JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1991, p. 1640-1644 Vol. 29, No. 80095-1137/91/081640-05$02.00/0Copyright © 1991, American Society for Microbiology

Strain Analysis of Hepatitis B Virus on the Basis of RestrictionEndonuclease Analysis of Polymerase Chain Reaction Products

J. WAI-KUO SHIH,l* LING C. CHEUNG,1 HARVEY J. ALTER,1 LEO M. LEE,2 AND JIAN REN GU3

Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health,Bethesda, Maryland 208921; Frederick Cancer Research and Development Center, National Cancer Institute,

Frederick, Maryland 217012; and Shanghai Cancer Institute, Shanghai, China3

Received 6 February 1991/Accepted 14 May 1991

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from thepolymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of thismethod was based on the selective sensitivity of the PCR product to digestion with different restrictionendonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis ofpublished nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-Sregion. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). Allof the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestionby a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, theserestriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samplesproduced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequenceof the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results inwhich they appeared to be a different subtype or were resistant to the restriction nuclease digestion. Thisgenetic alteration was consistent in the paired specimens from sexual transmission cases. These results showthat the described procedures are applicable to HBV strain assessment.

Human hepatitis B virus (HBV) is one of several viruses,termed hepadnaviruses, which have similar structural fea-tures and tissue tropism (19). Hepatitis B infection has aworldwide distribution, although its prevalence varies geo-graphically. There are distinct subtypes ofHBV indicative ofstrain heterogeneity. The subtypes ofHBV are distinguishedby antigenic determinants on the surface antigen (HBsAg)and their corresponding antibodies (11). There is a commongroup determinant, a, which appears in all HBsAg speci-mens. There are two sets of subdeterminants, d or y and w orr, which appear to be allelic or mutually exclusive (1) andwhich are used for the identification of subtypes. Thus, thereare at least four major groups into which HBsAg can beclassified: adw, adr, ayw and ayr. The distribution of sub-types among infected populations also varies geographically(3). It has been speculated that the subtype specificity ofHBV might affect the clinical consequences of the infection(13). Subtyping of HBV has proven to be a valuable markerin clinical and epidemiological studies.The genomic DNA of many strains of HBV has been

cloned and sequenced (5, 6, 18, 24). Comparisons of nucle-otide sequence homology between different isolates of thesame subtype (14) and between different subtypes have beenreported (6, 17). Since the introduction of the polymerasechain reaction (PCR) with a thermostable DNA polymerase(20), this technique has found extensive application in thediagnosis, genetic identification, and molecular cloning andanalysis ofDNA. The PCR technique has been applied to thedetection of HBV DNA in seronegative subjects who trans-mitted hepatitis B (22) and serum HBV DNA in patients withchronic hepatitis (9). PCR for the detection of HBV has beenshown to be slightly more sensitive than infectivity testing in

* Corresponding author.

the chimpanzee (23). A rapid and sensitive method for thedetection of serum HBV DNA, using PCR, has been re-ported (8). In this paper, we report the application of PCR tothe determination of strain variation among HBV-infectedpatient sera, and we compare strain classification based onnucleotide sequences with that based on serological analy-S1S.

MATERIALS AND METHODS

HBV-positive sera, subtyping reagents, and serological de-terminations. A panel of HBsAg-positive sera subtypedduring the International Workshop on HBs Antigen Sub-types, Paris, France, in 1975 (2) and hepatitis B patient serawhich were collected and subtyped previously at the Na-tional Institutes of Health (10) were used as source materialfor PCR typing. The preparation of HBsAg subtyping re-agents and the technique of serological subtyping weredescribed by Hoofnagle et al. (7). Briefly, monospecificanti-HBs/d, ly, or lw was prepared by absorption of poly-clonal anti-HBs/adw or layw with corresponding HBsAg-positive sera of the ayw, adw, or adr subtype. Inhibition ofmonospecific antibody activity by HBsAg-positive serum ina radioimmunoassay (Ausab; Abbott Laboratories, Chicago,Ill.) was utilized to indicate the presence of a specificsubtype determinant.Cloned HBV DNA sequences and selection of restriction

nucleases for the mapping of strains. The selection of primersets for synthesis was based on the published sequence dataof Gan et al. (6), Fujiyama et al. (4), Ono et al. (18),Valenzuela et al. (24), Lo et al. (12), and Galibert et al. (5).Primers for PCR amplification were chosen by the followingcriteria. (i) The fragments were located within a conservedregion so that the primers could be used to detect all possibleHBV subtypes (e.g., adr, adw, and ayw). (ii) The distance

1640

TYPING OF HBV BY PCR 1641

between upstream and downstream primer fragments wasless than a few hundred nucleotide bases within the se-quence. (iii) Primer fragments which allowed more than a3-base mismatch with any other documented sequence ingene banks were not utilized. (iv) Suitable restriction en-zymes that provided satisfactory site-specific cuts had to beavailable so that each of the subtypes could be identified.Upon completion of the sequence analysis and compari-

son, two consensus sequences were identified within thepreS-S region for each of the HBV subtypes; using thesequence numbering of Gan et al. (6), these resided betweenpositions 1601 and 1703 and positions 2201 and 2255. Partialnucleotide sequences for several HBV subtypes within thisregion are shown in Fig. 1. Primer pairs within the 3' end ofthe preSl gene and the 5' end of the major S gene wereselected. Primer 1679F 5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3' (25-mer) and primer 2254R 5'-GAAGATGAGGCATAGCAGCAGGAT-3' (24-mer) were synthesized. Theamplification by this primer pair begins at position 1679 andends at position 2254 of the HBV genome from an adr subtype(6). A PCR product of a 575-bp fragment containing threepotential restriction nuclease sites (BamHI [1849], HapII[1897], and XhoI [1948]) was expected. Susceptibility of thethree major subtypes to restriction enzyme digestion and theirexpected fragment sizes are summarized in Table 1.

Extraction of viral DNA from serum. A 100-pld portion ofserum was incubated at 65°C for 2 h in a mixture ofproteinase K (100 ,ug/ml), 0.5% sodium dodecyl sulfate, 5mM EDTA, and 10 mM Tris HCl, pH 8.0. The solution wasextracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol, followed byan ethanol precipitation. The precipitate was dissolved in100 pul of TE buffer (10 mM Tris HCl [pH 8.0], 0.1 mMEDTA).PCR amplification of HBV DNA. A 10-,u aliquot of serum

DNA was amplified in a 100-pul reaction mixture containing2.5 U of recombinant Taq DNA polymerase (AmpliTaq;Perkin-Elmer Cetus, Norwalk, Conn.), 200 puM each dATP,dGTP, dTTP, and dCTP, a 1 ,uM concentration of eachprimer, 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mMMgCl2, and 0.01% gelatin. The reaction mixture was overlaidwith 50 pAl of mineral oil.The reaction was performed for 35 cycles in a program-

mable thermal cycler (Perkin-Elmer Cetus). Samples wereheated to 95°C for 35 s (denaturation of DNA), cooled to55°C for 1 min (annealing to primer), and incubated at 72°Cfor 1 min (polymerase amplification reaction). All reagentswere tested to ensure that they were free of HBV DNAcontamination, and all experiments were performed in par-allel with positive and negative control sera.

Analysis of amplified DNA. A 20-,ul amplified DNA reac-tion mixture was analyzed by electrophoresis in 1% agaroseat 100 V for 2 h and visualized by UV fluorescence afterstaining with ethidium bromide. The specificity of the am-plified bands was confirmed by Southern blot and hybridiza-tion with radiolabeled vector-free HBV DNA probe.

Restriction nuclease analysis of amplified DNA. Restrictionnuclease digests of the amplified DNA were set up in parallelwith BamHI, HpaII, and XhoI in 30-pld reaction mixtures,each of which contained 20 pul of the amplified DNA sample,3 pul of lOx enzyme buffer, 3 pld of 10 mM dithiothreitol, 3 pldof bovine serum albumin (1 mg/ml), and 1 [lI of nuclease (10U). The reaction mixture was incubated at 370C for 2 h andfractionated by 1% agarose gel electrophoresis at 50 V for 4h. Restriction fragments were visualized after staining with

ethidium bromide, and specificity was confirmed by South-ern blot analysis with a cloned intact HBV DNA probe.

RESULTS

Restriction nuclease maps of the PCR products from spec-imens of predetermined subtypes. When the DNA of theprototype HBV of known serosubtype was amplified anddigested with the selected restriction nucleases by the de-scribed procedures, the predicted results were obtained (Fig.2). The amplified 575-bp DNA fragment of the adr subtypewas susceptible to HpaII and XhoI digestion and gaveproducts of 357/218 bp and 306/269 bp, respectively. The 306and 269 bp were not separated but merged as a wide band.The amplified DNA of adw subtype was digested by BamHIto give two fragments of 405 and 170 bp and was resistant toHpaII and XhoI. The amplified product of the ayw subtypewas susceptible to XhoI, giving fragments of 306 and 269 bp,and was resistant to BamHI and HpaII. In addition, thespecificity of the restriction nuclease fragments was con-firmed with a cloned HBV DNA probe in Southern blotanalysis (data not shown).

Application of PCR typing to HBV-positive sera. Table 2compares PCR typing results with those of the serologicallypredetermined subtype on selected patient sera. All eight adrserum samples gave the correct restriction fragments corre-sponding to the adr subtype prototype. Six of the nine adwsubtype serum samples gave the predicted restriction map,but three others gave a pattern similar to that of the aywsubtype. While five of the eight ayw subtypes gave the samefragmentation as the ayw prototype, the amplified productfrom two patients was totally resistant to digestion by theselected restriction endonucleases and one was susceptibleto all three nucleases. The results from patients 51, 43, and68, whose sera gave rise to unusual patterns, are shown inFig. 3. The PCR product or the restriction nuclease frag-ments were all HBV specific as determined by Southern blotanalysis (data not shown). It is of interest to note that two ofthe adw serological subtype serum samples which gave anayw PCR restriction pattern (patients 84 and 87) were sexualpartners in a case in which sexual transmission was pre-sumed.

DISCUSSION

We have demonstrated an alternative approach to classi-fying HBV on the basis of the pattern of restriction endonu-clease digests of amplified genetic material. The specificityof PCR subtyping was confirmed by the expected pattern ofdigestion and the correct size of the fragment obtained. Theexpected pattern and the sizes of the digested fragmentswere predicted from the cloned HBV nucleotide sequence ofknown serotypes. The HBV specificity of each fragment wasfurther confirmed by Southern blot analysis with a radiola-beled cloned HBV DNA probe. However, the categorizationof HBV by fragmentation of PCR products was not totallyconcordant with that of subtyping by serological analysis.This could be due to one or more of the following conditions:polymorphism of the restriction endonuclease sites; altera-tion or deletion of the specific site, nullifying the utility ofselected endonucleases; coinfection by more than one sub-type of HBV has been described by Siddiqui et al. (21), Onoet al. (18), and Okamoto et al. (14). In the case of patient 43,coinfection with more than one subtype of HBV was notlikely since no other cleavage product was observed in theSouthern blot analysis (data not shown). The failure to cut

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TYPING OF HBV BY PCR 1643

TABLE 1. Restriction nuclease susceptibility and expectedfragment size of different HBV subtypes

Enzyme restricted fragment size (bp)Subtype BamHI HpaII Xhol

(1849)a (1897) (1948)

adr 575 357, 218 306, 269adw 405, 170 575 575ayw 575 575 306, 269

a Number in parentheses indicates the susceptible nuclease digestion sitefrom the sequence determined by Gan et al. (6).

HBV DNA in patient 68 also suggests a mutation at therestriction nuclease site rather than coinfection, as evi-denced by the lack of mixed cleavage products, unless allamplified products were from resistant strains.The selection of regions for amplification was on the basis

ofHBV nucleotide sequence of predetermined subtypes, butthis region does not encompass the sequence encodingsubtypic determinants (15, 16). The observed general con-

sistency of the PCR DNA map within the same serologicalsubtype supports the notion that the extent of divergence inthe nucleotide sequences between two HBV genomes of thesame subtype (intrasubtypic divergence) is smaller than thatbetween two HBV genomes of different subtypes (intersub-typic divergence), as observed by other investigators (17).From the limited number of sera studied, sera of the adrsubtype gave the most consistent results, while sera of theadw subtype seemed to be more divergent. This result is alsoconsistent with the percent nucleotide difference of varioussubtypes calculated by Okamoto et al. (17).The most difficult task of this experiment was the selection

of appropriate regions of the genome for amplification andfragmentation. On the basis of criteria set for the selection ofprimers as described in Materials and Methods, a primer setwas selected to amplify the preS-S region of the S gene. Itencompassed the 3' portion of preSl, the entire preS2, andthe 5' portion of the major S gene. Three restriction nu-

clease-susceptible sites, BamHI, HpaII, and XhoI, were

observed within this region, and they gave a unique frag-mentation pattern for each subtype (Fig. 1). These restric-tion nuclease-susceptible sites are all located within thepreS2 gene. The BamHI site (GGATCC), which is specificfor the adw subtype, is located within the amino acidsequence 132 to 134 of the preS gene translational product.This site is formed by a switch code for amino acid 132,leucine (CTA) of the adr subtype to glutamine (CAG) of theadw subtype, and by a degenerate code for amino acid 133(aspartic acid as GAC in the adr subtype to GAT in the adwand ayw subtypes). This site is also eliminated in the aywsubtype as compared with the adw subtype due to a degen-

FIG. 1. Nucleotide sequence of different subtypes of HBV.Partial nucleotide sequences of the HBV genome, which includesthe region amplified, are shown. The sources of these sequences are

as follows: (1) Gan et al. (6), (2) Fujiyama et al. (4), and (3) Ono etal. (18) for the adr subtype; (4) Ono et al. (18), (5) Valenzuela et al.(24), and (6) Lo et al. (12) for the adw subtype; and (7) Galibert et al.(5) for the ayw subtype. The numbers in parentheses at the far rightare the nucleotide numbers at the end of each line in accord with the

original source. The shaded letters in the sequence indicate the

homology between all listed subtypes. The locations of primers andendonuclease restriction sites are indicated.

adr adw ayw

B H T' -WTHX 'BWi M

615 bp492369

246

FIG. 2. Agarose gel electrophoretic analysis of PCR products.DNAs from sera previously serologically subtyped as adr, adw, andayw were amplified for 35 cycles and then digested with BamHI (B),Hpall (H), and Xhol (X) and separated by electrophoresis in a 1.2%agarose gel at 100 V for 2 h. The unblotted product was visible byUV after ethidium bromide staining. The uncut fragment of 575 bpand the digested fragments were shown against a size marker (M),which was a 123-bp DNA ladder purchased from Life Technologies,Inc., Gaithersburg, Md.

erate code (CAA) for amino acid 132 in the ayw subtype. Thespecificity of the HpaII site (CCGG) at amino acids 148 and149 of the preS gene product for the adr subtype is formed byanother degenerate code for amino acid 148 (serine), TCC toTCA, in both the adw and ayw subtypes. The XhoI site(CTCGAG), comprising amino acids 165 to 167 of the preSgene, is eliminated from the adw subtype by switching codesfor amino acids 165 and 166. The predicted enzyme sensi-tivity and the fragment size of each HBV subtype are givenin Table 1.

Restriction enzyme fragmentation, as demonstrated in thisreport, could provide an alternative classification scheme.This procedure would not substitute for serological subtyp-ing, but rather would act as an alternate or additional means

TABLE 2. Comparison of typing by PCR and serology

Serotype Patient Restricted by: Type byno. BamHI HpaII XhoIPCRladr 46 - + + adr

49 - + + adr64 - + + adr65 - + + adr66 - + + adr67 - + + adr110 - + + adr111 - + + adr

adw 44 + - - adw48 + - - adw51 - - + ayw55 + - - adw56 + - - adw57 + - - adw84 - - + ayw85 + - - adw87 - - + ayw

ayw 41 - - - (Uncut)42 - - + ayw43 + + + (All cut)54 - - + ayw68 - - - (Uncut)69 - - + ayw70 - - + ayw79 - - + ayw

a Discrepant results between PCR typing and serotyping are in boldfacetype.

VOL. 29, 1991

1644 SHIH ET AL.

#51 #43 #68B H X B H X B H X

615bp

492

369

246

123

FIG. 3. Agarose gel electrophoretic analysis of PCR productsfrom selected samples of patients 51, 43, and 68 to show their uniquerestriction endonuclease digestion patterns. Patient 51 showed aswitch pattern from an adw serological determination to an ayw

restriction map of PCR products. The PCR product from patient 43was susceptible to all three endonuclease digestions, while that frompatient 68 was resistant to all.

to assess HBV strain variations. Its general utility andclinical application would require more extensive studiesand comparisons with serological tests. The value of classi-fication based on genetic variation was exemplified in theinstance of sexual transmission, in which the restrictionpattern differed from the serological pattern but was identi-cal in the sexual partners.

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