepted October 12, 1995. Supported by the Deutsche Forschungsgemeinschft (grant no. Schu NOD-3) and in part the Sondegorschungsbereich 172, C8. Address reprint requests to Volker Schuster, MD, Children’s Hospital, University of Wiirzburg, Josef-Schneider-Strasse 2, D-97080 Wiirzburg, Germany. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 V.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8704-0038$3.00/0 1579 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. SCHUSTER ET AL 1580 a 1 2 3 45 6 7 8 9l011 1213 141516 17 1819 IM patient.” The BL cell line Jijoye was used as a source of EBV type-:! prototype.” Polymerase chain reaction (PCR) of o portion of theEBNA-2 gene. Total DNAwas extracted from biopsy material, PBMCs, or cell lines and was then amplified in duplicates either with EBNA-2 primers specificfor EBV type 1 (S’-TCTTGATAGGGATCCGCTAGGATA-3’. nucleotide positions 48839-48862: and S’-ACCGTGGTTCTGGACTATCTGGATC-3’. nucleotide positions 49335-4931 I) or with EBNA-2 primers specific for EBV type 2 (S’-ACTGGATATGAATCCCCTGGGCAG-3’. nucleotide positions 48766-48789; and S‘-GAGTCCTGTACTATCAGAACTACAATG-3‘, nucleotidepositions 49231-4920S).’”.’2 The amplified products had a size of 497 bp (EBV type-l sequences) or 466 bp (EBVtype-2 sequences), respectively. PCR conditions have been reported eisewhere.” Southern blot h,vbridi:atior~ ona/ysi.r. To controlthespecificity ofEBNA-2 genotyping, PCR products were electrophoresed in a 2% agarosegelandwere Southem-blotted onto a nylonsupport (Gene Screen Plus; DuPont, Braunschweig, Germany). Membranes werethenhybridizedwith an internal”P-labeledEBNA-2 DNA probe that was either specfic for EBV type I or specific for EBV type 2. The internalprobespecificfor EBVtype 1 (155 bp) was 1 2 3 4 5 6 7 8 9 1011 1213 141518171819 generated by PCR using the primers S’-CAACCACTCATGATGCCACCAAGGC-3’ (nucleotide positions 49077-49101) and S’-GATGGTGTGGGTTGAAGTTCGGTAG-3’ (nucleotide positions 4923 149207) and as template EBV type-lprototypestrainB95-8. The internal probe specific for EBV type 2 (IS5 bp) was generated by S’4TGCCCATGGTAGCClTAGGAPCR using the primers CATA-3’(nucleotide positions 49004-49028) and S‘-AGACTTAGTTGATGCCCTAGTGTGA-3’ (nucleotide positions 49158-49134) and as template EBV type-2 prototype strain Jijoye. Hybridization probes were radiolabeled by inclusion of 2 pCi [a-“P]-deoxycytidine triphosphate (3.000 Cilmmol) in the PCR. PCR conditions were the same as indicated above.” After washing under stringentconditions blots were dried and subjected to autoradiography at -70°C with intensifying screens. Direct DNAseqrcencirtg uf EBNA-2 PCR products. Doublestranded PCR products were purified with PrimeErase Quick Push Columns (Stratagene, Heidelberg, Germany) and were directly cycle-sequenced by the dideoxy-termination method using ”S-deoxyadenosine triphosphate and theExo(-)Pfu Cyclist DNA Sequencing Fig 1. (a and b) Genotyping of clinical EBV isolates in the EBNAKit (Stratagene) according to the manufacturer’s protocol. Samples 2 gene. DNA was amplified in duplicates either by primers specific wereelectrophoresed on6% polyacrylamide/8.3 mol/L urea sefor EBV type 1 (A) or by primers specific for EBV type 2 (B)?’ Lane quencing gels (Roth, Karlsruhe, Germany), dried, andexposed to 1, 100 bp ladder; lane 2, EBV type-l prototype strain 895-8; lane 3, Kodak XAR-S films (Eastman-Kodak, Rochester. NY). EBV type-2 prototype strain Jijoye; lane 4, HP; lane 5, EBV-negative SSCP analysis .f theEBER region. Forgenotypingof EBV control DNA (BJAB); 14 different clinical isolates (tonsils, lanes 6-9 isolates in the EBER gene, which reveals 6 type-specific point mutaand 15-19; PBMC from children with IM, lanes 11-14; and HD-3, lane tions: we used SSCP analysis similar to that describede l s e ~ h e r e . ~ ” . ~ 10) ~ that all contain EBV type 1 are shown. (a) Ethidium bromide staining. (b) Southern blot hybridization with an internal (EBV typeThe genotype-specificpointmutationscaneasilybedetected by l-specific) probe (A); Southern blot hybridization with an internal characteristic shifts in mobility because of conformational changes fEBV type-2-specific) probe (B). of the (single-stranded) DNA sequences. PCR fragments with a size of190 bp were amplified from 200 ngof genomic DNA in a S0 pL mixture containing I pmol/L of both EBER-specific primers S’-GTGGTCCGCATGTTTTGATC-3‘ cyanol) and was heat-denatured for 10 minutes at 80°C. A total of (nucleotide positions 6780 - 6799) and S’-GCAACGGCTGTCCTG4 pL of themixture was electrophoresed in a nondenaturing 5% TTTGA-3’ (nucleotide positions 6969 - 6950),’1200 prnol/L of each polyacrylamidegel (Roth) containing S% glycerol in 0.SX Trisdeoxynucleotidetriphosphate, 0. I S pL “P-labeled deoxycytidine triborate-EDTA (TBE) buffer at room temperature for 7 hours at 10 phosphate (3.000 Cilmmol; Amersham Buchler, Braunschweig, GerW. The gel was dried and exposed to Kodak XAR-S films. many), 10 mmol/L Tris-HCI (pH 8.3). S0 mmol/L KCI, I .S mmol/L MgCI?, and 2.5 U of Taq DNA polymerase (Boehringer, Mannheim, RESULTS Germany). Initial denaturation was for S minutes at 94°C. SubseDNA was isolated from biopsy specimens of 18 malignant quently, 35 cycles with denaturation for 30 seconds at 94°C. annealtumors (AILD, B- and T-NHL, HD, and gastric carcinoma), ing for 30 seconds at 57°C. and extension for I minute at 72°C were from 10 tonsils (of EBV-seropositive childrenwho were performed in a PerkinElmer-Cetusthermocycler(PerkinElmertonsillectomized for recurrent chronic tonsillitis), from Cetus, Nonvalk. CT). A total of S pL of the final product was mixed PBMCs of 12 children with uncomplicated acute IM, and and diluted with 45 pL formamide-loading buffer (95% formamide, from PBMCs of a boy with SCAEBV and were examined 10 mmol/LEDTA, 0.05% bromophenolblue,and 0.05% xylene A B b A B From www.bloodjournal.org by guest on December 29, 2014. For personal use only. STRAINS COMMON EBV TYPE-l VARIANT Table 1. EBNA-2 Sequences and EBVGenotype in the EBER Region Present in 41 EBV Wild-Type lsolatw EBNA-2 Nucleotide‘ Sequences Group 1 895-8 (EBV type-l strain) B-NHL-1 (CB, 3844B9) B-NHL-2 (CB, 1005/90) B-NHL-3 (CB composite, 1826/91) T-NHL-1 (9594/89) HD-3 (379) IM (n = 4) Group 2 HD-2, B-NHL-4t AILD-2 (2769190). tonsils (n = 9) IM (n = 2) Group 3a (H209/89) AILD-1 (1966/91) AILD-3 B-NHL-5 (CB, H4088/91) B-NHL-6 (14760/91) IM (n = 4) SCAEBV Group 3b HD-1 (376) AILD-4 (24618/91) B-NHL-7 (H2141/91) B-NHL-8 (9945/92) GC-l (13811/92) GC-2 (17965/92)$ IM (n = 2) W91 (African BL)”,”§ Group 3c FWA (African BL)”§ Group 3d ODH (African BL)16§ (EBV AG876 type-2 strain) Gly Jijoye (EBV type-2 strain) Tonsils (n = 1) Ala 49113 49119 491W49137 49170 EBER Genotype A Thr C Leu - C Leu EBV type l Pro G Met T Ser A Thr C Leu C Leu EBV type 2 G Arg T Ile C T Ser G Val CTC Leu T Leu EBV type 2 Pro G T /le C Pro T Ser C Leu CTC Leu T Leu EBV type 2 49057 49091 A Gln G Met C A Gln Arg 49095 (W91 NT) G Arg T Ile C G Arg C A la T /le C C Pro Pro G C Pro C Leu CTC Leu T Leu NT G Thr A Thr C Leu C CTC Leu T Leu C Gln NT Pro - EBV type 2 Abbreviations: CB, centroblastic; GC, gastric carcinoma; SCAEBV, severe chronic active EBV infection. Numbering of nucleotides is rendered according to Baer et al?’ t Tumor biopsy specimens from two brothers withX-linked lymphoproliferative disease.” This EBV isolate shows an additional base exchange at position 49159 (G + A) leading to an amino acid substitution (Arg His). 5 Data are taken from the literature.”~’2~’E EBV isolates RNA and ODH show an additional base exchange at position 49110 (C T) leading to an amino acid substitution (Pro -,Sed. * - + for the presence of EBV. All but 1 of the biopsy samples contained EBV type- 1 genomes as determined by FCR using genotype-specific EBNA-2 primers (Figs l a and b). Only 1 isolate from benign tonsil tissue contained EBVtype 2. Double infections with both strains, EBV type 1 and EBV type 2, were not detected in any of the pathological specimens. To detect DNA sequence variations within the EBNA-2 gene that may possibly allow the definition of EBV type-l variant strains, EBNA-2 PCR products of all isolates were directly sequenced and compared with known EBNA-2 sequences of EBV type-l prototype strain B95-8andEBV type-2 prototype strains AG876 and Jijoye (Table 1).In addition, all isolates were simultaneously genotyped in a gene 40-kb distant, the EBER region. As shown in Table 1, the isolates could be classified into three different groups according to their individual EBNA-2 sequence pattern and their EBER genotype. Isolates showing a B95-&like pattern at both gene loci fall into group 1 (Fig 2%right [B-NHL-l]; Fig 2b, middle [T-NHL-l]; and Fig 3, lanes 12 and 9, respectively; see Table l]). EBV type-l substrains of group 2 were characterized by a nucleotide mismatch inthe EBNA-2 gene at position 49095 (C -+ T) leading toanamino acid exchange (Pro + Ser) and EBV type-2 sequences in the EBER region. Figure 2a (middle) shows the EBNA-2 sequence pattern of a group-2 isolate (AILD-2). Inthe EBER region, this isolate was found to contain EBV type-2 sequences (Fig 3, lane 10). Interestingly, in 2 brothers with X-linkedlymphoproliferative disease who developed histologically different malig- From www.bloodjournal.org by guest on December 29, 2014. For personal use only. SCHUSTERET 1582 a TCGA (C+A) 49091 * '4- 49198- b lT+C TCGA TCGA " 49095 (G-A 1 I" 491 98 AILD-1 TCGA AI LO-2 B-NHL-1 T C GTAC G A 49057* 1C+A 49091 AL Fig 2. (a) Nucleotide sequence pattern of EBNA2 PCR products of three different EBV type-l isolates (the noncoding, complementary strand is shown). Numbering of nucleotides is consistent with that used by Baer et 81.2" (Left panel) This EBV type-l isolate derived fromAILD-1 (H209/89) is a representative for the group3 EBV type-l substrain characterized by nucleotidechanges I") at positions 49091 (C A),49113 (T A), and 49170 (G A) and a 3-bp insertion (GAG) between position 49136 and 49137. In addition, a nucleotide mismatch was found C) in this isolate. (Middle at position 49119(G panel) This EBV type-l isolate derived from AILD-2 (2769/90) shows a nucleotide mismatch at position 49095 (G A), which ischaracteristic for thegroup2 EBV type-l substrain. (Right panel) The EBNA-2 nucleotide sequence of this EBV type-l isolate derived from centroblasticB-NHL-1 (3844/89) is identical with thecorresponding sequence of 895-8 prototype lie, group-l EBV type-l substrain). (b) Nucleotide sequence pattern of EBNA-2 PCR products of three different EBV type-l isolates (the noncoding, complementary strand is shown). Numbering of nucleotides is consistent with that used by Baer et 81.2' (Left and rightpanels) These EBV type1 isolates derived fromHD-1 (376)and AILD-3 (1966/ 91) are representatives for the group-3 EBV type-l substrains characterized by nucleotide changes (*I at positions 49091 IC A), 49113 (T -Al. and 49170 (G -A) and a 3-bp insertion(GAG) between position 49136 and 49137. The isolate from AILD-3 showed an additional nucleotide mismatch at position 49119 (G C). (Middle panel) The EBNA-2 nucleotide sequence of this EBV type-l isolate derived from TNHL-1 (9594/89) is identical with the corresponding sequence of 895-8 prototype lie, group-l EBV type1 substrain). - - * - - + L9136 I +GAG IG+Al - - HD-l T-NHL-1 AlLD- 3 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. STRAINS COMMON EBV TYPE-l VARIANT -ds DNA 12 3 4 5 6 7 8 9 10 11 12131415 16 17 18 Fig 3. PCR-SSCP analysis of EBER region. PCR products of 190 bp were amplified usingEBER-specific primers, were denatured, and were subjected t o SSCP analysis in a nondenaturing 5% polyacrylamide gel, as described in the Materials and Methods. Genotype-specific point mutations were visualized as shifts in mobility. The single-stranded DNA resolved into the threeupper bands, which is caused by thefaster mobility of the sense strand when compared with thatof the antisense strand." The reason for the third (fainter) upper band remains unclear. Probably, it contains a partially denaturedcomplex of senselantisense strands. The thick band with the fastest mobility represents the doublestranded DNA (dsDNA). Lane 1, 895-8 (EBV type-l prototype strain); lane 2, Jijoye (EBV t y p e 9 prototype strain); lane 3, BJAB (EBV-negative BL cell line); lane 4, Jijoye; lane 5, HD-1 (376); lane 6, HD-3 (379); lane 7, AILD-1 (H209/89); lane 8, T-NHL-2 (H101/90); lane 9, T-NHL-1 (95941 89); lane 10, AILD-2 (2769/90); lane 11, AILD-3 (1966/911; lane 12, B-NHL-1 (centroblastic, 3844/89); lane 13, B-NHL-3 (centroblastic composite, 1826/91); lane 14, B-NHL-6 (14760/91); lane 15, B-NHL-5 (polymorphic centroblastic, H4088/91); lane 16, B-NHL-2 (centroblastic, 1005/901; lane 17, double-stranded DNA control 1895-8); and lane 18, 895-8 (EBV type-l prototype). nant tumors, namely HD (HD-2) and small-cell lymphocytic B-NHLwith plasmacytoid features (B-NHL-4), the same EBV type-l substrain (group 2) was found in tumor biopsy specimens. This might suggest that the strain was transmitted from one family member to another. Somewhat surprisingly, all 9 EBV type-l isolates from tonsils of children with chronic tonsillitis examined so far could be classified as group 2 EBV type-l substrains. Group 3 is composed ofEBV isolates with EBNA-2 nucleotide mismatches at positions 49057 (A + G ) , 49091 (G + T), 491 13 (A T) and 49170 (C T), leading to three amino acid substitutions (Gln Arg,Met Ile, Thr + Ser) and an insertion of three nucleotides (CTC) between position 49 l36 and 49 137 resulting in an extra amino acid (Leu) (Figs 2a and b and Table 1). All these isolates contained EBV type-2 sequences in the EBER region (Fig 3). In 9 isolates (AILD-I; AILD-3; B-NHLJ; B-NHL-6; SCAEBV; IM, n = 4) an additional nucleotide mismatch was found at position 491 19 (C + G), leading to an amino exchange (Leu Val; see Fig 2a, left, andFig 2b, right, Table l). Recently, a region of the EBNA-2 gene of the African endemic BLs W91 ," FWA and ODH'" has been sequenced and compared with the corresponding region of EBV type1 strain B95-8. According to the characteristic nucleotide mismatches in EBNA-2, these three strains most probably belong to the category of group 3 substrains (Table l ) . However, genotyping in the EBER region has been not performed in any of these isolates. + + + + DISCUSSION Our results show that distinct stable EBV type-l variant strains are common in vivo. According to their characteristic sequence pattern in the EBNA-2 and the EBER genes, we can define three groups of EBV type-l substrains. Group 1 is composed of EBV isolates with a B95-8-like pattern at the EBNA-2 and the EBER gene locus. Group 2 isolates are characterized by one nucleotide mismatch (C T) in the EBNA-2 gene at position 49095 andbyEBV type-2-like sequences in the EBER region. Group 3 isolates show a W91-like pattern in the EBNA-2 gene and EBV type-2-like sequences in the EBER region. These EBV type-l variant strains seem not to be restricted to a certain clinical disorder or tumor type. We could also show that the same EBV type-l substrain (of group 2) occurred in histologically different malignant tumors of 2 brothers whoboth carried the defective gene of X-linked lymphoproliferative disease,"' suggesting intrafamilial transmission of this EBV substrain. During IM, the infection is not only restricted to B cells, which morphologically include Reed-Stemberg-like cells, immunoblasts, medium-sized lymphoid cells, andplasma cells. In addition, varying numbers of T lymphocytes are also infected by EBV.'"' Therefore, it is tempting to assume that these EBV-infected B- and T-cell populations in IM might represent thebenign counterparts of EBV-infected tumor cells in BL, HD, and different B- and T-cell lymphomas. Our findings that the EBV type-l variant strains found in benign clinical conditions (such as 1M) matched the EBV type-l variant strains isolated from different malignant tumors might support this suggestion. In addition, EBV variant strains with certain deletions/mutations within the gene of EBVlatent membrane protein-l have beenfound in both malignant andbenign EBV-associated diseases." These findings and our findings might suggest that these EBV variant strains may represent the most prevalent circulating EBV + From www.bloodjournal.org by guest on December 29, 2014. For personal use only. 1584 SCHUSTER ET AL strains to which the patient population is exposed. However, analysis of more EBV isolates from benign and malignant lesions must showwhether more EBV type-l substrains exist in vivo. The biological consequences of EBV variant strains in vivo are still not clear. Because the W91 strain has a “normal” transforming capacity in vitro”.” and in vivo,” we suggest that other EBV isolates from group 3 also do not significantly differ in their biological behavior when compared with B95-8-like group-l EBV type-l strains. Intactand deleted W91 strain-like EBNA-2 sequences have been recently identified in two patients with oral hairy leukoplakia,**suggesting that group-3 (and most probably also group-2) EBV type-l variant strains may also persist within the epithelial compartment. It has been suggested that the sequences of the EBNA-2 and EBER genes normally belong to the same genotype.’ However, all isolates investigated in this study and classified as belonging to group 2 and 3 of EBV type-l variant strains carried EBV type-l sequences inthe EBNA-2 gene while maintaining EBV type-2 sequences in the EBER region. Similar “discrepant” results of genotyping atboth loci, EBNA-2 and EBER, have been recently described in some BL and nasopharyngeal carcinoma cell lines and in a higher percentage ofbiopsy HD and posttransplantation lymphoproliferative lesions.29It is conceivable that these “mixed” isolates, when sequenced in the EBNA-2 gene, may also represent EBV type-1 variant strains of either group 2 or group 3 . It isvery likely thatall these mixedEBV variant strains with type-l sequences in the EBNA-2 gene show the biological behavior of the EBV type-l prototype strain B95-8. The pathogenesis of EBV-associated diseases is complex, involving viral, immunologic, and genetic factors. To what extent certain EBV type-l variant strains may influence the course of EBV infection is not known at present. Functional assays are needed to test and compare the biological consequences of EBV type-l variant strains. ACKNOWLEDGMENT We would like to thank Prof Helms (Department of Otorhinolaryngology, University of Wurzburg, Germany) for supplying tonsil tissue from tonsillectomized children. EBNA-2 primers were kindly provided from Prof Jilg (Department of Medical Microbiology, University of Regensburg, Germany). We thank Dr Johnston (Department of Virology, University of Wurzburg, Germany) for critically reading the manuscript. REFERENCES I . Zur Hausen H, Schulte-HolthausenT, Klein G, Henle W, Henle G, Clifford P, Santesson L: EBVDNAin biopsies of Burkitt’s tumors and anaplastic carcinomas of the nasopharynx. Nature 228: 1056, 1970 2. Hanto DW, Frizzera G , Gajl-Peczalska KJ, Simmons RL: Epstein-Barr virus, immunodeficiency, and B cell lymphoproliferation. Transplantation 39:461, 1985 3. Ott G, Ott MM, Feller AC, Seidl S, Muller-Hermelink HK: Prevalence of Epstein-Barr virus DNA in different T-cell lymphoma entities in a European population. Int J Cancer 51:562, 1992 4. Weiss LM, Strickler JG, Warnke RA, Purtilo DT, Sklar J: Epstein-Barr viralDNAin tissues of Hodgkin’s’s disease. Am J Pathol 129:86, 1987 5. Ott G, Kirchner Th, Muller-Hermelink HK: Monoclonal Epstein-Barr virus genomes but lack of EBV-related protein expression in different types of gastric carcinoma. Histopathology 25:323, 1994 6. Rowe M, Young LS, Cadwallader K, Petti L, Kieff E, Rickinson AB: Distinction between Epstein-Barr virus type A (EBNA2A) and type B (EBNA2B) isolates extends to the EBNA 3 family of nuclear proteins. J Virol 63: l03 1, 1989 7. Sculley TB, Apolloni A, Stumm R, Moss DJ, Mueller-Lantzsch N, Misko IS, Cooper DA: Expression of Epstein-Barr virus nuclear antigens 3,4, and 6 are altered in cell lines containing B-type virus. Virology 171:401, 1989 8. Sample J, Young L, Martin B, Chatman T, Kieff E, Rickinson AB, Kieff E: Epstein-Barr virus types 1 and 2 differ in their EBNA3A, EBNA-3B, and EBNA-3C genes. J Virol 64:4084, 1990 9. Arrand JR, Young LS, Tugwood JD: Two families of sequences inthe small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B. J Virol 63:983. 1989 IO. Lin JC, De BK, Lin SC: Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products. J Virol Methods 43:233, 1993 1 1. Cohen JI, Wang F, Kieff E: Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation. J Virol 65:2545, 1991 12. Cohen JI, Kieff E: An Epstein-Barr virus nuclear protein 2 domain essential for transformation is a direct transcriptional activator. J Virol 65:5880, 1991 13. Cohen JI, Picchio GR, Mosier DE: Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro. J Virol 66:7555, 1992 14. 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For personal use only. 1996 87: 1579-1585 Common Epstein-Barr virus (EBV) type-1 variant strains in both malignant and benign EBV-associated disorders V Schuster, G Ott, S Seidenspinner and HW Kreth Updated information and services can be found at: http://www.bloodjournal.org/content/87/4/1579.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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