Proc. Nati. Acad. Sci. USAVol. 89, pp. 559-563, January 1992Medical Sciences

Characterization of a prototype strain of hepatitis E virus(anti-hepatitis E virus antibodies/PCR/genome sequence comparison)

SERGEI A. TSAREV*tt, SUZANNE U. EMERSON*, GREGORY R. REYES§, TATIANA S. TSAREVA*,LLEWELLYN J. LEGTERSI, IFTIKHAR A. MALIK II, MUHAMMAD IQBAL II, AND ROBERT H. PURCELL**Hepatitis Viruses Section, Laboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,MD 20892; tShemyakin Institute of Bioorganic Chemistry, Moscow 117871, Russia, U.S.S.R.; §Molecular Virology Department, Genelabs Inc., Redwood City,CA 94063; 1Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; and'lPakistan-U.S. Laboratory for Sero-Epidemiology, Army Medical College, Ralwalpind, Pakistan

Contributed by Robert H. Purcell, October 10, 1991

ABSTRACT A strain of hepatitis E virus (SAR-55) impli-cated in an epidemic of enterically transmitted non-A, non-Bhepatitis, now called hepatitis E, was characterized extensively.Six cynomolgus monkeys (Macaca fascicularis) were infectedwith a strain of hepatitis E virus from Pakistan. Reversetranscription-polymerase chain reaction was used to determinethe pattern of virus shedding in feces, bile, and serum relativeto hepatitis and induction of specific antibodies. Virtually theentire genome of SAR-55 (7195 nucleotides) was sequenced.Comparison of the sequence of SAR-55 with that of a Burmesestrain revealed a high level of homology except for one regionencoding 100 amino acids of a putative nonstructural polypro-tein. Identification of this region as hypervariable was obtainedby partial sequencing of a third isolate of hepatitis E virus fromKirgizia.

Epidemics of enterically transmitted non-A, non-B hepatitis(hepatitis E) have been reported in Asia, Africa, and NorthAmerica (for reviews, see refs. 1-4). Similar cases of spo-radic hepatitis, presumed to be hepatitis E, account for up to90o of reported hepatitis in countries where hepatitis E isendemic. Furthermore, hepatitis E has been implicated infulminant hepatitis of pregnancy: up to 20o of pregnantwomen with hepatitis E die oftheir disease. That a viral agentwas responsible for hepatitis E epidemics was first shown byBalayan et al. (5). It was provisionally called hepatitis E virus(HEV) (2). On the basis of electron microscopy and othercharacterization it was proposed that HEV belongs to thecalicivirus family (6, 7). A partial sequence (8) and later theentire sequence (9) of an HEV genome have been reported.Comparisons ofHEV sequences with sequences available incomputerized data bases, including those of caliciviruses(10), showed little similarity between HEV and any otherknown viruses.

Several species of primates have been experimentallyinfected with HEV (5, 11-19). Although HEV infection is lesssevere in monkeys than in humans, it is sufficiently similarthat monkeys can serve as a reasonable and reproducibleanimal model to provide a more complete characterization ofthis infection.

Determination of the pattern of HEV shedding in feces,bile, and sera during infection is important for characteriza-tion of the disease and elucidation of the epidemiology ofHEV. In some cases, the presence of HEV in a sample hasbeen demonstrated by transmission to animals (5, 11-19) butsuch transmission studies cannot be used for routine inves-tigation. Until recently, the primary method available forcharacterization of virus shedding was immune electronmicroscopy (IEM) (for reviews, see refs. 1-4). However,

because the level of HEV in feces and bile is very low (5,11-19), the sensitivity of IEM is inadequate for completecharacterization of HEV infection. A more sensitive tech-nique, detection of the virus genome by reverse transcrip-tion-polymerase chain reaction (RT-PCR), was used in thisstudy to correlate the presence of HEV in serum, bile, andfeces ofan experimentally infected cynomolgus monkey withbiochemical evidence of hepatitis and development of anti-bodies to HEV (anti-HEV). In addition, we obtained struc-tural information about the virus genome through sequencingof the PCR products themselves or their cloned deriva-tives.**

MATERIALS AND METHODSVirus Samples and Inoculation of Primates. Feces contain-

ing HEV SAR-55 were collected from a patient during ahepatitis E outbreak in Sargodha, Pakistan (20, 21). Approx-imately 0.5 ml per monkey ofa 10% (wt/vol) stool suspensionof feces in fetal calf serum was used for intravenous inocu-lation of six cynomolgus monkeys (Macaca fascicularis).Blood samples from these monkeys were taken approxi-mately twice weekly before and after inoculation and testedby Metpath (Rockville, MD) for biochemical evidence ofhepatitis by measuring levels of serum alanine aminotrans-ferase, isocitric dehydrogenase, and y-glutamyltransferase.Fecal and bile samples were also collected from one monkey.For daily collection of bile, implantation surgery was per-formed on the 7th day after inoculation to establish anindwelling T-tube into the bile duct.

Bile from a cynomolgus monkey infected with anotherstrain of HEV (OSH-1852) was kindly provided by MichaelBalayan (Institute of Poliomyelitis and Viral Encephalitis,Moscow). This cynomolgus monkey had been inoculatedwith feces collected during a hepatitis E outbreak in 1988 inOsh, Kirgizia, U.S.S.R. Bile was collected on the 14th dayafter infection.

Detection of Anti-HEV Antibodies. A modified ELISAprotocol (22) was used in this study. Recombinant HEVantigens for use in the ELISA were derived from Mexicanand Burmese strains (23, 24) and were produced in thepGEX1 vector system (25). Cynomolgus sera were diluted1:100 in 1% gelatin/phosphate-buffered saline (PBS). Alka-line phosphatase-conjugated goat anti-human IgG was usedas a second antibody.

Abbreviations: HEV, hepatitis E virus; IEM, immune electronmicroscopy; RT, reverse transcription; nt, nucleotide(s); ORF, openreading frame.tTo whom reprint requests should be addressed at: Laboratory ofInfectious Disease, National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Building 7, Room 200, 9000Rockville Pike, Bethesda, MD 20892.**The sequences reported in this paper have been deposited in theGenBank data base (accession nos. M80581 and M81415).

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Primers. Ninety-two primers, 21-40 nucleotides (nt) long,and complementary to plus or minus strands of the genomeof a strain ofHEV from Burma (BUR-121) (9) or the SAR-55genome were synthesized using an Applied Biosystemsmodel 391 DNA synthesizer. For cloning ofPCR fragments,EcoRI, BamHI, or Bgl II restriction sites preceded by 3-7 ntwere added to the 5' end of primers.For detection of the HEV genome in samples of bile, sera,

and feces, two sets of "nested" primers were used thatrepresented sequences from the 3' region (ORF-2) of theSAR-55 genome. Primers for RT and the first PCR were5'-GTATAACGGATCCACATCTCCCCTTACCTC-3' and5'-TACAGATCTATACAACTTAACAGTCGG-3' and forthe second PCR were 5'-GCGGCAGATCTCACCGACAC-CATTAGTAC-3' and 5'-TAACCTGGATCCTTATGCCGC-CCCTCTTAG-3'.

Preparation of Virus RNA Template for PCR. Bile (10 sul),20%6 (wt/vol) SDS (to a final concentration of 1%), proteinaseK (10 mg/ml; to a final concentration of 1 mg/ml), 1 A.l oftRNA (10 mg/ml), and 3 A.l of 0.5 M EDTA were mixed in afinal volume of250 Ala and incubated for 30 min at 550C. Totalnucleic acids were extracted from bile twice with phenol/chloroform, 1:1 (vol/vol), at 65TC and once with chloroform,then precipitated by ethanol, washed with 95% ethanol, andused for RT-PCR. RT-PCR amplification ofHEV RNA fromfeces and especially from sera was more efficient when RNAwas more extensively purified. Serum (100 ,l) or a 10%6 fecalsuspension (200 ,ul) was treated as above with proteinase K.After a 30-min incubation, 300 ,ul of CHAOS buffer (4.2 Mguanidine thiocyanate/0.5 N-lauroylsarcosine/0.025 MTris HCI, pH 8.0) was added. Nucleic acids were extractedtwice with phenol/chloroform at 65°C followed by chloro-form extraction at room temperature. Then 7.5 M ammoniumacetate (225 ,ul) was added to the upper phase and nucleicacids were precipitated with 0.68 ml of 2-propanol. The pelletwas dissolved in 300 ,lA ofCHAOS buffer and 100 ,ld ofwaterwas added. Chloroform extraction and 2-propanol precipita-tion were repeated. Nucleic acids were dissolved in water,precipitated with ethanol, washed with 95% ethanol, andused for RT-PCR.RT-PCR. The usual 100-Al RT-PCR mixture contained

template, 10 mM Tris HCl (pH 8.4), 50 mM KCl, 2.5 mM

MgCl2, all four dNTPs (each at 0.2 mM), 50 pmol of directprimer, 50 pmol of reverse primer, 40 units of RNasin(Promega), 16 units of avian myeloblastosis virus reversetranscriptase (Promega), 4 units of AmpliTaq (Cetus), under100 ,ul of light mineral oil. The mixture was incubated 1 h at42°C and then amplified by 35 PCR cycles: 1 min at 94°C, 1min at 45°C, and 1 min at 72°C. The PCR products wereanalyzed on 1% agarose gels.

Cloning of PCR Fragments. PCR fragments containingrestriction sites at the ends were digested with EcoRI andBamHI or EcoRI and Bgl II restriction enzymes and clonedin EcoRI/BamHI-digested pBR322 or pGEM-3Z (Promega).Alternatively, PCR fragments were cloned into pCR1000using the TA cloning kit (Invitrogen, San Diego).

Sequencing of PCR Fragments and Plasmids. PCR frag-ments were excised from 1% agarose gels and purified byGeneclean (Bio 101, La Jolla, CA). Double-stranded PCRfragments were sequenced by using Sequenase (UnitedStates Biochemical) as described (26). Double-stranded plas-mids purified through CsCl gradients were sequenced with aSequenase kit (United States Biochemical).Computer Analysis of Sequences. Nucleotide sequences of

HEV strains were compared using the Genetics ComputerGroup (Madison, WI) software package (27), version 7.5, ona VAX 8650 computer (at the National Cancer Institute,Frederick, MD). This software package was also used togenerate the hydropathy plots.

RESULTSBiological, Serological, and Virological Characterization of

Infection. Six cynomolgus monkeys were intravenously in-oculated with a 10% suspension of human feces containingthe SAR-55 strain of HEV. Biochemical and serologicalassays confirmed that all six inoculated animals were in-fected. Although data are shown for only one monkey (Fig.1), all six monkeys displayed an increase in alanine ami-notransferase, isocitric dehydrogenase, and 'y-glutamyltrans-ferase activity, indicative of acute hepatitis. All six animalsalso developed antibody to recombinant-derived HEV anti-gens. These data demonstrated that the SAR-55 strain of

HEV genome- - + + + + + - -- - - - - serum- -- -++ +---++ +++ + ++++++ ++ - - - - - feces

_ _ _

-6 0 10 20 30

Day post-inoculation

+ + + +

40 50 60

FIG. 1. Evidence for SAR-55infection of Cyno-376. Serum ala-nine aminotransferase (ALT) lev-els are plotted as units/liter andthe presence (+) or absence (-) ofviral genomes or antibody to HEVis indicated. Viral genomes in thefeces and serum were detected byRT-PCR. The presence of totalserum antibodies to HEV was

monitored by ELISA using re-combinant antigens from both theMexican and Burmese strains ofHEV (23, 24).

Anti-HEV

125

100 _

-j 75

50

25 -

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A.,,e

FIG. 2. IEM detection ofSAR-55 virions in the bile ofCyno-376. IEM was performed asdescribed (13). Viral aggregateswere identified in the bile by reac-tion of diluted bile with anti-HEVfrom a chimpanzee that had beeninfected with a strain ofHEV fromIndia (13). (x 150,000.)

HEV was able to consistently infect and induce hepatitis E incynomolgus monkeys.To correlate the distribution of virus with an increase in

virus-specific antibody and alanine aminotransferase levels,a sensitive nested RT-PCR protocol was performed on fecal,serum, and bile samples from cynomolgus monkey 376(Cyno-376). Control experiments in which we assayed dilu-tions of feces of known infectivity demonstrated that onaverage the HEV genome could be detected by RT-PCR infeces, with a sensitivity approximately equal to that of anassay based on transmission ofHEV to cynomolgus monkeys(unpublished results). All bile, serum, and fecal samples weretested by RT-PCR multiple times to ensure that positivesamples were identified.Although Cyno-376 was inoculated with at least 1 x 103

infectious doses intravenously, we were not able to detectvirus in serum on the day of infection or for several daysthereafter (Fig. 1). However, we were able to detect viralgenomes in feces and bile as early as day 6 and day 7,respectively, confirming that excretion of virus was an earlyindicator of infection. All samples of bile collected (days7-41) were positive for HEV RNA by RT-PCR. Unfortu-nately, due to the indwelling catheter method of bile collec-tion, residual contamination from earlier samples could notbe eliminated so the end point for virus presence in the bilecould not be determined by PCR. However, when the less-sensitive IEM technique was used in the analysis of the bile,virus was detected sporadically from day 26 to day 37 with apeak on day 32 (data not shown). The HEV particles resem-bled those described previously (Fig. 2).

Viral excretion in feces could be documented more pre-cisely by PCR, and excretion began on day 6 and ended byday 35 (Fig. 1). Three samples, taken between days 10 and 15,were repeatedly negative for viral RNA. The inability todetect viral RNA in these three samples suggested that thelevel of excreted particles was low. Virus was first detectedin the serum on day 9, appeared to be present at higher titer(based on the quantity of PCR product) from days 14 to 23,then disappeared by day 28 (Fig. 1). A similar pattern ofviremia in a cynomolgus monkey infected with HEV wasobserved by Uchida et al. (28).

Molecular Characterization of the SAR-55 Genome. RT-PCR amplification of bile from Cyno-376 was employed togenerate six cDNA fragments encompassing the entire ge-nome of SAR-55. The PCR-generated fragments were eithersequenced directly or were cloned into individual plasmidsand sequenced subsequent to amplification in Escherichiacoli. Direct sequencing of the PCR product provided 45% ofthe genome sequence as a consensus sequence. Both strandsof cloned cDNAs representing 87% of the genome were

sequenced to provide the remainder of the sequence and toconfirm the consensus sequence. The sequence of the entire7.195-kilobase genome, with the exception of 30 and 27 nt ofthe 5' and 3' termini, respectively, was obtained.

Since one other isolate of HEV, BUR-121 (9), had beentotally sequenced, computer analyses were performed todetermine the relatedness of SAR-55 to BUR-121. Uniquenucleotide insertions or deletions were not detected and thesame three ORFs identified in BUR-121 were found inSAR-55. Overall, the genomes were quite similar and differedby only 6.7% in nucleotide sequence and by 1% in deducedamino acid sequence (Table 1). As might be expected, themost conserved region was located in the overlap of ORF-2and ORF-3 (bases 5147-5477). Against this overall pattern ofsequence relatedness, the region between bases 2011 and2325 in ORF-1 appeared unique (Fig. 3). Although the sum ofthe nucleotide differences in this region was only 2% higherthan the average for the entire genome, the sum ofamino aciddifferences was 14% or 14 times higher than the average.Because the sequence of this region in the SAR-55 genomediffered so greatly from that of BUR-121, it was also ampli-fied by RT-PCR directly from the original human feces. Thesequence obtained from virus in the human fecal sample wasidentical to that obtained from virus in the cynomolgus bile.

Identification of a Hypervariable Region. To determine ifthis extreme divergence of sequence between nt 2011 and2325 was unique to these two isolates or had a more universalsignificance, sequences from two regions of a third strain ofHEV were derived. Fragments of cDNA from the RT-PCRamplification of the OSH-1852 strain of HEV correspondingto positions 2002-2424 and 4424-4800 of the genome weresequenced. Both the nucleotide and amino acid identities inthe region (positions 4424-4800) encoding the putative RNA-directed RNA polymerase were high and did not differsubstantially among the three strains (Table 2 and Fig. 3). Incontrast, between nt 2002 and 2424 there was significantdivergence in nucleotide sequences and even greater diver-gence in amino acid sequences among the three strains.

Table 1. Comparison of BUR-121 and SAR-55 sequencesNucleotides Amino acids

Length, Identity, Length, Identity,Region no. % no. %ORF-1 5079 93.1 1693 98.8ORF-2 1980 93.8 660 99.4ORF-3 369 98.9 123 100Total 7138* 93.3 2476 99.0

*Length of the primers is not included.

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Non structural region (nucleotide binding protein?)

H T F1 7Y 7

A O R L S L T G H F W F H2002 GCCCAGCGCCTTTCGCTGACCGGTAATTTTTGGTTCCAT 2040

A T C AA

L L7 I

P EG L L G P F A P F S P G H V W E S A N P F C G E S T L Y T R T W S E V D A V

A A T C a T C CA T G G T

S R A P P L L L P P

P S P A O P D L G F T SE P SI P S R AA T P T P A A P L P P P A P D P S P T L

T G T Tr. G T C cc CT C G T c T C TTT T C C

LA S - GS AP A R G E P A P G A T A R A P A I T HO0 T AR H R R L L F T Y P D G S KV F

C T C T G a T C C AC GT Tr. C C T T A T

A G S L F E S TGCCGGCTCGCTGTTTGAGTCGACA 2424

C

FMA-dependlent EWA polymerase region

S T O NHF4423 TCCACCCAGAATAATTTT

C

S L G L E C A I M E EC G M PO0W L I R L Y H L I R S AW I L 0 A P K E S L R G

TC T C T CT TA

2160

2280

2400

444

4560

C

L7

F W K K H S G E P G T L L W H T V W H M A V I T H C Y 0 F R D L O VA A F KIGDTTTTGAAGAACCTCCGTGACCCGCACCTTCGTGAATATGTTGGACATGCCGTATCCCCATGTATGATTCGCGACTGCGGTGCTGCTTTAAGTG 46808

T A T T C T TT A T T C

D S I V L C S E Y ROQ S P G A AV L I A G C G L K L K V D F R P IG L Y A G V V

T T AT

CC-- T CT C

V A P G L G AL P D V V R F AG R L T E K H W G P GGTGGCCCCCGGCCTTGGCGCGCTTCCTGATGTCGTGCGCTTCGCCGGTCGGCTTACTGAGAAGAATTGGGGCCCTGGC

C TC C

C C

4800A C C T

T

4878

FIG. 3. Partial cDNA nucleotide sequence and the deduced amino acid sequence of SAR-55. Nucleotides and amino acids that differ in the

BUR-121 (boldface type) (9) or OSH-1852 (boldface underlined type) strains are indicated.

Therefore, the region between nt 2011 and 2325 probably

represents a relatively hypervariable region of the HEV

genome.

DISCUSSION

HEY Infection of Cynomolgus Monkeys. Previous studies

have shown that cynomolgus monkeys are useful for exper-

imental HEV infection (5, 11-13, 16, 18, 19). Extension ofour

original study demonstrated that all six cynomolgus monkeys

inoculated with human feces containing HEV SAR-55 devel-

oped hepatitis E, as defined by liver enzyme elevation, and

developed anti-HEV antibodies. Since this virus was able to

consistently infect cynomolgus monkeys and cause hepatitis,

it appeared to be suitable for selection as a prototype virus for

biological and molecular characterization. Therefore, infec-

tion of a cynomolgus monkey with the SAR-55 strain ofHEV

was intensively monitored. Detection of the viral genome in

bile and feces suggested that the virus replicated in the liver

during the first week after infection. This finding is in good

agreement with previous studies in which virus was detectedby IEM in feces and bile as early as 9 days after infection (5,11, 19). Although the monkey was inoculated intravenouslywith the virus, we were not able to detect virus in serum on

Table 2. Comparison of SAR-55, BUR-121, and OSH-1852sequences in two regions of ORF-1

Identity, %Nucleotides Amino acids

Region BUR-121 OSH-1852 BUR-121 OSH-1852nt 2002-2424SAR-55 92.0 92.9 89.4 91.5BUR-121 100 91.0 100 92.2

nt 4423-4878SAR-55 93.2 96.3 99.3 100BUR-121 100 92.6 100 99.3

Regions compared are nt 2002-2424 (predicted nonstructural pro-teins) and nt 4423-4878 (predicted RNA-directed RNA polymerase).

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FIG. 4. Hydropathy plot of the hypervariable region shown inFig. 3 was plotted for each strain using the method of Kyte andDoolittle (30) and profiles were superimposed for comparison. SAR-55, bold line; BUR-121, dotted line; OSH-1852, dashed line.

the day of infection or thereafter until 2-3 days after virus wasfirst detected in bile and feces. Virus excretion started by 1week after infection and 3 weeks before evidence of diseasewas observed. The amount of virus excreted at this time wasnot great, which probably explains why we were not able todetect the HEV genome in fecal samples taken 12-14 daysafter infection even though samples taken prior to and afterthis time were positive. Viremia was first observed on the 9thday of the infection and appeared to intensify between 2 and3 weeks after infection. Elevation of liver enzymes was firstnoted as viremia ceased. It is probable that viremia is theresult of viral replication in the liver although nonhepaticsites of replication have not been ruled out.Comparison of SAR-55 Genome with That of Other Strains.

We found that the sequence of SAR-55 was quite similar tothat of BUR-121 over the entire length of the genome (Table1). This result is not unexpected since the two viruses wereisolated from geographically contiguous regions at about thesame time. Differences between the two sequences weredistributed approximately uniformly throughout the genomeexcept for one hypervariable domain located in the putativenonstructural ORF (9) (Fig. 3 and Table 2). This region ofhypervariability preceded the nucleotide triphosphate bind-ing domain and also the RNA-directed RNA polymerasemotifs recognized in ORF-1 (8, 9, 29). That this region washypervariable was confirmed when another strain from Asiawas partly sequenced. The genome of the OSH-1852 strainfrom Kirgizia differed in the hypervariable region to approx-imately the same extent from the Pakistani and Burmesestrains as those strains differed from each other (Table 2).However, all three strains did not differ significantly fromeach other in the region coding for RNA-directed RNApolymerase. The finding of a hypervariable region in apresumed nonstructural protein is surprising, since suchregions are usually found in structural proteins of viruses,where they are exposed to selection by the immune responseof the host. A hydropathy analysis of the region revealed thatall three strains displayed a similar profile in spite of theirextensive sequence differences (Fig. 4). However, the sig-nificance of this pattern remains to be determined. Wepredict that the existence of this hypervariable region willallow even closely related strains to be distinguished by theapplication of molecular epidemiological approaches. Pre-sumably, the sequence of the hypervariable region could beused for more detailed molecular classification of HEVstrains, and the sequence from the much more conserved

RNA polymerase region could be used for designing PCRprimers to detect a spectrum of HEV strains with differentorigins.

We are grateful to Mrs. Marianne Lewis, Mr. Terry Popkin, andMs. Katherine Gabor for technical assistance and to Drs. MichaelBalayan, Joe Bryan, and Stephen Denny for providing materialcontaining HEV. Excellent animal care was provided by the staff ofBioqual, Gaithersburg, MD.'We acknowledge the National CancerInstitute for allocation of computing time and staff support at theAdvanced Scientific Computing Laboratory of the Frederick CancerResearch and Development Center. This study was supported in partby a grant from the World Health Organization Programme forVaccine Development and Contrict N01-AI-05069.

1. Balayan, M. S. (1987) Virol. Rev. 2, Sect. E, 235-261.2. Purcell, R. H. & Ticehurst, J. R. (1988) in Viral Hepatitis and Liver

Disease, ed. Zuckerman, A. J. (Liss, New York), pp. 131-137.3. Bradley, D. W. (1990) Br. Med. Bull. 46, 442-461.4. Ticehurst, J. R. (1991) in Viral Hepatitis and Liver Disease, eds.

Hollinger, F. B., Lemon, S. M. & Margolis, H. S. (Williams & Wilkins,Baltimore), pp. 501-513.

5. Balayan, M. S., Andjaparidze, A. G., Savinskaya, S. S., Ketiladze,A. S., Bragipsky, D. M., Savinov, A. P. & Poleschuk, V. F. (1983)Intervirology 211, 23-31.

6. Bradley, D. W. & Baiayan, M. S. (1988) Lancet i, 819.7. Bradley, D. W., Andjaparidze, A. G., Cook, P. H., McCaustland, K.,

Balayan, M., Steeler, H., Velazques, O., Robertson, B., Humphrey, C.,Kane, M. & Weisfuse, I. (1988) J. Gen. Virol. 69, 731-738.

8. Reyes, G. R., Purdy, M. A., Kim, J., Luk, K.-C., Young, L. M., Fry,K. E. & Bradley, D. W. (1990) Science 247, 1335-1339.

9. Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C., Bradley, D. W.,Fry, K. E. & Reyes, V4. R. (1991) Virology 185, 120-131.

10. Neil, J. D. (1990) Virus Res. 17, 145-160.11. Andjaparidze, A. G., Balayan, M. P., Savinov, A. P., Braginsky, D. M.,

Poleschuk, V. F. & Zamyatina, N. A. (1986) Vopr. Virusol. 31, 73-81.12. Bradley, D. W., Krawczynski, Cook, E. H., Jr., McCaustland, K. A.,

Humpherey, C. D., Spelbring, J. E., Myint, H. & Maynard, J. E. (1987)Proc. Nati. Acad. Sci. USA 84, 6277-6281.

13. Arankalle, V. A., Ticehursf, J. R., Sreenivasan, M. A., Kapikian, A. Z.,Popper, H., Payri, K. M. & Purcell, R. H. (1988) Lancet i, 550-554.

14. Gupta, H., Tandon, B. N., Sriramachpri, S., Joshi, Y. K. & Iyenger, B.(1990) Indian J. Med. Rex. Sect. A 91, 87-90.

15. Krawczynski, K. & Bradley, D. W. (1989) J. Infect. Dis. 159,1042-1049.16. Soe, S., Uchida, T., Suzuki, K., Komatsu, K., Azumi, J., Okuda, Y.,

fida, F., Shikata, T., Rikihisa, T., Mizuno, K., Win, K. M. & Tin, K. M.(1989) Liver 9, 135-145.

17. Panda, S. K., Datta, R., Kaur, J., Zuckerman, A. J. & Nayak, N. C.(1989) Hepatology 10, 466-472.

18. Uchida, T., Win, K. M., Suzuki, K., Komatsu, K., fida, F., Shikata, T.,Rikihisa, T., Mizuno, K., Soc, S., Myint, H., Tin, K. M. & Nakane, K.(1990) Jpn. J. Fxp. Med. 60, 13-21.

19. Uchida, T., Suzuki, K., Komatsu, K., fida, F., Shikata, T., Rikihisa, T.,Mizuno, K., Soe, S., Win, K. M. & Nakane, K. (1990) Jpn. J. Exp. Med.60, 23-29.

20. Iqbal, M., Ahmed, A., Qamar, A., Dixon, K., Duncan, J. F., Islam,N. U., Rauf, A., Bryan, J. P., Malik, I. A. & Legters, L. J. (1989) Am.J. Trop. Med. Hyg. 40, 438-443.

21. Ticehurst, J., Popkin, T. J., Bryan, J. P., Innis, B. L., Duncan, J. F.,Ahmed, A., Iqbal, M., Malik, I., Kapikian, A. Z., Legters, L. J. &Purcell, R. H., J. Med. Virol., in press.

22. Skidmore, S. J., Yarbough, P. O., Gabor, K. A., Tam, A. W., Reyes,G. R. & Flower, A. J. E. (1991) Lancet 337, 1541.

23. Reyes, G. R., Yarbough, P. b., Tam, A. W., Purdy, M. A., Huang, C.,Kim, J., Bradley, D. W. & Fry, K. E. (1991) Gastroenterol. Jpn. 26,142-147.

24. Yarbough, P. O., Tam, A. W., Fry, K. E., Krawczynski, K., McCaust-land, K. A., Bradley, D. W. & Reyes, G. R. (1991) J. Virol. 65, 5790-5797.

25. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40.26. Winship, P. R. (1989) Nucleic Acids Res. 17, 1260.27. Devereux, J., Haeberly, P. & Smithies, 0. (1984) Nucleic Acids Res. 12,

387-395.28. Uchida, T., Suzuki, K., Iida, F., Shikata, T., Araki, M., Ichikawa, M.,

Rikihisa, T., Mizuno, K., Soe, S. & Win, K. M. (1991) in Viral Hepatitisand Liver Disease, eds. Hollinger, F. B., Lemon, S. M. & Margolis,H. S. (Williams & Wilkins, Baltimore), pp. 526-527.

29. Fry, K., Tam, A. W., Smith, N.; M., Kim, J. P., Luk, K.-E., Young,L. M., Piatak, M., Feldman, R. A., Yun, K. Y., Purdy, M. A., Mc-Caustland, K., Bradley, D. W. & Reyes, G. R. (1991) Virus Genes, inpress.

30. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.

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