Proc. Nati. Acad. Sci. USAVol. 87, pp. 2867-2871, April 1990Medical Sciences

Molecular epidemiology of human hepatitis A virus defined by anantigen-capture polymerase chain reaction method

(immunoassay/viral diagosis/picornavuses/sequencing/strain comparisons)

ROBERT W. JANSEN*t, GUNTER SIEGLt, AND STANLEY M. LEMONttDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030; and tInstitut fur Hygiene und MedizinischeMikrobiologic, Universitat Bern, Bern, Switzerland

Communicated by Michael G. Rossmann, January 16, 1990 (receivedfor review September 29, 1989)

ABSTRACT We describe an immunoaffinit-linked nu-cleic acid amplification system (antigen-capture/polymerasechain reaction, or AC/PCR) for detection of viruses in clinicalspecimens and its application to the study of the molecularepidemiology of a picornavirus, hepatitis A virus (HAV).Immunoaffinity capture of virus, synthesis of viral cDNA, andamplification of cDNA by a polymerase chain reaction (PCR)were carried out sequentially in a single reaction vessel. Thisapproach simplified sample preparation and enhanced thespecificity of conventional PCR. AC/PCR detected less thanone cell culture infectious unit of virus in 80 jai of sample.Sequencing ofAC/PCR reaction products from 34 virus strainsdemonstrated remarkable conservation at the nucleotide levelamong most strains but revealed hitherto unsuspected geneticdiversity among human isolates. Epidemiologically relatedstrains were identical or closely related in sequence. Virusstrains recovered from epidemics of hepatitis A in the UnitedStates and Germany were identical in sequence, provdingevidence for a previously unrecognized epidemiologic linkbetween these outbreaks.

Type A viral hepatitis, historically a common infectiousdisease in many underdeveloped regions, is recognized to bea continuing problem in the United States and some WesternEuropean countries. However, current understanding of theepidemiology of the picornavirus responsible for this disease(hepatitis A virus, HAV) has been limited by the absence ofstraightforward methods for isolating virus and distinguishingbetween virus strains. Certain differentiation between HAVstrains has been accomplished only by "RNA fingerprinting"or by a comparison of nucleotide sequences derived frommolecularly cloned viral cDNA (1, 2). Such approachesrequire considerable quantities of virus or the adaptation ofvirus to growth in cell culture, in itself a difficult task (3, 4)and one that may lead to mutations within genomic RNA(5-7). These labor-intensive methods of distinguishing be-tween viral strains are not directly applicable to the smallquantities of virus usually present in clinical specimens andhave thus been of limited use in epidemiologic studies. Toovercome these limitations and develop a molecular ap-proach to studying the epidemiology of HAV, we turned tothe polymerase chain reaction (PCR) as a means of amplify-ing viral nucleic acid present in clinical specimens (28).PCR involves the selective, enzymatic amplification of a

segment of a DNA molecule and may effect a >106-foldenrichment of a target DNA sequence (8, 9). The exquisitesensitivity of this procedure makes it an attractive approachto viral diagnosis but also leads to potential problems withrespect to specificity. Contamination of clinical specimenswith miniscule quantities ofrecombinant nucleic acid presentin the laboratory environment may lead to false-positive PCR

results, and in some cases random priming of complexmixtures of nonspecific nucleic acids may lead to amplifica-tion of stochastic reaction products. Moreover, typical meth-ods for preparing viral nucleic acid for PCR are tedious,involve multiple manipulations, and are a potential source ofcontamination. To reduce these problems, we developed aprocedure (antigen-capture/PCR, or AC/PCR) for selectiveamplification of antigen-associated viral nucleic acid. Viruspresent in crude fecal suspensions was captured by mono-clonal antibody coating the walls of a reaction tube anddenatured in situ by heat treatment. Virion RNA was re-leased into a universal buffer permitting reverse tran-scriptase-mediated cDNA synthesis to be followed directlyby Taq polymerase (Thermus aquaticus DNA polymerase)-directed PCR, without isolation of viral RNA or primarycDNA reaction products. Application of AC/PCR to humanand cell culture-derived materials has provided an alternativeview of the molecular epidemiology of HAV.

MATERIALS AND METHODSVirus Samples. Fecal specimens were obtained from an owl

monkey (Aotus trivirgatus) inoculated with cell culture-adapted HAV (10), volunteers undergoing experimental in-fection with HAV (11), or patients with hepatitis A in NorthCarolina, Kansas, or the Federal Republic of Germany (12).Additional fecal materials and cell culture isolates of HAVwere provided by the HAV Strain Bank of the World HealthOrganization's Programme for Vaccine Development. Fecalsamples were tested as clarified 5-10%o suspensions.AC/PCR Procedure. Sterile 1.5-ml polypropylene micro-

centrifuge tubes were coated with a 1:300 dilution of anti-HAV monoclonal ascitic fluid (K32F2) and then "blocked"with 1% bovine serum albumin as described for immunoaf-finity hybridization (13). The antibody-coated tubes werewashed three times with 300 Al of phosphate-buffered saline(PBS) containing 0.05% Tween-80 and 0.02% sodium azide,loaded with virus-containing samples (50-80 Al), and heldovernight at 40C. Tubes were then washed six times with 300.lI of 20 mM Tris, pH 8.4/75 mM KCl/2.5 mM MgCl2, withcare taken to avoid cross-contamination (disposable Com-bitips, Eppendorf). Universal buffer [80 AI; 25 mM Tris, pH8.4/75 mM KCl/2.5 mM MgCI2/0.25 mM (each) dATP,dCTP, dGTP, and TTP] was added to each virus-coated tubealong with 5 A.l (100 pmol) of a HAV negative-strand primer.Following denaturation of virus at 950C for 5 min and coolingto 420C, reverse transcriptase was added [either Life Sci-ence's (Saint Petersburg, FL) avian myeloblastosis virus or

Abbreviations: HAV, hepatitis A virus; PCR, polymerase chainreaction; AC/PCR, antigen-capture/PCR; RFU, radioimmunofocus-forming unit(s); PV1, poliovirus type 1; RSV, respiratory syncytialvirus.*To whom reprints should be addressed at: Division of InfectiousDiseases, CB#7030, 547 Burnett-Womack Clinical Sciences Build-ing, Chapel Hill, NC 27599-7030.

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BRL's Moloney murine leukemia virus; 2 units in 5 A.l ofH20]. Reaction mixtures were held at 420C for 30-60 min,after which 5 A.l (100 pmol) of positive-strand primer and 5 A.lcontaining 2 units of Taq polymerase (Perkin-Elmer/Cetus)and 20 ,ug ofgelatin was added. Reaction mixes were overlaidwith mineral oil and subjected to 30 automated cycles ofdenaturation at 950C for 15 sec, annealing at 370C for 5 sec,and extension at 70'C for 1 min, with an additional 5-minincubation at 70'C during the final extension. Reaction prod-ucts were ethanol precipitated and resuspended in 15-30 plof sterile H20.Two regions of the HAV genome were selected for ampli-

fication: a highly conserved region encoding the carboxylterminus of the capsid protein VP3 (positive-strand primerHAV+2020, 5'-ACAGGTATACAAAGTCAG, and nega-tive-strand primer HAV-2211, 5'-CTCCAGAATGATC-TCC) and a less conserved region (14) encoding the carboxylterminus of VP1 and amino terminus of protein 2A (primerswere HAV+ 2984, 5'-TCCCAGAGCTCCATTGAA, andHAV-3265, 5'-CATTATl-CATGCTCCTCAG).

Analysis ofAC/PCR Amplification Products. A 30% aliquotof the amplified products was electrophoresed through 3%NuSieve GTG/1% SeaKem ME (FMC) agarose gels in Trisacetate/EDTA buffer and visualized by staining with ethid-ium bromide. Products amplified with primers +2020/-2211were subjected to oligonucleotide hybridization. AmplifiedDNA products were transferred to nitrocellulose membranesby conventional methods (15). Prehybridization was carriedout at 37°C in 6x SSC (lx SSC is 0.15 M NaCl/0.015 Msodium citrate), 5x Denhardt's solution [lx Denhardt's is0.02% (each) bovine serum albumin, Ficoll, and polyvi-nylpyrrolidone], 0.5% SDS, 0.05% sodium pyrophosphate,and 100 ,ug ofcalfthymus DNA per ml. Hybridization was for20 hr at 37°C in a similar buffer containing lx Denhardt'ssolution and 0.2 pmol (5 ACi/pmol; 1 Ci = 37 GBq) of aHAV-specific oligonucleotide (HAV+2189, 5'-TATGGAT-GTTACTACACA) per ml, 5' end-labeled with T4 polynu-cleotide kinase (Promega Biotec). Blots were washed fivetimes at room temperature in 6x SSC/0.05% sodium pyro-phosphate and exposed to XAR-5 film (Eastman Kodak) for16 hr.

Nucleotide Sequence ofAC/PCR Products. AC/PCR prod-ucts were purified from 3% NuSieve GTG agarose gels byphenol extraction and ethanol precipitation and sequenced bythe dideoxy method (16) using the Sequenase kit (UnitedStates Biochemical). DNA (10-100 ng), 5 x buffer, and 1-2pmol of 32P-end-labeled positive-strand primer were dilutedto 18 ,u, heated to 97°C for 3 min, and cooled quickly on ice.While at 4°C, 4 IlI was dispensed into each of four microplatewells containing 2 Al of dideoxy-/deoxynucleotide triphos-phate mix and 2 ,l of Sequenase mix (0.4 units of SequenaseV2.0 per ,l and 5 mM dithiothreitol in Tris/EDTA, pH 8.0).Reactions were mixed by centrifugation at room temperaturefor a total of2 min, placed at 37°C for 8 min, and then stoppedby addition of 4 ,ul of stop mix. After heating at 80°C for 10min, 2 ,ul was electrophoresed through 6% polyacrylamide/7M urea gels, which were then exposed to XAR-5 film (Kodak)for 1-4 days.

RESULTSSensitivity of AC/PCR. To evaluate the sensitivity of

AC/PCR, we compared its ability to detect viral nucleic acidwith a conventional PCR method involving the enzymaticamplification ofcDNA made from HAV RNA that had beenprepared by phenol/chloroform extraction following incuba-tion of virus with SDS and proteinase K (17). Dilutions madefrom a suspension of feces collected from an infected owlmonkey were tested by AC/PCR, whereas samples contain-ing SDS/proteinase K-extracted RNA from identical dilu-

tions were subjected to PCR with the VP3 primer set +2020/-2211 (Fig. lA). In replicate experiments, AC/PCR andconventional PCR yielded a single amplified reaction productof the expected length, confirmed to contain the appropriateHAV sequence by Southern hybridization (Fig. 1 B and C).The end-point dilutions were similar for both methods, indi-cating that heat-denatured, affinity-isolated virus providedtemplate of sufficient purity for subsequent polymerase re-actions.

Since the fecal specimen tested in this experiment wascollected from an owl monkey that had been inoculated witha cell culture-adapted HAV variant (10, 18), we were able tocompare the detection limits of AC/PCR with the infectioustiter of virus determined by radioimmunofocus assay (107-RFU of virus per g in this fecal sample). With serial dilutionsofthe fecal suspension down to that containing as little as 0.47RFU in the tested sample, AC/PCR resulted in progressivelylesser amounts of the amplified reaction product (Fig. 1B). Aweak signal was detected at 0.047 RFU. An isolated positiveresult was obtained at 0.0047 RFU, probably representing arandom aggregate of virus particles present in this dilution ofthe sample. PCR amplification of cDNA derived from phe-nol/chloroform-extracted virus RNA appeared similarlyquantitative with respect to input virus, with reaction prod-ucts consistently detected down to 0.047 RFU (Fig. 1C). Asprevious experiments have suggested that the particle/infectivity ratio of cell culture-adapted HAV is -60:1 (7), weestimate the detection limit of either technique to be 3-30virus particles.

Specificity of AC/PCR. We assessed the contribution ofimmunoaffinity capture of virus to the overall specificity ofAC/PCR. Virus samples were placed into reaction tubescoated with monoclonal antibodies to HAV, poliovirus type1 (PV1), or respiratory syncytial virus (RSV) for assay byAC/PCR. Strong hybridization signals were obtained withproducts from reaction tubes coated with anti-HAV mono-clonal antibody, whereas virus particles sticking to the wallsof reaction vessels coated with nonspecific antibodies ledonly occasionally to low levels of amplified HAV reactionproducts detectable by Southern hybridization (Fig. 2). Thus,monoclonal antibody capture provided a relatively high levelof specificity, despite the sensitivity of the subsequent PCRamplification procedure.

AVP4 VP2

*-2020 6°p -_221!VP3 I ,Vpit

o b c d e f g h I k a b c d e f g h j_

_r~~~~~~~~~

B C

FIG. 1. AC/PCR detection ofHAV RNA encoding the carboxyl-terminal region of capsid protein VP3. (A) P1 (capsid protein-encoding) region of the HAV genome showing locations of PCRprimers HAV+2020 and HAV-2211. bp, Base pairs. (B and C)Southern hybridizations ofagarose gel-fractionated AC/PCR (B) andconventional PCR (C) products made from a primate fecal suspen-sion with HAV+2020/HAV-2211 primers. Lanes b-k contain re-action products made from serial half-logarithmic dilutions of a fecalsample collected from an owl monkey infected with a cell culture-adapted variant ofHM175 virus. Titrations of this fecal sample in cellculture indicated that lanes b contained products made from 0.0047radioimmunofocus-forming units (RFU) of infectious virus (10).Lanes: c, 0.015 RFU; d, 0.047 RFU; e, 0.15 RFU; f, 0.47 RFU; g, 1.5RFU; h, 4.7 RFU; i, 15 RFU; j, 47 RFU; and k, 150 RFU. Lanes acontain PCR reaction products made from PBS only.

f.7 ..n-111,11-11,11, --- '',I,--":; ul"'I"'I",

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b c d e

FIG. 2. Southern hybridization of agarose gel-fractionated AC/PCR reaction products obtained with capture antibodies havingdifferent specificities. Reaction tubes were coated with 1:100 dilu-tions of ascitic fluids containing monoclonal antibodies to HAV (lanea, monoclonal K32F2), PV1 (lane b, monoclonal 956, and lane c,monoclonal 234), or RSV (lane d) or with carbonate buffer only (lanee), prior to loading with a HAV-positive l1o human fecal suspension(GR-1).

Comparison of AC/PCR with Conventional Methods forDetection of HAV. We applied AC/PCR to fecal specimenscollected during the first week of illness from 21 personsinvolved in a common source outbreak of hepatitis A (12).Seventeen of 21 (81%) specimens yielded reaction productsthat were visualized in an ethidium bromide-stained agarosegel (Fig. 3). Southern hybridization confirmed the HAV-specific nature of these products, with 19 of the 21 specimens(90%) yielding positive results (data not shown). The sensi-tivity of AC/PCR was thus considerably greater than solid-phase radioimmunoassay (19) (50%o positive) or cDNA RNAhybridization (13) (65% positive), two methods commonlyused for detection of HAV in clinical samples. The hybrid-ization blot intensity and radioimmunoassay cpm value cor-related with the quantity of PCR-amplified product obtainedfrom each positive fecal sample (not shown), suggesting thatAC/PCR is at least semiquantitative.Molecular Epidemiology of HAV. Reaction products am-

plified by AC/PCR from clinical samples (or cell culturematerials) containing HAV obtained in different epidemio-logic settings were sequenced by primer extension. Wecompared collinear sequences from 36 virus strains betweennucleotides 2056 and 2208 (carboxyl terminus of VP3) and3020 and 3191 (VP1/2A region), within the genomic regionsamplified by the primers described in Materials and Meth-ods. These regions encode maximally and minimally con-

served domains in the capsid proteins (14) and togetherrepresent -5% of the entire viral genome.We found no differences between the AC/PCR sequences

of HM175, CR326, or HAS15 strains of HAV and thesequences of these viruses derived from molecularly clonedcDNA (20-22), demonstrating the accuracy of sequence dataderived from viral RNA by AC/PCR.§ As expected fromprevious analyses of cloned cDNA (20-24), most HAVstrains shared a high degree of nucleotide identity (>92%)(Fig. 4).¶ However, unexpectedly large differences (up to14% in the VP3 region and 24% in the VP1/2A region) werefound with two strains (H122 and CF53) recovered fromdifferent epidemiologic settings. This relatively high degreeof sequence divergence led to problems with PCR primersderived from the HM175 sequence, as H122 cDNA amplifiedonly with the +2984/-3265 primer set. Although the absolutedegree of difference between strains was greater in theVP1/2A region, dendrograms depicting the relatedness ofdifferent strains (27) within each of the two genomic regions(VP3 or VP1/2A) were similar (not shown), indicating thatthe sample size was sufficient for reliable measures of relat-edness.

DISCUSSIONThe data presented above demonstrate the utility ofAC/PCRas a diagnostic procedure and support its use in character-izing the molecular epidemiology of viruses such as HAV.Important benefits derive from combining the specificity ofan immunoaffinity capture step with the sensitivity of PCRfor virus detection, including rapid confirmation of the spec-ificity of amplified DNA products (Fig. 2). AC/PCR involvesfewer manipulations than conventional PCR and is techni-cally relatively simple, features that should reduce the risk ofsample contamination. The technical simplicity of AC/PCRmakes it applicable to the testing of substantial numbers ofspecimens in epidemiologic or clinical studies, includingfuture HAV vaccine trials. The general method is not re-stricted to use with HAV and has been carried out withviruses as diverse as Epstein-Barr virus and hepatitis deltavirus (R.W.J. and S.M.L., unpublished observations). Thus

§The AC/PCR-derived sequence of strain MBB fecal material dif-fered from the reported cDNA-derived sequence (23) at a singlebase position.SIndividual nucleotide sequences are available upon request.

a bc d e f g h j k mn o p q r s t u v w x y z

Uot.

206bp m-'N _ _*.I i-

FIG. 3. Agarose gel analysis of AC/PCR products obtained with fecal samples collected during a common-source outbreak of hepatitis A(12), using the HAV+2020/HAV-2211 primer set. Reaction products were visualized by ethidium bromide staining. Lanes: a and b, PBS control(a) and GR-7 fecal suspension (b) immunoaffinity-captured by PV1-specific monoclonal antibody 956; c-w, acute hepatitis A fecal suspensionscaptured by HAV specific monoclonal K32F2 (lane h is GR-7); x, prechallenge fecal suspension from human volunteer; y and z, additional PBSPCR controls. The difference in signal between lanes b and h is due to specific antibody capture. In this experiment, tubes were washed threetimes before addition of universal buffer. Further enhancement of specificity without loss of sensitivity was obtained by increasing washing tosix cycles (see Fig. 2).

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H-122PA-21AgIlAg5978Ag60I4Ag6084H*175MBBH-152JandaBR 5j/7190/1CF-979SM-iS"-YICLF SGBMLSH/SH-153H-141S85-ICR-326MS-LHAS-lSLCDCNC-IKM"-lFR-ALLAOR-IOR-7LU-BECPRDJCARICF-53

p2 Swedenp7 Panama

GreeceGreeceGreeceGreece

p16 AustraliaNorth Africa

p5 FR Gernanwp11 CSSRp1 DR Gernanwp2 Greecep1 USAp6 PR Chinap5 PR Chinap1 SwitzerlandpI FR Germanwp1 Englandp3 North AfricaP4 India

Chinap6 Costs Rica

USA, Nyp20 USA, AZp20 China

USA. NCSwitzerland

p5 USA. AKp7 USA. CA

FR GernanwFR GernanwUSA. KS

p4 Englandp3 Brasiliap4 Italwp1O France

1979___19801983198319831983 r1976197819811982198219871979197819781983197619831981197919851966 --196419791984 F19891979 -F1976 --__197?1982198219821976198019851979

VP3 + VP1/2A 100I I I95 90 85

x IDENTITYFIG. 4. Dendrogram constructed by comparative subset averaging (25) of% nucleotide identity data derived by AC/PCR from 34HAV strains

representing epidemiologically diverse sources. The geographic location and date of collection of each strain are noted, as is the passage levelin cell culture for those sequences derived from cell culture-adapted viruses. Where sequence of a virus differed markedly between virus in fecalmaterial and a cell culture-adapted isolate (suggesting that the isolate was in fact a contaminant), only the fecal virus sequence is shown (KMW-1,S85-1). Differences shown represent those between collinear sequences spanning bases 2056-2208 and 3020-3191 (325 total), except for H122,where sequence could be obtained only in the region 3020-3191 (see text). For completeness, PA21 (14) and LA (24) virus sequences derivedfrom cloned cDNA have been included. Similarly, as CR326 did not prime with the VP3 primer set, published sequence from cDNA (21) wasincluded in this region. The approximate degree of nucleotide identity between any two strains is represented by the distance from the left ofthe diagram to the first common node. Dashed lines indicate special conditions, as noted above.

we consider AC/PCR to be a potentially important steptoward the reduction ofPCR methodology to general clinicallaboratory usage.

Analysis ofAC/PCR products demonstrated a high level ofsequence conservation among most human HAV strains (Fig.4). However, it was surprising to find that eight cell culture-adapted virus isolates were identical in sequence to the MBBstrain. We suspect that many of these isolates representcontamination of cell lines with MBB virus, as this was oneof the first HAV cell culture isolates (26) and was carried asa reference strain in many laboratories. We know this is thecase with a "KMW-1" cell culture isolate (not shown in Fig.4), as its sequence was identical to MBB, whereas sequencederived by AC/PCR from the original KMW-1 fecal specimenwas unique and had only 92.6% identity. Similar evidenceindicates that a cell culture-adapted "S85-1" isolate is anHM175 strain containment. Such contamination appears tohave occurred in a number of laboratories engaged in theisolation of HAV in cell cultures, indicating that firm con-clusions concerning the epidemiologic relatedness of strainssharing identical sequences may only be based on sequencesderived directly from virus present in clinical materials. Theability ofAC/PCR to rapidly generate such data directly fromclinical specimens represents a further advantage of thisapproach.As might be expected, viruses present in fecal samples

collected from three American soldiers (GR-1, GR-7, andGR-CL) involved in a 1982 outbreak of hepatitis A in theFederal Republic of Germany (12) shared a common nucle-otide sequence. Similarly, fecal specimens collected fromfour cases of endemic hepatitis A occurring in central Greece

during 1983 (AG11, AG5978, AG6014, and AG6084) con-tained viruses that were identical or differed at only one baseposition. Although this suggests the presence of a single viruscirculating in Greece at that time, the 9.8% difference be-tween the sequences of viruses recovered in Greece and inGermany (Fig. 4) indicates the existence of distinct strains ofHAV in Northern and Southern Europe during 1982-1983. Ofparticular interest, however, was the fact that a fecal sample(LV-BE) collected from an American soldier involved in anepidemic of hepatitis A in Kansas during 1982 (12) containedvirus with a sequence that was identical to that of the viruscausing the disease outbreak in Germany. These sequencedata, obtained directly from viruses present in feces, estab-lish a clear but previously unrecognized epidemiologic linkbetween the two epidemics and demonstrate the power ofthismolecular approach to epidemiology.Rico-Hesse and coworkers (27) have shown that contem-

porary isolates of single poliovirus serotypes commonlyshare <85% nucleotide identity and have employed thiscriterion to define distinct viral genotypes. Although HAVshares many features in common with the polioviruses, we

found considerably greater genetic relatedness among mosthuman HAV strains. Although substantial changes haveoccurred in the nucleotide sequences of polioviruses sinceisolation of the Sabin strains several decades ago (27), thesequences of the MS1 and CR326 HAV strains (recovered in1964 and 1966, respectively) are 96.6% and 96.9%o identicalwith that of the NC1 strain recovered in 1989 (Fig. 4).Although conservation of the primary structure of viralcapsid proteins could reflect structural constraints imposedon the evolutionary process, this would not explain the

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exceptionally high degree of nucleotide relatedness evidentamong most human HAV genomes. Moreover, 5-6 aminoacid substitutions are present within the carboxyl-terminal 29residues of VP1 in PA21 and H122 viruses (not shown),suggesting that extreme conservation of this capsid proteindomain is not essential to virus survival. Possible biologicalvariables that could account for a slower rate ofaccumulatedmutations within the HAV genome include a lower number ofviral replication cycles completed during natural infection orfewer infection cycles per year. Intrinsic differences in themutational rates of HAV and polioviruses would seem lesslikely.Although most human HAV strains were closely related,

H122 and CF53 were strikingly different. These viruses,recovered from patients in Sweden and France during 1979,differ from each other and the remaining human HAV strainsat 16-24% of base positions sequenced. The H122 virus,however, shared 97% nucleotide identity with PA21 virus(Fig. 4), which was recovered from a captive owl monkey andhas been considered to be of simian origin (14). AlthoughH122 and CF53 strains show considerable genetic diver-gence, they were detected by AC/PCR employing a HAV-specific monoclonal antibody and thus appear to be closelyrelated serologically to other human strains. The biologicalsignificance of these newly recognized HAV genotypes re-mains uncertain and will require further investigation. Thegenetic divergence manifested by these strains cannot berelated to geographic site, to disease expression, or to riskfactors for acquisition of hepatitis A given presently availabledata.

We thank C. Hutchison for oligonucleotide synthesis, J. Sninskyfor his generous gift of reagents, and P. Minor and E. Hu for PV1 andRSV monoclonal antibodies. This work was supported in part bygrant R01-AI22279 from the U.S Public Health Service, GrantsDAMD17-85C-5272 and DAMD17-89Z-9022 from the Army MedicalResearch and Development Command, and Technical ServicesAgreements with the Programme for Vaccine Development of theWorld Health Organization.

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