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Copyright ©1991, American SocietyforMicrobiology

Strain Analysis

of

Hepatitis B Virus

on

the Basis of Restriction

Endonuclease

Analysis

of

Polymerase

Chain Reaction Products

J. WAI-KUO SHIH,l* LING C.

CHEUNG,1

HARVEY J. ALTER,1 LEO M. LEE,2AND JIAN REN GU3 Department ofTransfusion Medicine, Warren GrantMagnuson ClinicalCenter, National Institutesof Health,

Bethesda, Maryland208921;Frederick Cancer Research andDevelopment Center, National Cancer Institute, Frederick, Maryland 217012; andShanghai Cancer Institute, Shanghai, China3

Received 6February 1991/Accepted 14 May1991

To assess the value ofclassifying hepatitisB virus(HBV)strainsatthegenomiclevel withproductsfromthe polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design ofthis method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size ofthe fragment resulting from aspecific nuclease digestion. On the basis of published nucleotide sequences ofdifferent HBV subtypes, a set ofprimers was selected within the preS-S region. One oftheprimerswas 1679F containing25 nucleotides(5'-GGGTGGAGCCCTCAGGCTCAGGGC A-3'), and the other was 2254R containing24nucleotides(5'-GAAGATGAGGCATAGCAGCAGGAT-3').All ofthe reactive seraproducedthe same sized575-bpidentification band. Theproductwassubjectedtodigestion bya selected panel of restrictionendonucleases. Byusing prototypeHBV of knownsubtypeas amodel,these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nucleasefragmentation in accordance with the nucleotidesequence ofthe prototype virus. Occasionally, samplesof either the adw or theaywsubtype gave discrepantresultsin which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration wasconsistent in thepaired specimens from sexual transmission cases. These resultsshow thatthedescribed proceduresareapplicable toHBV strain assessment.

Humanhepatitis B virus (HBV) isoneof severalviruses, termed hepadnaviruses, which have similar structural fea-tures and tissue tropism (19). Hepatitis B infection has a worldwide distribution, although its prevalence varies geo-graphically. There are distinct subtypes of HBV indicative of strainheterogeneity. The subtypes ofHBV aredistinguished by antigenic determinants on the surface antigen (HBsAg) and theircorrespondingantibodies(11). There isa common group determinant, a, which appears in all HBsAg speci-mens.There are two sets ofsubdeterminants, d or y andwor r, which appear to be allelic or mutually exclusive (1) and which are used for the identification of subtypes. Thus, there are at least four major groups into which HBsAg can be classified: adw, adr, ayw and ayr. The distribution of sub-types amonginfected populationsalso varies geographically (3). It has been speculated that the subtype specificity of HBVmightaffect the clinical consequences of the infection (13). Subtypingof HBV has proven to be a valuable marker in 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 the samesubtype (14) and between different subtypes have been reported (6, 17). Since the introduction of the polymerase chain reaction (PCR) with a thermostable DNA polymerase (20), this technique has found extensive application in the diagnosis, genetic identification, and molecular cloning and analysis of DNA. The PCR technique has been applied to the detection of HBV DNA in seronegative subjects who trans-mittedhepatitis B (22) and serum HBV DNA in patients with chronichepatitis (9). PCR for the detection of HBV has been shown to be slightly more sensitive than infectivity testing in

* Correspondingauthor.

the chimpanzee (23). A rapid and sensitive method for the detection of serum HBV DNA, using PCR, has been re-ported (8). In this paper,wereporttheapplication of PCR to the determination of strain variation among HBV-infected patient sera, and we compare strain classification basedon

nucleotide sequences with that based on serological analy-S1S.

MATERIALS ANDMETHODS

HBV-positive sera, subtyping reagents, and serological de-terminations. A panel of HBsAg-positive sera subtyped during the International Workshop on HBs Antigen Sub-types, Paris, France,in 1975 (2) andhepatitis Bpatientsera which were collected and subtyped previously at the Na-tional InstitutesofHealth(10) wereused assourcematerial for PCR typing. The preparation ofHBsAg subtyping re-agents and the technique of serological subtyping were described by Hoofnagle et al. (7). Briefly, monospecific anti-HBs/d, ly, or lw was prepared by absorption of poly-clonal anti-HBs/adw or layw with corresponding HBsAg-positive seraof the ayw, adw, or adr subtype. Inhibition of monospecificantibody activity by HBsAg-positive serum in aradioimmunoassay (Ausab; Abbott Laboratories, Chicago, Ill.) was utilized to indicate the presence of a specific subtype determinant.

Cloned HBV DNA sequences and selection of restriction nucleases for the mapping of strains. The selection of primer setsfor synthesis was based on the published sequence data of 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). Primersfor PCR amplification were chosen by the following criteria. (i) The fragments were located within a conserved region so that the primers could be used to detect all possible HBV subtypes (e.g., adr, adw, and ayw). (ii) The distance

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TYPING OF HBV BY PCR 1641

between upstream and downstream primer fragments was less than a few hundred nucleotide bases within the se-quence. (iii) Primer fragments which allowed more than a 3-base mismatch with any other documented sequence in gene banks were not utilized. (iv) Suitable restriction en-zymes that provided satisfactory site-specific cuts had to be available sothat each of the subtypes could be identified.

Upon completion of the sequence analysis and compari-son, two consensus sequences were identified within the preS-S region for each of the HBV subtypes; using the sequencenumbering of Gan et al. (6), these resided between positions 1601and 1703 and positions 2201 and 2255. Partial nucleotide sequences for several HBV subtypes within this region are shown in Fig. 1. Primer pairs within the 3' end of the preSl gene and the 5' end of the major S gene were selected. Primer 1679F5'-GGGTGGAGCCCTCAGGCTCA GGGCA-3' (25-mer) and primer 2254R5'-GAAGATGAGG CATAGCAGCAGGAT-3' (24-mer) were synthesized. The amplification by this primer pair begins at position 1679 and ends at position 2254 of the HBV genome from an adr subtype (6). A PCR product of a 575-bp fragment containing three potential restriction nuclease sites (BamHI [1849], HapII [1897], andXhoI [1948]) was expected. Susceptibility of the threemajorsubtypes to restriction enzyme digestion and their expected fragment sizes aresummarized in Table 1.

Extraction of viral DNA from serum. A 100-pld portion of serum was incubated at 65°C for 2 h in a mixture of proteinase K (100 ,ug/ml), 0.5% sodium dodecyl sulfate, 5 mMEDTA,and 10 mM Tris HCl, pH 8.0. The solution was extractedtwicewithphenol-chloroform-isoamyl alcohol (25: 24:1)and oncewith chloroform-isoamyl alcohol, followed by an ethanol precipitation. The precipitate was dissolved in 100

pul

of TE buffer (10 mM Tris HCl [pH 8.0], 0.1 mM EDTA).

PCRamplificationofHBVDNA. A 10-,u aliquot of serum DNA wasamplified in a

100-pul

reaction mixture containing 2.5 U of recombinant Taq DNA polymerase (AmpliTaq; Perkin-ElmerCetus, Norwalk, Conn.), 200

puM

each dATP, dGTP, dTTP, and dCTP, a 1 ,uM concentration of each primer, 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mM

MgCl2,

and0.01% gelatin.Thereactionmixturewasoverlaid with 50

pAl

of mineral oil.

The reaction wasperformed for 35 cycles in a program-mable thermal cycler (Perkin-Elmer Cetus). Samples were

heated to 95°C for 35 s (denaturation of DNA), cooled to

55°C for1 min

(annealing

toprimer), and incubatedat

72°C

for 1 min (polymerase

amplification

reaction). All reagents were tested to ensure that they were free of HBV DNA contamination, andall experiments were

performed

in par-allel withpositiveand negative controlsera.

Analysis ofamplifiedDNA. A 20-,ul

amplified

DNA

reac-tion mixturewas

analyzed by electrophoresis

in 1% agarose at 100 V for 2 h and visualized by UV fluorescence after staining with ethidium bromide. The

specificity

of the

am-plified bandswasconfirmedby Southernblot and

hybridiza-tion withradiolabeled vector-free HBV DNA

probe.

Restriction nucleaseanalysis ofamplifiedDNA. Restriction nucleasedigests ofthe

amplified

DNAwere setupin

parallel

withBamHI, HpaII, and XhoI in

30-pld

reaction

mixtures,

eachof whichcontained20

pul

ofthe

amplified

DNA

sample,

3

pul

of lOx enzyme

buffer,

3

pld

of10mM

dithiothreitol,

3 pld of bovineserumalbumin(1

mg/ml),

and 1

[lI

of nuclease

(10

U).Thereactionmixturewasincubatedat370C for 2 h and fractionatedby 1% agarose

gel

electrophoresis

at50V for 4 h. Restrictionfragments werevisualized after

staining

with

ethidiumbromide, and specificitywas confirmed by

South-ernblotanalysis withacloned intact HBV DNA probe. RESULTS

Restriction nuclease maps ofthe PCR productsfrom spec-imens of predetermined subtypes. When the DNA of the prototype HBV of known serosubtype was amplified and digested with the selected restriction nucleases by the de-scribedprocedures,thepredictedresults wereobtained (Fig. 2). The amplified 575-bp DNAfragmentof the adr subtype was susceptible to

HpaII

and XhoI digestion and gave products of 357/218 bpand306/269 bp, respectively.The 306 and 269 bp were not separatedbut merged as a wide band. TheamplifiedDNAof adwsubtypewasdigested byBamHI

to givetwofragments of405 and 170bpand wasresistantto

HpaII

andXhoI. The amplified product ofthe ayw subtype

wassusceptibletoXhoI,giving fragments of 306 and 269 bp, and was resistant to BamHI and

HpaII.

In addition, the

specificity

of the restriction nuclease fragments was con-firmed with a cloned HBV DNA probe in Southern blot

analysis

(datanot shown).

Application of PCR typing to HBV-positive sera. Table 2 comparesPCR

typing

results with thoseofthe

serologically

predetermined

subtypeonselectedpatientsera.Alleightadr serum

samples

gave the correctrestriction

fragments

corre-sponding

totheadr subtypeprototype. Six ofthe nine adw subtype serum

samples

gave the

predicted

restriction map, but three others gave a

pattern

similar to that ofthe ayw subtype.While five of the

eight

aywsubtypesgavethesame

fragmentation

as the ayw prototype, the

amplified product

from two

patients

was

totally

resistant to

digestion by

the selectedrestriction endonucleases andone was

susceptible

to all three nucleases. Theresultsfrom

patients

51, 43, and 68, whose sera gaverise to unusual

patterns,

are shown in

Fig.

3. The PCR

product

or the restriction nuclease

frag-mentswereall HBV

specific

asdetermined

by

Southernblot

analysis (data

notshown).Itisofinteresttonote thattwoof the adw

serological subtype

serum

samples

whichgave an

aywPCRrestrictionpattern

(patients

84and87)weresexual partners in a case in which sexual transmission was pre-sumed.

DISCUSSION

We have demonstrated an alternative

approach

to

classi-fying

HBVonthebasisofthe

pattern

ofrestriction endonu-clease

digests

of

amplified

genetic

material. The

specificity

ofPCR

subtyping

wasconfirmed

by

the

expected

patternof

digestion

and the correctsizeof the

fragment

obtained. The

expected

pattern

and the sizes of the

digested fragments

were

predicted

from theclonedHBVnucleotidesequenceof known serotypes. The HBV

specificity

of each

fragment

was further confirmed

by

Southern blot

analysis

witha radiola-beled clonedHBV DNA

probe.

However,

the

categorization

ofHBV

by

fragmentation

ofPCR

products

was not

totally

concordant with that of

subtyping

by

serological

analysis.

Thiscould be dueto one ormoreof the

following

conditions:

polymorphism

of the restriction endonuclease

sites;

altera-tion or deletion of the

specific site,

nullifying

the

utility

of selected

endonucleases;

coinfection

by

more than one

sub-typeof HBV has beendescribed

by

Siddiqui

etal.

(21),

Ono

etal.

(18),

and Okamotoetal.

(14).

Inthecaseof

patient

43,

coinfection with more than one

subtype

of HBV was not

likely

since no other

cleavage

product

was observedin the Southern blot

analysis (data

not

shown).

The failureto cut

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TYPING OF HBV BY PCR 1643

TABLE 1. Restriction nuclease susceptibility and expected

fragmentsize ofdifferent HBV subtypes

Enzymerestrictedfragment size (bp)

Subtype BamHI HpaII Xhol

(1849)a (1897) (1948)

adr 575 357, 218 306, 269

adw 405, 170 575 575

ayw 575 575 306, 269

a Number inparentheses indicates the susceptible nuclease digestion site

from thesequence determined by Gan et al. (6).

HBV DNA in patient 68 also suggests a mutation at the restriction nuclease site rather than coinfection, as

evi-denced by the lack of mixed cleavage products, unless all amplified products werefrom resistantstrains.

Theselection of regions for amplificationwas onthebasis

ofHBVnucleotidesequenceof predetermined subtypes,but

this region does not encompass the sequence encoding

subtypic determinants (15, 16). The observed general

con-sistencyof the PCR DNA map within the same serological

subtypesupportsthenotion that theextentof divergence in thenucleotidesequencesbetweentwoHBVgenomesofthe samesubtype (intrasubtypic divergence) is smaller thanthat

betweentwo HBVgenomes of different subtypes

(intersub-typic divergence), as observed by other investigators (17).

From the limited number ofsera studied, sera of the adr

subtype gave the most consistent results, while seraofthe

adwsubtype seemedtobemoredivergent. This result is also

consistent with thepercentnucleotide difference of various subtypes calculated by Okamotoetal. (17).

Themostdifficult task of thisexperimentwastheselection

ofappropriate regions of the genome for amplification and

fragmentation.On the basis of criteriasetfor the selection of primersasdescribed inMaterials and Methods, aprimerset

was selected toamplify the preS-S region of theS gene. It

encompassed the 3' portion of preSl, the entire preS2, and the 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 patternfor each subtype (Fig. 1). These restric-tion nuclease-susceptible sites are all located within the

preS2gene. The BamHI site (GGATCC), which is specific

for the adw subtype, is located within the amino acid

sequence132 to134of the preSgene translational product.

This site is formed by a switch code for amino acid 132,

leucine(CTA) of the adr subtype toglutamine (CAG) of the adwsubtype, and by adegenerate code for amino acid 133

(aspartic acidasGAC in the adrsubtypetoGAT inthe adw and ayw subtypes). This site is also eliminated in the ayw

subtype ascompared withtheadw subtypeduetoa

degen-FIG. 1. Nucleotide sequence of different subtypes of HBV.

Partial nucleotide sequences of the HBVgenome, which includes

theregionamplified,areshown. Thesourcesof thesesequencesare asfollows: (1)Ganetal.(6), (2) Fujiyamaetal.(4),and(3)Ono et

al.(18)for the adrsubtype;(4)Onoet al.(18), (5)Valenzuelaet al.

(24), and (6)Loetal.(12)for the adwsubtype;and(7)Galibert et al.

(5)for theaywsubtype.Thenumbers inparenthesesatthe farright

arethenucleotide numbersattheend of each line in accord withthe

original source. The shaded letters in the sequence indicate the

homologybetween all listedsubtypes.Thelocations ofprimersand

endonucleaserestriction sitesareindicated.

adr adw ayw

B H T' -WTHX 'BWi M

615bp 492 369 246

FIG. 2. Agarose gel electrophoretic analysis of PCR products. DNAsfromserapreviously serologically subtyped as adr, adw, and ayw wereamplifiedfor 35 cycles and then digested withBamHI(B), Hpall(H), and Xhol (X) and separated byelectrophoresisin a1.2%

agarosegelat 100 Vfor2 h. Theunblotted product was visible by UVafterethidiumbromide staining. The uncut fragment of 575 bp

and the digestedfragments were shownagainst a size marker (M), which was a 123-bp DNA ladderpurchasedfrom LifeTechnologies,

Inc.,Gaithersburg,Md.

eratecode (CAA) foraminoacid132 in the aywsubtype.The specificity ofthe

HpaII

site (CCGG)atamino acids 148 and 149of thepreSgene product for the adr subtype is formed by anotherdegenerate codefor aminoacid148(serine), TCCto TCA, in both the adw and ayw subtypes. The XhoI site (CTCGAG), comprising amino acids 165 to 167 of thepreS gene,is eliminated fromtheadwsubtypebyswitchingcodes for amino acids 165 and 166. The predicted enzyme sensi-tivity and the fragment sizeofeach HBV subtype aregiven in Table 1.

Restrictionenzymefragmentation,asdemonstratedin this report, could provide an alternative classification scheme. This procedurewould not substituteforserological subtyp-ing,but rather would act as analternate or additional means

TABLE 2. Comparison of typing by PCR and serology

Serotype Patient Restricted by: Type by

no. BamHI HpaII XhoI

PCRl

adr 46 - + + adr

49 - + + adr

64 - + + adr

65 - + + adr

66 - + + adr

67 - + + adr

110 - + + adr

111 - + + adr

adw 44 + - - adw

48 + - - adw

51 - - + ayw

55 + - - adw

56 + - - adw

57 + - - adw

84 - - + ayw

85 + - - adw

87 - - + ayw

ayw 41 - - - (Uncut)

42 - - + ayw

43 + + + (All cut)

54 - - + ayw

68 - - - (Uncut)

69 - - + ayw

70 - - + ayw

79 - - + ayw

aDiscrepant resultsbetweenPCRtypingand serotypingarein boldface

type.

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#51 #43 #68

B H X B H X B H X

615bp

492 369

246

123

FIG. 3. Agarose gel electrophoretic analysis of PCR products fromselected samples of patients 51, 43, and 68toshow their unique restriction endonuclease digestion patterns. Patient 51 showed a

switch pattern from an adw serological determination to an ayw

restrictionmapof PCR products. ThePCR product from patient 43 wassusceptibletoall three endonucleasedigestions, while that from patient68wasresistanttoall.

to assess HBV strain variations. Its general utility and clinical application would require more extensive studies

andcomparisons with serologicaltests. The value of classi-fication based on genetic variation was exemplified in the

instance of sexual transmission, in which the restriction

pattern differedfrom the serological patternbutwas

identi-cal inthe sexualpartners.

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