Mar 21, 1988 - Infectious Mononucleosis Patients and Healthy Carriers ... infectious mononucleosis and from healthy subjects from California, Hawaii, and ...
Vol. 62, No. 10
JOURNAL OF VIROLOGY, OCt. 1988, p. 3862-3866 0022-538X/88/103862-05$02.00/0 Copyright © 1988, American Society for Microbiology
Genetic Polymorphism of Natural Epstein-Barr Virus Isolates from Infectious Mononucleosis Patients and Healthy Carriers MARIA LI LUNG,'* R. SHIHMAN CHANG,2 AND JOHN H. JONES3 Department of Microbiology, University of Hong Kong, Hong Kong,' and Department of Medical Microbiology and Immunology2 and Student Health Center,3 University of California, Davis, California 95616 Received 21 March 1988/Accepted 10 June 1988
We analyzed Epstein-Barr virus (EBV) genomes from lymphoblastoid cell lines isolated from patients with infectious mononucleosis and from healthy subjects from California, Hawaii, and Hong Kong between 1970 and 1987. Using genetic polymorphism as epidemiological markers, we found that several genotypes of EBV cocirculate in a community and that although most EBV strains isolated from California and Southern China may be differentiated genotypically, there was no specific association between genotype and disease or time of isolation.
M, S, and A), and yet other regions of the genome are highly polymorphic (BamHI regions H, K, and I). Figure 1A shows representative results. Sixteen IM isolates obtained in the 1970s and 1980s were probed with B95-8 BamHI fragments
Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis (IM) (16) and is associated with two human malignancies (17), Burkitt's lymphoma and nasopharyngeal carcinoma. EBV infection is ubiquitous, and the virus has been detected in oropharyngeal secretions (29) and peripheral blood (25) of healthy individuals. We studied the restriction fragment length polymorphisms of EBV freshly isolated from the oropharynges and peripheral blood of 21 IM patients and 25 healthy carriers in California, Hawaii, and Hong Kong between 1970 and 1987. Lymphoblastoid cell lines (LCLs) were established as described previously (7, 8) by infecting B lymphocytes with frozen or fresh throat washings from the following groups of individuals: nine IM patients who were students at the University of California at Davis (UCD) from 1970 to 1973 (4, 6, 13), seven IM patients at UCD in 1986, seven carriers who were healthy employees or patients with minor surgical ailments at a UCD clinic from 1970 to 1973 (4, 7), eight healthy students at UCD in 1987, three healthy students at the University of Hawaii in 1977 (5), and seven healthy students at the Chinese University of Hong Kong in 1985 (R. S. Chang, R. Dan, and R. C. K. Chan, J. Am. Coll. Health, in press). B lymphocytes from an adult EBVseronegative donor were used for all transformations except those from Hong Kong, for which cord blood lymphocytes were used. Spontaneous transformants from the peripheral blood of 11 IM patients from California in 1986 were also established into LCLs by standard procedures (9) but with cyclosporin A (1) instead of T-lymphocyte depletion. Seven of these patients had LCLs established from throat washings. To study the genetic polymorphism among natural EBV isolates harbored in the LCLs from these 46 individuals, we purified cellular DNAs (23) and analyzed them by Southern blotting (27, 30) with Amersham Hybond N transfer membranes. EBV prototype LCLs used as positive controls on all blots included B95-8, P3HR-1, Raji, W91, and MABA. The BJAB cell line served as an EBV-negative control. The nine cloned BamHI fragments of B95-8 used as probes are depicted in Fig. 1A. Our analysis showed that certain regions of the genome are highly conserved (BamHI regions W, L, and E), other regions have unique variants (BamHI regions *
W, L, and E. The viruses were homogeneous in these regions. The uniform pattern of fragments revealed by the BamHI L probe is representative of results seen with BamHI W and E probes (data not shown). REM 7 and 12 are variants detected in the BamHI M/S and A regions, respectively. When probed with B95-8 BamHI fragments H, I, and K, a high degree of heterogeneity is observed among viruses. These polymorphic regions of the virus were used as epidemiological markers for studying natural EBV isolates associated with IM patients and healthy individuals. Viruses isolated from the throat wash and peripheral blood of the same patients are genotypically indistinguishable. Figure 1B shows that pairs of isolates obtained from five IM patients in 1986 are identical after being probed with three separate polymorphic viral markers. Much of the genetic polymorphism detected is probably attributed to minor changes such as different numbers of repeats in the IR2 (BamHI fragment H), IR3 (BamHI fragment K), and IR4 (BamHI fragment I) regions (2, 3, 10, 14, 21, 22, 24, 31). In the BamHI H region, at least 20 subtypes of EBV were observed among the 46 specimens analyzed. Probes representative of the 5' and 3' ends and the middle of the BamHI H region were prepared by Hinfl digestion of the BamHI H fragment (Fig. 2). Probing Hinfl digests of specimens obtained from four IM patients and heterogeneous in the BamHI H region with these smaller probes showed that the variability among isolates is not confined to the middle region containing the HpaII repeats. In addition to these differences, REM 3 showed heterogeneity in the 3' end and REM 1 showed heterogeneity in the 5' end. In the BamHI K region, the 46 viral isolates were confined to eight observable restriction patterns (Fig. 1A). The variability observed in the 46 isolates was localized to the 5' end of the BamHI K fragment, which is known to encode Epstein-Barr nuclear antigen 1 (12, 15, 18, 19) and contains a number of tandem repeats. The BamHI K fragment was cleaved into three smaller fragments by digestion with HindIII (Fig. 3). These probes, representative of the 5' and 3' ends and middle region. were used to analyze four heterogeneous isolates cleaved with two enzymes, BamHI and
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FIG. 1. (A) Schematic representation of the restriction map of B95-8 virus and Southern blots of IM specimens evaluated for genetic polymorphism. The nine BamHI fragments used as probes in this study are designated with asterisks. Sixteen LCLs established from UCD IM patients from 1970 to 1973 (REM 1, REM 3, REM 5, REM 7, REM 9, REM 11, REM 12, and REM 15) and 1987 (PBL 4, PBL 8, PBL 10, PBL 12, PBL 13, PBL 20, PBL 22, and PBL 28) were digested with BamHI and probed with B95-8 BamHI fragments H, I, M, and L or were digested with HindIII and probed with B95-8 BamHI fragments K and A. Fragments recognized by the probes are designated at the right of the figures. Note that BamHI fragment H recognizes two fragments, since the sequences are reiterated in the DSR. With HindlIl digests, the BamHI probes recognize several fragments. The positions of lambda HindlIl digest molecular weight markers are designated on the left of the figures. A high degree of heterogeneity is observed among the 16 IM viruses studied with the BamHI H, K, and I probes. A unique isolate, REM 7, is revealed by probing the BamHI digest of these specimens with BamHI fragment M. REM 12 is the only isolate polymorphic in the BamHI A region. The BamHI L region is highly conserved in the isolates studied. (B) Southern blot of DNAs from paired LCLs established from throat washing (TW) and peripheral blood (PBL) of five IM patients in 1986. Upper left: DNAs were digested with BamHI and probed with B95-8 BamHI fragment H; upper right: DNAs were digested with Hinfl and probed with B95-8 BamHI fragment H; lower left: DNAs were digested with HindIll and probed with B95-8 BamHI fragment K; lower right: DNAs were digested with BamHI and probed with B95-8 BamHI fragment I. Fragments recognized by the probes are designated to the right of the figure, and molecular weight markers (in thousands) are shown at the left. Although a great deal of heterogeneity from patient to patient is present, each pair of specimens from a single patient contains viruses with identical genotypes. This is confirmed for the BamHI H region by the Hinfl digest of these specimens.
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HindIII. Heterogeneity was confined to the 5' end, which encodes the tandem repeats, and was confirmed with a 0.77-kilobase HpaII probe described by Heller et al. (15) (data not shown). The third region which was investigated and found to be heterogeneous in sequence is the BamHI I region. Among the prototype viruses, differences in this region have been
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documented (14) and are illustrated in Fig. 4A. The B95-8 virus has a major deletion in this region of the genome (26) and therefore contains only the I fragment. W91 and Raji are similar to each other and have the B1* and 1* fragments. P3HR-1 is missing the BamHI site between W1* and I* present in W91 and therefore has a fused W1*I* band. The viruses analyzed in this study are basically of two types. For ease of description, viruses having the fused W1*I* band are designated type C. They lack the BamHI site between fragments W1* and I* as seen in P3HR-1 after BamHI analysis and probing with BamHI fragment I. Those having separate B1* and I* fragments as seen in W91 are termed type D. Of the 46 viruses, 30 are type D and 14 are type C. The remaining two isolates, REM 72 and REM 8717, have B1* fragments of lower mobility which presumably carry large deletions. Figure 4B shows a blot of representative viruses. None resembles B95-8, which contains a major deletion in this region (26). Of 21 IM viruses studied, 18 (86%) are type D) and only 3 (14%) are type C. Of the viruses isolated from the 25 healthy individuals 12 (48%) are type D, 11 (44%) are type C, and 2 (8%) are of neither type. Grouping the isolates by geographical origin, it is apparent there is a geographical predominance of type D viruses in the continental United States. Of the 15 isolates from California, 11 (73%) are type D, 2 (13%o) are type C, and 2 (13%) are of neither subtype. The seven Hong Kong viruses studied were all type C. This difference between California and Hong Kong isolates is highly significant (P < 0.001 by critical ratio or x2 analysis). We have only three isolates from Hawaii; one isolate was type D, and two were type C. The finding of a geographical prevalence of type C strains in Asia is strengthened by the results of an analysis of 26 more strains from Hong Kong and the People's Republic of China, in which 25 of 26 isolates were similar to P3HR-1 in the BamHI
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I region and only 1 isolate was type D (M. L. Lung, R. S. Chang, M. L. Huang, H. Y. Guo, S. Y. Tsao, P. Cheng, D. Choy, M. H. Ng, unpublished data). The possibility of a geographical prevalence of one type of EBV strain is not surprising, since Zimber et al. (31) have shown that these viruses may be typed as A or B according to the presence or absence of unique sequences in the BamHI H region. All strains in this study are type A (data not shown) with the BamHI I probe. Thus, type A virus may be further differentiated as A-C or A-D according to polymorphism of the BamHI I region of the viral genome. Using the regions of genetic polymorphism as epidemiological markers, we observed a great deal of heterogeneity among natural EBV strains associated with IM patients and healthy individuals, confirming earlier studies on recently established LCLs (22, 28). In a university community, viruses of several genotypes are circulating simultaneously. Figure 5 shows Southern blots of seven isolates collected from healthy individuals at UCD and probed with the BamHI H fragment. Six of these isolates were collected at the same clinic on the same day by the same physician from healthy students undergoing routine physical examination. Note that REM 8726, an LCL established from a healthy individual in 1987, appears to be dually infected. Of the 46 isolates obtained from clinical specimens, only this one isolate, collected from a throat wash of a healthy UCD student, harbored viruses of two distinct genotypes. Related patients do not necessarily harbor viruses of identical genotypes. Figure 6 illustrates Southern blots of isolates obtained from a father and son (REM 15 and REM 16); the father was suffering from acute IM and his son was healthy. Their viruses were genotypically distinguishable in the BamHI H region. In specimens obtained from another father-son pair (REM 17 and REM 18), both of whom were healthy, identical viruses were isolated. This virus differed from those detected in REM 15 and REM 16. Reinfection of carriers with exogenous virus, although rare, may occur, and the interaction of this virus with the latent virus may provide further opportunity for generating genetic diversity. Identical viruses are responsible for initiating the infection in the oropharynx and subsequently establishing latent infection in the individuals (Fig. 1B). Only
FIG. 6. Southern blot of DNAs from LCLs established from throat washings collected from two father-son pairs. REM 15 was an isolate from an IM patient in the 1970s, and REM 16 was an isolate from his healthy son. REM 17 and REM 18 were isolated from a healthy father-son pair in 1970. Variation between viruses is evident after (A) BamHI digestion or (B) Hinfl digestion and probing with B95-8 BamHI fragment H. Fragments recognized by the probe are designated at the right of the figure, and molecular weight markers (in thousands) are shown at the left. Virus transmission may occur between father and son, as seen in the REM 17 and REM 18 pair. The viruses detected in REM 15 and REM 16 are distinct from one another and from REM 17 and REM 18. one individual in this study showed evidence of dual infection (Fig. 5). This finding is at variance with the multiple isolates associated with IM patients and immunocompromised patients in a recent report (22) and may reflect the higher incidence of dual infections with EBV among immunocompromised hosts. In conclusion, several EBV markers are available for assessing the identity of EBV strains from different clinical sources. These markers are useful in epidemiological studies of EBV strains present in IM patients and healthy individuals. No dominant prevailing strain of EBV is associated with IM. Identical genotypes are present in IM patients and healthy individuals. No dominant prevailing strain of EBV is associated with IM. Identical genotypes are present in IM patients and healthy carriers, and viruses isolated from throat washes and peripheral blood of IM patients are indistinguishable. It is clear that many genotypically distinct strains of EBV are circulating in a given community. Related patients may somethimes harbor the same genotype. Most EBV strains may be differentiated in the BamHI I region as type C or type D viruses. No specific association between genotype and disease or time of isolation was detected. This work was supported in part by the Croucher Foundation and by Public Health Service grant CA43051 awarded by the National Cancer Institute. We acknowledge the technical assistance of Y. Y. Chang, M. S. Cheng, W. P. Lam, and Y. K. Pang. We thank B. Griffin and T. Lindahl for supplying us with B95-8 recombinant plasmids, G. Miller for the B95-8 and W91 cells, D. Crawford for the MABA cells, and G. Bornkamm for the MABA pM-Bam H2 recombinant plasmids. LITERATURE CITED 1. Bird, A. G., and S. M. McLachlan. 1980. Cyclosporin A and Epstein-Barr virus. Lancet ii:418. 2. Bornkamm, G. W., H. Delius, U. Zimber, and J. Hudewentz. 1980. Epstein MA. Comparison of Epstein-Barr virus strains of
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14. 15. 16.
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