Heterogeneity and evolution rates of delta virus RNA sequences.

Heterogeneity and Evolution

Rates

of

Delta Virus RNA

Sequences

FUMIO IMAZEKI, MASAOOMATA,* ANDMASAO OHTO

FirstDepartmentof Medicine, Chiba University School of Medicine, Chiba 280,Japan

Received 24 May 1990/Accepted14August1990

To investigate the geographical divergenceofdelta virus RNAsequences,868 nucleotides(nt), including the

deltaantigen-coding region,weredetermined in isolates fromtwoJapanese patients,MandS, by polymerase

chain reaction and directsequencing andcomparedwith threepreviously reportednucleotidesequences. The sequenceobtained for hepatitisdelta virus RNAfrom patientMwasapproximately92% identicaltosequences

previously obtained fortwoother strains of hepatitis deltavirus,whereasthesequenceofhepatitisdeltavirus RNA obtained from patient S was approximately 81% identical to the previously sequenced strains. This suggeststhat deltaagentinJapanhasaheterogeneousoriginand the delta virus RNAsequencefromJapanese patientS is the mostdivergentdelta virus isolateyetanalyzed.Tostudythe evolutionrateof delta virusRNA,

viral isolates obtained 3 and 4yearsapartfromeachoftwopatientswerealsosequenced.Itwasestimated that the substitution rateof viral RNAwas0.57 x 10-3ntpersiteper yearinpatientMand 0.64 x 10-3ntper

siteper year inpatient Sfor the deltaantigen gene.

Delta agentwas firstdiscovered by Rizzettoetal. (20)as a novel antigen localized in the nuclei of hepatocytes in

patients infected with hepatitis B virus. This agent was

experimentally transmitted to chimpanzees (21) and

wood-chucks (17) in association with hepadnaviruses. Hepatitis delta virus (HDV) is a 36-nm particle containing acircular RNAgenomeof 1.7 kilobases (2, 4, 9, 19). HDV isendemic

throughout the world; itoccurs eitheras aresult of superin-fection of the hepatitis B virus carrier state or as an acute

coinfection, and delta virus infection is known to cause severe liver damage (24). Approximately, 1% ofJapanese

hepatitisB viruscarrierswereinfectedwith deltaagent(14).

However, there was no information on the nucleic acid

structureofdeltaagentderived from Japanese patients. Recently, complete nucleotide sequences of delta virus

RNAwerereported from three laboratories. Wangetal. (27, 28) analyzed an isolate from the serumofa chimpanzee to

which the serumofanItalian patient wastransmitted, Kuo

etal.(11)examinedanisolate from the liver ofawoodchuck towhich theserumofthesamepatientwastransmitted, and Makino et al. (13) analyzed an isolate directly from the serum ofan Americanpatient. In thisreport, partial RNA

sequences 868nucleotides long, includingthe delta antigen-coding regions of isolates fromtwoJapanese patients,were determined to investigate the geographical divergence of viral RNA, and viral isolates obtained 3 and 4 years apart from each oftwopatients were also examined tostudy the evolution of delta virus RNA.

MATERIALS AND METHODS

Virus isolates. Two HDV isolates were obtained on two differentoccasions from Japanese patient M, a 25-year-old malewithchronic active hepatitis.Japanese M-1was avirus

isolate obtained on 20 September 1986, and Japanese M-2 wasobtained on 6 July 1989. Similarly, two HDV isolates were obtained from Japanesepatient S, a 39-year-old male

withchronic active hepatitis. JapaneseS-1isa virus isolate

obtainedon 18July 1983, and Japanese S-2wasobtainedon 6August 1987. The seraof both patients were positive for

*Corresponding author.

hepatitis B surface antigen and hepatitis B eantibody, and the patients were born and grew up in Japan and had no

history ofbloodtransfusion,intravenousdruguse,or homo-sexualactivity.

Extraction of HDV RNA. Serumsamples (100,ul)obtained

from the patients were incubated into a 400-,u solution

containing 10 mM Tris hydrochloride (pH 7.5), 10 mM

EDTA,0.2% sodiumdodecyl sulfate,and 1mgofproteinase

K per ml at 50°C for 3 h. HDV RNA was then extracted twice with 1 volume of phenol-chloroform, followed by

ethanol precipitation in the presence of salt and carrier dextran.

cDNAsynthesisand PCR. Samples (30to50

[L)

ofserum weredenaturedat65°Cfor 3minandreversetranscribed in 20,ul ofasolutioncontaining50 mMTrishydrochloride (pH

8.3),100 mMKCl,10 mMMgCl2,10 mMdithiothreitol,50 U of human placental RNase inhibitor (Takara Shuzo Co., Kyoto, Japan), 1 mM deoxynucleoside triphosphate, 1 ,uM

antisense primer,and 25 U ofreverse transcriptase(Takara ShuzoCo.)at42°Cfor 60min. cDNAproductswereusedas

templates for polymerase chain reactions (PCR) (22). PCR wereperformedin 100 pl ofasolution containing 10mM Tris

hydrochloride (pH 8.4), 50 mMKCl,2.5 mMMgCl2,0.2mg

ofgelatinper ml, 1 ,uM senseprimer, and 4U ofTaqDNA

polymerase (New England BioLabs, Inc., Beverly, Mass.)

overlaid with 100 pul of mineral oil withan automatic

ther-mocycler.The thermalprofileinvolved35cycles of denatur-ationat95°Cfor 2min, primer annealingat42°C for2min,

and extension at 70°C for 2 min. Oligonucleotide primers

were synthesized by a DNA synthesizer (380B; Applied

Biosystems, Inc., Foster, Calif.) and purified with high-performance liquid chromatography. Synthetic primersused for cDNAsynthesisandPCRarelisted in Table 1.Theyare basedonthe nucleotidesequenceinformation ofWangetal.

(27, 28) or sequencing data obtained with isolates from

patients M and S. PCR products were electrophoresed on 8%polyacrylamide gel, andthegelwas stained with

ethid-ium bromide,followedby confirmationthat thebandwasof the estimated size.

Directsequencing.PCRproductsweredirectly sequenced bidirectionally by using the appropriate 32P-end-labeled

se-quencing primerand thedideoxy-chain termination method

5594 0022-538X/90/115594-06$02.00/0

Copyright © 1990,American Society for Microbiology

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TABLE 1. Syntheticoligonucleotides used for cDNA synthesis and PCR'

* ~~~~~~~~~~~~~~Nucleotide

Primer Sequence position

positions DELP4 5'-CGGGGGCGGCTTCGTCCCCA-3' 1110-1091

DELP7 5'-TAATGGCGAATGGGAC-3' 754-769

DELP25 5'-GAGGTTGACCGAGGAAGACG-3' 1206-1187 DELP3 5'-GGTCGACAACTCTGGGGAGA-3' 961-980 DELP24 5'-CCGGACCTAGGAAGAGGC-3' 1351-1334 DELP30 5'-TTGGGGACGAAGCCGCC-3' 1090-1107 DELP11 5'-AGCAGTCTCCTCTTTACAGA-3' 1657-1638 DELP9 5'-TCTTGTTCTCGAGGGCCTTC-3' 1265-1284 DELP37 5'-AAGGAAAAGAAGAGTAGCCG-3' 1182-1163 DELP34 5'-TGGGTCCCCCTGATGTCCAG-3' 1024-1043

"Thenotationis that ofWangetal.(27,28).Thesynthetic oligonucleotides for DELP4, DELP7, DELP25,DELP3,DELP24, DELP9, and DELP11are based onthe information ofWang et al. (27, 28), andthose for DELP30,

DELP37, and DELP34 are based on data obtained from Japanese patients M andS.

Patient M

5

-31 125

4.~. 37

38

301[ 124

*~. 29

e-25

31 -5 ---13

--71 1~4 9

< - 6 *4 32

19 4-12

5'

500 1000 --4---'1500 169 bp3'

<~ HDAg

Patient S

afterpurificationwith a Centricon 30 (Amicon Div. of W. R.

Grace & Co., Danvers, Mass.) (25). The oligonucleotide

primers used for sequencing are listed in Table 2, and the

strategies used for PCR and sequencing of the HDV

ge-nomes ofpatients M and S are shown in Fig. 1.

Analysis ofsequence identity. HDV RNA sequences ob-tained with isolates from the Japanese patients were com-paredwith those reported previously and analyzed with the

SDC Genetyx system (Software Development Co., Tokyo,

Japan).

Nucleotide sequence accession numbers. The nucleotide

sequence data reported here will appear in the DDBJ,

EMBL, and GenBank nucleotide sequence data bases under

accession numbers D90190, D90191, D90192, and D90193.

RESULTS

cDNAsequence of HDV RNA and comparison of sequence

identities. Eight hundred sixty-eight nucleotides, from

posi-TABLE 2. Syntheticoligonucleotides used for directsequencing"

Nucleotide

Primer Sequence poiton

positions DELP5 5'-TCGGCTGGGAAGAGTATATC-3' 991-1010 DELP6 5'-AGTCCCGGAGTCCCCCTTC-3' 1080-1062 DELP10 5'-TTCGTCGGTGATCCTGCCTC-3' 1286-1305 DELP12 5'-AGAAGGATAAGGATGGAGAG-3' 1420-1401 DELP13 5'-TCTAACTTCTTTCTTCCG-3' 1516-1533 DELP19 5'-CGCGGTCCGACCTGGGCATC-3' 874-855 DELP20 5'-GGAGTACACTCGAGGAGTGG-3' 961-980 DELP22 5'-TTCTTCCTCGAGTTTCTTGA-3' 1458-1477 DELP25 5'-GAGGTTGACCGAGGAAGACG-3' 1206-1187 DELP26 5'-AGGAAGAAAATCCCTGGCTG-3' 1465-1446 DELP29 5'-TTCACCGACAAGGAGAGG-3' 1319-1302 DELP31 5'-CAGAACTCTCTCTAGATTCC-3' 771-790 DELP32 5'-AAAGAGTAAGAGTACTGAGG-3' 1637-1618 DELP33 5'-AAAGAATAGAGAGAACTGAG-3' 1637-1618 DELP35 5'-CTCCCCCGTCCGAGAGAAGG-3' 1047-1066 DELP36 5'-CCCGCGGGTTGGGGATGTGA-3' 1161-1142 DELP38 5'-GGTTCACATCCCCAACC-3' 1139-1155

aThe notation is that ofWangetal.(27,28).Thesyntheticoligonucleotides for DELP5, DELP6, DELP10, DELP12, DELP13, DELP19, DELP20, DELP25, DELP29,and DELP31 are based on the information ofWangetal.

(27, 28), and those for DELP22, DELP26, DELP32, DELP33, DELP35, DELP36,andDELP38 are based on data obtained fromJapanesepatientsM andS.

35

-34 E 37

-4-- 36

36-4

301 124

4-~29

4--25

20 -. 2

4-.-6 4.. 33

4-l . we

5'

S00 1000 1300 I6>9

bp

FIG. 1. Strategies used for PCRandsequencing of the genomes of HDV isolates from patients M and S shown relative to the 1679-nucleotide full-length HDV genome and delta antigen-coding frame. The notation is that of Wang et al. (27, 28). The open rectangles indicatecDNA segmentsamplified by PCR. The numbers onboth sides of the rectangles indicate the synthetic oligonucleo-tidesusedforPCR (Table 1). The arrows indicatetheorientations and regions sequenced. The numbers by the arrows indicate the syntheticprimers used for direct sequencing (Table 2). HDAg, HDV antigen; bp,basepairs.

tions770 to1637,asdesignated by Wang et al. (27, 28),were

determinedinJapanesevirusisolatesM-1, M-2,S-1,andS-2 (Fig. 2). Theopen readingframe of deltaantigen, the only knownprotein of HDV, is included in this region. ThecDNA sequence of HDV RNA from patient S showed not only single-base changes but six-base deletions and four-base insertions from positions 770 to 1637 compared with the sequence reported by Wang et al. (27, 28), whereas only single-base changes werefound in HDV RNAfrom patient

M(Fig. 2).

Thepercent identities of nucleicacids in thisregionamong

five virus isolates are shown in Table 3. The highest nucle-otide identity (98.8%), as expected, wasobserved between two isolates from chimpanzee-adapted (27, 28) and

wood-chuck-adapted (11) HDV genomes from an Italian patient (Table 3). The cDNA sequenceidentitybetweenthe

Italian-derived (27, 28) and American-derived (13) HDV genomes

was89% in the entire region and 92.4% in the regionfrom

positions 770 to 1637. Similar degrees of cDNA sequence identity (90.8 to92.3%)wereobserved between HDVRNA frompatientM and HDV RNAs derived from the Italianand American patients (Table 3). In contrast, the cDNA

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770

Japanese M-1 CCAGAAATCTCTCTAGATTC CCAGAGAGAA TCGAGAGAAAACTGGCTCTC CCTTAGCCAT Japanese M-2 --- ---JapaneseS-1 ---C---.--__

---Japanese S-2 ---C---.-.---__-

_--_---Italian ---C---G-T---G---

---830

JapaneseM-1 CCGAGTGGACGTCTGTCCTC CTTCGGATGC CCAGGTCGGA CCGCGAGGAG GTGGAGATGC

JapaneseM-2 ---

---JapaneseS-1 ---TC--- --A---

---Japanese S-2 ---TC---A--- ---Italian ---GC---

---890

JapaneseM-1 CATGCCGACC CGAAGAGGAA AGAAGGACGC GAGACACGAACCCGTGAGTG GAAACCCGCT JapaneseM-2 --- --- ---JapaneseS-1 ---A--T--x---G---- x---x---TGA

Japanese S-2 --- ---A--T--x---G----x---x---TGA

Italian ---G-A--

--T---950

Japanese M-1 TTATTCACTGGGGTCGACAA CTCTGGGGAG AAAAGGGAGG ATCGGCTGGA AAGAGTATAT JapaneseM-2 --- ---

---JapaneseS-1 -CC--T-T---AGT---C TCGA--A-T- G--G-C-TT- T-G--GG--- G-TG-GC-TA

JapaneseS-2 -CC--T-T-- ---AGT---C TCGA--A-T-G--G-C-TT-T-G--GG---G-TG-GC-TA

Italian ---C---G

---1010

JapaneseM-1 CCTATGGGAA TCCCAGGTTTCCCGTCACGT CCAGCCCCTCCCCGGTCCTG GTGAAGGGGG Japanese M-2 ---

---Japanese S-1 ----C--- ----T--G-C ---C-G-T-- ----T--- ---C----GA -A---A-Japanese S-2 ----C--- ----T--G-C---C-G-T---T--- ---C----GA -A---A-Italian --C----A-- ----C--- ---C-G-T---GA

-A---1070

Japanese M-1 ACTCCGGGACGCCTTGCATG TTGGGGACAAAGCCGCCCCC GGGCGCTCCC CTCGGTCCAC JapaneseM-2 --- ---JapaneseS-1 ---A-- T---GC---T-GA---G---A-- --- ---A--T-Japanese S-2 ---A--T---GC---T-GA---G---A---

---A--T-Italian ---T--C----GA C---A---

----A---1130

Japanese M-1 CTTCGAGGGG GTTCACACCCCCAACCGACG GGCCGGCTACTCTTCTTTCC CTTCTCTCGT Japanese M-2 ---

---Japanese S-1 --CT-GA---T-- ---CG---T- ---TC---JapaneseS-2 --CT-GA---T---CG---T- ---TC---Italian --- -A---G---

---_-1190

JapaneseM-1 CTTCCTCGGT CAACCTCTTA AGTTCCTCTTCTTCTTCCTTGCTGAGGTTC TTCCCTCCCG

Japanese M-2 ---__

-Japanese S-1 ----A---G---TC-G---C--- ---A--

--T---JapaneseS-2 ----A--- --G---TC-G---C--- ---.A- --T---Italian ---C-G---C---

--G---1250

Japanese M-1 CGGCCAGTTG CTTCCTCTTGTTCTCGAGGGCCTTCCTTCGTCGGTGATCC TGCCTCTCCT JapaneseM-2 ---A---T--- ---Japanese S-1 ---AG--C-- ----T---T G--- -C---JapaneseS-2 ---AG--C-- ----T---TG---

-C---Italian -C-AT--C-- ----T--- ---

---1310

JapaneseM-1 TGTCGGAGAACCCTCCCCTGAGAGGCCTCTTCCTAGGCCCGGTGTCTACCTCCATCTGGT

JapaneseM-2 --- ---JapaneseS-1 ---T--- ---G-T-T-- T--- ---C---T-- --A---G---

---A-JapaneseS-2 ---T--- ---G-T-T-- T--- ---C---T-- --A---G--- ---A-Italian ---T---T---T-- --A---

---1370

Japanese M-1 CTGTTCGGGC CCTCTTCGCCGGGGGAGCCCCCTCTCCATCCTTATCCTTC TTTCCGAGAA

Japanese M-2 --- ---JapaneseS-1 -C--C----G.---C--G-- ---xx--C-x

----T--TT-Japanese S-2 -C--C----G.---C--G-- ---xx--C-x

----T--TT-Italian -C.--- ---1430

Japanese M-1 TTCCTTTGACGTTTCCCAGC CAGGGATTTTCTTCCTCAAG TTTCTTGATT TTCTTCTTAA Japanese M-2 --- ---Japanese S-1 ----GAC--T ---C---G---GG---C-GG Japanese S-2 ----GAG--T ---C---G---GG---C--G

Italian ---T ---C---G---TC--T----G- ---TG 1490

JapaneseM-1 TCTTCCGGAGGTCCCTCTCG AGTTCCTCTA ACTTCTTTCTTCCGTCCACC CACTGCTCGA JapaneseM-2 --- ---JapaneseS-1 --- A--TT--- --C---CGC---T--C---G-TGTG-T- --T-T---Japanese S-2 C---A--TT---C---CGC---T--C---G-TGTG-T- --T-T---Italian ---T---G---

---1550

JapaneseM-1 GGATCTGCTCCCTTCCGGCG CxGCTTCCCCT xxTCGACTCGGAACGGCTCATC TCGACAAGAG

JapaneseM-2 ---x---xx--- ---Japanese S-1 --G---C---GTTC-C-TT-G---T--CC---T---TT---T CTC-GGTCG-Japanese S-2 --G---C---GTTC-C -TT-G---T--CC---T--- TT---T CTC-GGTCG-Italian ---CT--T--C--TC-- -x-G---TT-Cxx---C---

---G-T----1610

Japanese M-1 GCGGCAGTCCTCAGTACTCTxTACTCTTT

JapaneseM-2 ---G---G----x

Japanese S-1 ---AG .---T----

C--T---Japanese S-2 ---AGG---T---- C--T---Italian ---

x---FIG. 2. Nucleic acid sequence alignment of five virus isolates

from two Japanese patients and an Italian patient (27, 28) at

nucleotide positions 770 to 1637. An x indicates deletion of the

sequence. The designations are thoseof Wangetal. (27, 28). The

Japanese M-1 isolate obtained from patient M was isolated on20

September 1986; the Japanese M-2 isolate obtained from patientM

wasisolatedon6July1989;the Japanese S-1isolate obtained from

patient S was isolated on 18 July 1983; the Japanese S-2 isolate

obtained from Japanesepatient Swasisolatedon6August 1987; the

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Italianisolate obtained fromanItalianpatientwasreported byWang etal.(27, 28).

TABLE 3. Comparisonofnucleicacidsequenceidentity inthe region between positions 770and 1637

%Identitywith:

Isolate Italian Italian Japanese

(chimpanzee)' (woodchuck)b American' M_jd Italian (wood- 98.8 100

chuck)b

Americanc 92.4 92.2 100

JapaneseM_1d 92.3 92.2 90.8 100

JapaneseS-V' 81.4 81.7 81.0 80.2

" Chimpanzee-adapted HDVgenomefrom anItalianpatient,reported by

Wang et al. (27, 28).

bWoodchuck-adapted HDV genome from the same Italian patient,

re-ported byKuo etal.(11).

' Obtainedfrom anAmerican patient,reported byMakino et al.(13). dObtained fromJapanesepatientM(virus isolatedon 20September 1986).

eObtainedfromJapanesepatient S (virus isolatedon 18July 1983).

quences of isolates from patient S showed rather weak

identities (80.2 to 81.7%) with the other isolates (Table 3). Deltaantigenwascomposed of 214 amino acids (aa) in the reports of Wang et al. (27, 28) and Makino et al. (13). However, the delta antigen of Japanese patients M and S

was supposed tobe 195 aa because of a G-to-Asubstitution

atposition 1012,asin the reportfromKuo etal. (11)(Fig. 2).

A comparison of nucleic acid and aa sequence identities

amongdeltaantigen-codingframesof HDV isolates is shown in Table 4. The percent identities of the aa of deltaantigen predicted by the nucleic acid sequences were 85 to 88%

among isolates from the Italian patient, the American pa-tient, and JapanesepatientM(Table 4). When the aaof delta

antigenderived from Japanesepatient S were compared with thosefrom the Italianpatient(27, 28), the Americanpatient

(13), and Japanese patient M, there was only 75 to 79%

sequenceidentity.

Evolution of HDV RNA. The sequences of two virus

isolates obtained 3 and4years apartfrom patients MandS,

respectively,werecompared. Of 868 nucleotides, therewere

substitutions at positions 1253, 1264, 1615, and 1625 in the isolates frompatientMandpositions 1436, 1488, and 1490 in theisolates from patient S (Fig. 2). Two of four nucleotide substitutions in the isolatesfrom patient Mand all three in theisolatesfrom patient S occurred within the delta

antigen-coding region,which is 585 nucleotides long. The

substitu-tion rate of HDV RNA for the delta antigen gene was

calculated(5)as0.57x 10-3nucleotides per site per year for

patientMand 0.64 x 10-3 nucleotides persite per year for

patient S. Thefour-of-five nucleic acid substitutions in the deltaantigen-codingregion occurred at position 1 or 2ofa

codon, and this resulted infour aminoacidchanges.

DISCUSSION

Five groups have reported the molecular cloning and

sequencing of either partor all of the HDV genome (9, 11, 13, 23, 27, 28). Three of them determined the complete nucleotide sequence of thevirus and, hence, determined the open reading frame of delta antigen (11, 13, 27, 28). We obtained partial sequences of delta virus RNAs from two Japanesepatients,including the delta antigen-coding region, and compared them with the previous reports (11, 13, 27, 28). The percent identities of the nucleic acids from positions 770to1637were91to92%amongthe three deltavirus RNA sequences,namely, thatofanisolate from an Italian patient reported by Wanget al. (27, 28), that of an isolate from an

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TABLE 4. Comparison of nucleic acid and aa sequence identities in the delta antigen-coding region'

%Identity of nucleic acids/aa

Isolate Italian Italian A Japanese

(chimpanzee) (woodchuck) American M-1

Italian (wood- 98.6/98.5 100/100 chuck)

American 91.3/86.9 91.0/86.2 100/100

Japanese M-1 90.8/87.2 91.0/88.2 89.3/84.6 100/100 Japanese S-1 82.4/77.6 83.1/78.6 81.4/75.0 80.5/74.5

aNucleotides954 to1598in the American andchimpanzee-adaptedItalian

isolatesand nucleotides 1011 to 1598inthe woodchuck-adapted Italian isolate

and theJapanese M-1andS-1 isolateswerecompared because ofa stop codon atnucleotides1011 to 1013.

American patient reported by Makino et al. (13), and that of an isolate from Japanese patient M. There was only 80% identity of nucleic acid sequences between Japanese patient S and the other three patients. Thus, the nucleic acid sequenceobtained from patient S was the most divergent of those studied. Several subtypes of delta virus may exist in

the world, and HDV in Japan may have a heterogeneous

origin.

When the aa and nucleic acid sequences in the delta

antigen-codingregions of five virus isolates were compared, the percent identities of aa were lower than those of nucleic

acids (Table 4). The nucleic acid substitutions in the delta

antigen-codingregion occurred more frequently at positions 1and 2 of a codon than at position 3. Nucleotide

substitu-tions atposition 1 or 2usually change the codingat the aa level, while nucleotide substitutions at position 3 usually do not.Ingeneral,coding at the aa level is more conserved than

atthenucleic acid level in RNA viruses like human influenza A virus (1), hepatitis C virus (10), and retroviruses (5). In

these viruses, most of the nucleotide substitutions within a

subtype occur at position 3 in a codon. HDV may be a

uniqueRNAvirus in this regard.

Therateofevolution of HDVwasestimated to bearound 10-3 nucleotides per site per year, and this value is almost thesame asthatof the nonstructural (NS) gene of influenza Avirus(3) and the envelope gene of human T-cell

lympho-tropicvirus type III(6); 10to

102-fold

higher than that of the surface, core, X, and polymerase genes of hepatitis B virus (16);and

106-fold

higher than that ofmostDNA genomes(5, 7). This highevolutionrate of HDVseems to bepartly due

tolack of theproofreading enzymes like otherRNAviruses (7). The HDV genome may existas amixture of viruses with

many microheterogeneities of the nucleotide sequence in

oneindividual. The nucleotide sequence reported byWang

et al. actually contains an ambiguity arising from clonal

heterogeneity of single nucleotides at 16 different positions (27, 28), and such aresult makes the calculations of

evolu-tion rates, such as thoseoffered here, seem inappropriate.

However,weusedamethodof directsequencingafter PCR,

instead of ordinarycDNA cloning,toestimatetheevolution

rateofthe HDVgenomeonthebasis oftwopairs of isolates. Although these results apply only to changes in which the averagevalueofthenucleotideat apositionwaschanged,it is possible to determine the nucleotide sequence ofa virus genomewhich is dominant inone individual and, hence, to

know its transition based on two pairs of virus isolates. In

previousreports of estimates of the evolutionrates of virus

genomes, nucleotide sequences of the NS gene of human

influenza A viruses were determined by direct RNA

se-quencingofpurified viral RNA (3)or nucleotide sequences

of sequentially isolated human T-cell lymphotropic virus type III in one individual were determined by molecular

cloning and only one clone for each virus isolate was

analyzed and compared (6). Because virus genomes may

exist as a heterogeneous population in one individual, it

seemsimportanttodetermine the nucleotide sequence of the virus isolate which isdominant,eitherby sequencingmany clones after ordinary

cloning

orby direct

sequencing

after

PCR,as wedid,tocalculate and estimate the evolutionrates

of genomes.

Recently, Luo et al. reported that RNA duplex

unwin-dase,which deaminatesadenosineandconvertsittoinosine, mightbe involved innucleic acid substitutions in the HDV genome(12). Theeffect isreplacement ofAwith G on one

strand and T with C on the complementary strand. They

found thatAwasreplaced byGat

position

1012,which leads

to translation of the large 214-aa delta antigen,

during

replication

ofthe HDV genome. Inour

study,

4and 3of868

nucleotides were substituted in

patient

M over a 3-year

period and in patient S over a 4-year

period, respectively.

Conversion ofA to G orT toC was found in two of four nucleotidechangesinpatientMandoneof three inpatientS. This value, three of seven, is

higher

than that

expected,

although conversion of A to G at

position

1012 was not

detected in eitherpatient. Theseresults suggest that unwin-daseplays some importantrole innucleic acid substitutions of the HDV genome in

humans,

but not all of the

changes

followedthe

proposed

rule. More

study

is

needed,

becausea

minorpopulation of virus genomesmight be missed

by

our

method ofdirect sequencingafterPCR.

Analysis ofHDV RNA sequences showed 5 to 11 open

readingframeslargerthan 100aawithin HDV RNA and the

complementary anti-HDV RNA strand(11, 13, 27,

28).

The

delta

antigen

is theonly viral

protein

knowntobe

expressed

during HDV infection in vivo and encoded from the

antige-nomic strand ofHDV. Wang et al. (27, 28) predicted two

delta antigen coding frame

species

214 and 195 aa

long

because of theambiguity arisingfrom clonal

heterogeneity.

Makinoetal.(13)andKuoetal. (11)

predicted only

the

214-and 195-aa

species,

respectively.

In our

study,

only

the 195-aa delta

antigen-coding

frame

species

was

predicted

in

HDVisolates from bothJapanese

patients.

HDV is consideredtobe

directly

cytopathic

(18),but the mechanism of

pathogenesis

remains to be elucidated.

Re-cently, Negro et al. found that extensive

complementarity

between human 7SL RNA and

antigenomic

HDV RNA

exists andthey

presented

the

hypothesis

thatan

antigenomic

form of HDV may anneal to the human 7SL RNA and

formation of

hybrids

maybeafactor in disease

(15).

Indeed,

nucleotides 858 to 899 in HDV sequences from Japanese

patients

M and S were very well conserved

(in fact,

100%

matched in this 42-nucleotide segment;

Fig. 2),

like those fromanItalian

patient

andanAmerican

patient (13, 27, 28).

HDV has some characteristic structures in common with

viroid or virusoid (27). The virusoid consensus sequence,

GATTTT,

which may be

important

for

replication

of

viru-soids,

exists in HDV from Japanese

patients

M and S at

nucleotides 1454 to 1459, as

pointed

out in HDV isolates froman Italian

patient

andanAmerican

patient (13, 27,

28).

Wanget al. determined the

immunogenic

domains of HDV

antigen by using

the

synthetic hexapeptides

spanning

the entire214-aaresiduesof the

protein (26). They

reported

that the domains

recognized by

antibodies present in sera from

humanchronic carriers of HDV included residues 2to

7,

63 to74, 86to 91,94to 100, 159to

172,

174to

195,

and 197to 207. When these

regions

were

compared

among five virus

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Italian1 Italian2

American

JapaneseM-1

Japanese S-1

Italian Italian

American

Japanese M-1

Japanese S-1

Italian Italian American

JapaneseM-1

Japanese S-1

10 20 30 40 50 60

MSRSESRKxNRGGREEILEQW VAGRKKLEELERDLRKTKKK LKKIEDENPWLGNIKGILGK ---R--xD- ----D--- -S--- ---L--- I--L-ED--- ---I---KGxk- A---Q--- -D--- ---I--- I--L-E---- ---V---Q--T-RGR- -T---T--K- ITA---A--- -K---R-T I--L-E---- ----V--IxR 70 80 90 100 110 120 KDKDGEGAPPAKRARTDQME VDSGPRKRPL RGGFTDKERQ DHRRRKALEN KKKQLSAGGK ---KL-M---- I-A--- --- -R----S---.-.---.T---S--- --- -R---A---G--- ---P--- ---G---H KS---E--- ---130 140 150 160 170 180 NLSKEEEEEL RRLTEEDERRERRVAGPPVG GVIPLEGGSR GAPGGGFVPS LQGVPESPFS

--- --- --N--- ---N

---S--R.---K---K- ---I---S--_-N--- ---M---A --- K---S-- --N---P- ---N M---T I---D---E-K---KR-- D-N-SR--P- ---Q

MA---190 200 210 Italian RTGEGLDIRG NRGFPWDILF PADPPFSPQS CRPQ Italian ---

-Q---American --- SQ.--- ----Japanese M-1 ---VT-

-L---Japanese S-1 ---

TQ---FIG. 3. Alignment of theaasequencesof the deltaantigengenes of five virus isolates. The single-letter aa code is used. An x

indicates deletion of thesequence.The Italian'sequenceis that ofa

chimpanzee-adapted HDVgenomefromanItalianpatient, reported by Wangetal. (27,28); the Italian2sequenceis thatofa woodchuck-adapted HDV genomefrom the same Italianpatient, reported by Kuoetal.(11);the Americansequence wasreportedby Makinoet al.(13); the Japanese M-1sequenceobtained fromJapanese patient M wasisolated on20 September 1986; the JapaneseS-1 sequence obtained fromJapanese patient Swasisolatedon18July1983.

isolates, residues 63to72, 160to 169,174to 179,and 181 to 187were completely conserved (Fig. 3). These results sug-gest that sequences in these functional domains were well conserved in each of the virus isolates.

We used the PCR technique instead of ordinary cDNA

cloningtoexaminethevariationinHDV andtoestimate the evolution rates ofgenomes. This method saves time, and very small amounts of infected serum are needed to

deter-mine the amplified cDNA sequences (about 30 to 50 ,ul of serum per PCR in our study). The nucleotide substitutions

shown in our study might be due to an artifact of PCR

amplification (8, 22), but they were reproducible and were confirmed by bidirectional sequencing. This method is also usefulfordiagnosisof delta virus infectionbycDNA ampli-ficationby using synthetic primerslocated inveryconserved

regions of HDV sequences.

In conclusion, the sequence obtained for hepatitis delta virus RNAfrompatientMwasapproximately92% identical

to sequences previously obtained for two other strains of

hepatitis deltavirus, whereas thesequenceofhepatitisdelta

virusRNAobtained from patient Swasapproximately 81%

identicaltothepreviously sequenced strains and 80% iden-tical to the sequence obtained from patient M. These data suggestthatdeltaagentinJapan hasaheterogeneous origin. The delta virus from patient S may be adifferent strain of deltaagent. Complete sequencing of delta virus RNA from

our patients and comparison with other sequences derived

from different areasof the world may elucidate the signifi-cance of theobserved heterogeneity.

ACKNOWLEDGMENTS

Thisstudywassupported inpartbyJapaneseMinistry of Educa-tiongrants(B)58480215, (C)60570310, and(I)59010029.

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