Vol. 25, No. 10JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1987, p. 1822-18290095-1137/87/101822-08$02.00/0Copyright C 1987, American Society for Microbiology

Detection of Hepatitis A Virus by Extraction of Viral RNA andMolecular Hybridization

JOHN R. TICEHURST,1t* STEPHEN M. FEINSTONE,' TAYLOR CHESTNUT,' NICOLAOS C. TASSOPOULOS,'tHANS POPPER,2 AND ROBERT H. PURCELL'

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892,1 andDepartment of Pathology, Mt. Sinai Hospital, New York, New York 100292

Received 15 September 1986/Accepted 15 June 1987

Hepatitis A virus (HAV) RNA was extracted from cell culture, serum, liver, and feces and then detected bymolecular hybridization with cloned HAV cDNA. Hybridization was approximately 10-fold more sensitive thanimmune electron microscopy or radioimmunoassay was and less sensitive than was assays of HAV infectivityin primates or in cell culture. As little as 103 50% infective doses of HAV, or approximately 0.1 pg of viral RNA,was detected by this method. Analysis of fecal specimens from an experimentally infected marmoset and an

epidemic of hepatitis A showed that HAV excretion could often be detected later in the illness by hybridizationthan by radioimmunoassay. This technique should be widely applicable for detection and analysis of HAVRNA.

Hepatitis A virus (HAV) is endemic in developing coun-tries and causes approximately 105 cases of hepatitis in theUnited States annually (for a review, see reference 16). It isusually spread via the fecal-oral route. HAV replicates in theliver and is excreted into the feces via the biliary system.Viremia has been documented prior to or during acutehepatitis A.

Tests for detecting HAV include, in order of increasingsensitivity, immune electron microscopy (IEM; 8), enzyme-linked immunoassay (21) or radioimmunoassay (RIA; 26),and inoculation into susceptible primates (25). Most wild-type HAV strains grow very slowly if at all in cell culture,and because they are not cytolytic, they must be detected byimmunologic methods. Antibody to HAV (anti-HAV) isusually present during the acute phase of illness and mayinterfere with viral isolation or detection.

Detection of HAV RNA would be useful for laboratorystudies and possibly for clinical studies. Molecular hybrid-ization techniques have been described for the detection ofmany viruses, including HAV, in a variety of specimens (1,9, 12-14, 17, 18, 22, 29-32). In this study, we describe apractical method for the extraction and detection of HAVRNA from virus in crude suspensions. We then apply thismethod of extraction and hybridization to the analysis ofexperimental and natural HAV infections, as previouslyreported in preliminary form (25).

MATERIALS AND METHODS

Equipment, enzymes, and reagents. Materials and theirsources were as follows: Cotworth Stomacher 80, A. J.Seward, London, United Kingdom; nitrocellulose paper(type BA-85), Elutip-d columns, and Minifold Il manifold,Schleicher & Schuell, Inc., Keene, N.H.; redistilled phenol,vanadyl ribonucleoside complex, low-melting-point agarose,

* Corresponding author.t Present address: Department of Virus Diseases, Walter Reed

Army Institute of Research, Washington, DC 20307.t Present address: National Center for Viral Hepatitis, Athens

School of Hygiene, Athens, Greece.

nick translation kits, restriction endonucleases, and NACS-prepac columns, Bethesda Research Laboratories, Gaithers-burg, Md.; restriction endonucleases, New England Bio-Labs, Beverly, Mass.; proteinase K, Boehringer MannheimBiochemicals, Indianapolis, Ind.; RNase A (type IIIA) andsalmon testis DNA (type III), Sigma Chemical Co., St.Louis, Mo.; [a.-32P]dCTP (3,000 Ci/mmol), AmershamCorp., Arlington Heights, III.; formamide, EM Science,Gibbstown, N.J.; HAVAB RIA kits, Abbott Laboratories,North Chicago, III.; XAR-5 film and diethyloxydiformate,Eastman Kodak Co., Rochester, N.Y.; Cronex Lightning-Plus screens, Du Pont Co., Wilmington, Del.; formaldehyde(AnalaR grade), BDH, Poole, United Kingdom; DNase I(Worthington DPRF), Cooper Biomedical, Inc., WestChester Pa.; RNasin, JEM Research, Kensington, Md.;Ficoll 400, Pharmacia, Uppsala, Sweden; DU-8 spectro-photometer/densitometer, Beckman Instruments, Inc., PaloAlto, Calif.

Cell culture. Primary African or continuous (Buffalo)green monkey kidney cells were infected with HAV HM-175(10, 25) or used as uninfected cells.

Clinical specimens. Stool was collected daily from a mar-moset that was experimentally infected with HAV HM-175.Fecal suspensions, 20% (wt/vol) in phosphate-buffered sa-line (PBS; 3 mM Na2HPO4-1 mM KH2PO4 [pH 7.4]-0.15 MNaCI-7.7 mM NaN3), were prepared by homogenization in astomacher for 30 s and centrifugation at 2,000 x g for 30 min.Selected samples were diluted to _5% (wt/vol) with 10 mMTris hydrochloride (pH 7.5)-0.1 M NaCl-0.1% (wt/vol)Nonidet P-40 and used to establish optimal conditions forHAV RNA extraction. Homogenates of liver (20% [wt/vol]in PBS) were prepared from marmosets that were uninfectedor experimentally infected with HAV HM-175. Stool suspen-sions and sera from experimental studies of HAV MS-1 inchildren (15) and adult volunteers (2) and from outbreaks ofstrain SD-11 in U.S. naval recruits (6, 27) and strain HM-175in an Australian family (10) were stored at -70°C.RIA. HAV antigen was detected by a solid-phase RIA

(26). Sample/negative control count-per-minute ratios of'2.1 were considered to be positive assays for HAV anti-gen. Serum anti-HAV was assayed by HAVAB RIA.

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DETECTION OF HAV BY EXTRACTION-HYBRIDIZATION

Extraction of HAV RNA. Cytoplasmic RNA was isolatedfrom cultured cells as previously described (20), except thatDNase I digestion was omitted (31). This technique wasslightly modified for extraction of HAV RNA from clinicalspecimens. Tubes and micropipette tips were sterilized withethylene oxide gas. Reagent solutions were treated with0.1% (vol/vol) diethyloxydiformate (20). Volumes were se-lected to confine extraction steps to 1.5-ml microcentrifugetubes. Typically, 160 pil of sample was mixed with 160 ,uI of0.2 M Tris hydrochloride (pH 7.5)-25 mM EDTA (ethylene-diaminetetraacetic acid)-0.3 M NaCl-2% (wt/vol) sodiumdodecyl sulfate (SDS)-400 ,ug of proteinase K per ml andincubated at 37°C for 30 min. Phenol (320 ,u) was added andshaken with the sample for 1 min, followed by centrifugationat 12,000 x g for 1 min, addition of CHC13 (320 ,ul), shaking,and centrifugation for 2.5 min. The aqueous phase (340 ,ul)was removed to a new tube containing 340 ,uI of CHCl3,shaken, and centrifuged for 2.5 min. The aqueous phase wasremoved to a new tube and mixed with 1.02 ml (3 volumes)of 10x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate [pH 7.0])-6.2 M formaldehyde and stored at -70°C.

After being heated at 65°C for 15 min, 50% of the extract(680 pil) was filtered from a manifold well through nitrocel-lulose paper that had been equilibrated with 10x SSC (G.Wahl, Sequences Application Update 371, Schleicher &Schuell). Wells were washed with an equal volume of 10xSSC. Papers were dried and then baked under vacuum at80°C for 2 h. Standard controls were extracts of marmosetfeces collected 1 day prior (negative) and 9 days after(positive) inoculation with HAV. Other negative controlswere extracts of uninfected cell culture, serum from ahealthy adult human, and liver from an uninfected marmo-set. Dilution series of denatured plasmid DNAs (22) wereused for quantitation.DNA probes. HAV probes were made from recombinants

of pBR322 with wild-type strain HM-175 cDNA inserts (31).The insert of pHAVLB 228, 228i (2.4 to 4.6 kilobases [kb]from the 5' end of the HAV genome), was isolated bydigestion with PstI, electrophoresis in two successive low-melting-point agarose gels, and purification over prepack-aged resin columns. Other HAV probes were Pi (0.74 to 3.5kb) from pHAV/J and P2-P3 (3.0 to 7.0 kb) from pHAV/L2(J. Ticehurst, J. I. Cohen, T. Chestnut, and R. H. Purcell,submitted for publication). pBR322 was used to determine ifclinical specimens contained homologous procaryotic nu-cleic acid. DNAs were labeled with [a-32P]dCTP by nicktranslation to specific activities of 2 x 108 to 10 x 108 dpm/,ugof DNA.

Hybridization. Papers were prehybridized (0.1 ml/cm2 ofpaper) at 42°C for 16 to 24 h in 50% (vol/vol) formamide-5 xSSPE-0.2% (wt/vol) SDS-5x Denhardt solution-100 ,ug ofdenatured sheared salmon testis DNA per ml (ix SSPE is 10mM sodium phosphate [pH 7.4], 0.18 M NaCI, and 1 mMEDTA, and ix Denhardt solution is 0.02% [wt/vol] eachbovine serum albumin, polyvinylpyrrolidone, and Ficoll).For hybridization at 42°C, fresh solution was used (0.05ml/cm2): 50% (vol/vol) formamide-5x SSPE-0.2% (wt/vol)SDS-2x Denhardt solution-100 ,ug of denatured shearedsalmon testis DNA per ml-10 ng of denatured [32P]DNAprobe per ml. Probes were annealed with specimens to reachapproximately 3 x Cot112 (20): 26 h for 228i, 33 h for Pi, or 48h for P2-P3. pBR322 was hybridized as long as the HAVprobe to which it was being compared. Papers were washedtwice in 2x SSPE-0.1% (wt/vol) SDS at room temperaturefor 10 min and then five to eight times in 0.1x SSPE-0.1%(wt/vol) SDS at 50°C for 15 min, until Cerenkov radiation

from 10 ml of wash buffer was _30 cpm. Papers were thenexposed to X-ray film at -70°C with two intensifyingscreens. For rehybridization, probes were removed at 68°Cfor 2 h in 50% (vol/vol) formamide-3 x SSPE (24).

Immunoaffinity-hybridization. Selected fecal samples werealso assayed after adsorption to anti-HAV (13). Monoclonalanti-HAV K3-2F2 (19) was the generous gift of A. G.Coulepis and I. D. Gust, Fairfield Hospital, Melbourne,Australia. Serum from a chimpanzee that was experimen-tally infected with HAV MS-1 (11, 21) was also used.

Autoradiography. Films were visually evaluated for auto-radiographic signals resulting from hybridization of extractswith probes. All positive results from specimens negative byIEM and RIA were confirmed with at least one additionalHAV probe. HAV RNA mass in selected extracts wasestimated from signal intensities, measured as area bydensitometry, produced by the sample extracts (As) andpHAV DNA standards (AD). For each AS, an apparent DNAmass (mA) was read from a plot of AD versus DNA mass.HAV RNA mass was calculated from mA by including afactor to account for the different molecular weights of targetRNA and DNA. It was assumed that both targets were fullydenatured and formed stable hybrids with the completesequence representation of the probe, except that HAVRNA hybridized only with the negative strand. HAV RNAmass was proportional to the ratio of target lengths(7,500/LD), where LD is the length in nucleotides of thepHAV DNA, because the twofold increase in probe thathybridized with pHAV DNA was offset by its twofold highermass per base. An additional factor accounted for thedifferent specific activities of probe DNA strands. Becausesignal intensity varied directly with the content of labeledcytosine (%C) in the probe, HAV RNA mass was inverselyproportional to %C_/%C+ (-, negative strand; ±, doublestrand). Then, HAV RNA approximately equals mA X(7,500/LD) X (%C+/%C_).For extracts that hybridized with HAV and pBR322

probes, specificity was assessed by comparing exposures inwhich the detection of target pBR322 was approximatelyequal. Densitometry measurements were used to calculate aratio of normalized signals, P1/pBR, where Pi and pBR areAs/Ar for the HAV Pi and pBR322 probes, respectively, andAp is the signal from 100 pg of target pBR322. When P1/pBRwas >1, indicating greater signal intensity with the HAVprobe, the extract was considered to contain HAV RNA.

RESULTS

Extraction of HAV RNA from clinical specimens. A tech-nique for the extraction of cytoplasmic RNA containinggenomic HAV RNA (20, 31) was optimal for extracting HAVRNA from virions in fecal suspensions. Results were readfrom autoradiograms exposed for 16 to 96 h. so that a 10-3dilution of the positive control was easily visualized, while a10-4 dilution of the positive control and the undilutednegative control had no signals (Fig. 1C). Extracts that weremixed in 7.5x SSC-4.6 M formaldehyde (see Materials andMethods) or precipitated in ethanol produced similar signals.There was also no effect when suspensions were treated withtrifluorotrichloroethane or when RNasin or vanadyl ribonu-cleoside complex was added prior to extraction (data notshown). When the extraction was performed without pro-teinase K and SDS or without organic solvents, substantiallyless HAV RNA was obtained. There was no detectable lossofHAV RNA from extracts after 2 years of storage. Extracts

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1824 TICEHURST ET AL.

A

pHAV/Jpg

I 1

3.3

1.1

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J. CLIN. MICROBIOL.

Densi tometer t racing

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B

HAV

HM-175

log ID10 50

2.5

2.0

1. 5

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c-

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3.4282

2. 0883

0.4349

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3.1268

0.1709

<0 .1

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4.0410

1 2299

0.2704

0.0601

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7.8459

4. 5158

1 .5557

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12

8.0

3.3

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0.80

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0.56

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DETECTION OF HAV BY EXTRACTION-HYBRIDIZATION 1825

Assay fo

IEM RIA*/Sq S/N

1 ND 41

2 -9 35

3 ND 0.6

4 O 2.5

5 2 1.7

27.3 27+

12.7 21+

- 1.0

- 1. 5

6 -1 7.7 8.3+

27 0115 «»

'_NA 3.1 2.0

3.9 8.5+ am5.3 14 q_

2.1 5.6 9.6 M"

- 6 8 7.3 _ -

21.3 35+

1 ng -IIIb100 pg

10 pg

FIG. 2. Detection of HAV RNA and procaryotic nucleic acid in fecal specimens and HAV RNA in marmoset liver. (A) Suspensions (2%[wt/vol]) were prepared from feces of naval recruits with type A hepatitis (6, 27). Pt, Patient; Day, day of feces collection relative to the onsetofjaundice; ND, day not determined. IEM #/sq, virus particles per grid square (average from two to five squares); -, no particles visualized(data of J. Dienstag, summarized in references 6, 21, and 27); NA, not assayed. RIA S/N, Sample/negative control; -, ratio of < 2.1. RNA,Mass of HAV RNA in 680 ,ul of extract; -, <0.1 pg of HAV RNA. Signals from extracts with >8 pg (+) exceeded the linear range ofdensitometer readings. The same paper was first hybridized with HAV probe Pi, stripped of probe (24), and hybridized with pBR322. Extractsfrom patients 3 to 7 and patient 9 on day 9 did not hybridize with pBR322 (data not shown). Autoradiograms of extracts that hybridized withboth probes were compared by densitometry (Pi/pBR; see Materials and Methods). All of these samples, except those from patient 5 andpatient 9 on day 9, were considered to contain HAV RNA. (B) Homogenate (20% [wt/vol]) of marmoset liver. (C) Denatured pBR322 DNA.

of stool suspensions ranging from 0.005 to 0.5% (wt/vol) andcomparably diluted extracts of 5% (wt/vol) suspensionsyielded similar results.

This extraction-hybridization (EH) technique was usedwith other types of specimens. Viral RNA was readilydetected in an extract of liver from a marmoset infected withstrain HM-175 (Fig. 2B) and in human serum containingstrain MS-1 (Table 1).Autoradiograms were analyzed by densitometry to enable

estimation ofHAV RNA mass (Fig. 1). Cloned HAV cDNAswere used as standards because HAV RNA is difficult topurify in measurable quantities. The weakest signals were

calculated to contain approximately 0.1 pg, or 2 x 104genome equivalents, of HAV RNA. The largest amounts of

HAV RNA in fecal extracts were approximately 670 ng.These were determined after dilution, because strong signalsexceeded the linear range of densitometer readings (Fig. 1B;HM-175, 102.5 50% infective doses [ID50s]).EH results were compared with ID50 titrations of three

HAV strains (Fig. 1 and Table 1). The lower limit ofdetection by EH was approximately 103 ID50s but varied in a

pattern that was paralleled by RIA results. The wild-typestrain HM-175, from which the probes were derived, was themost sensitively detected. However, each strain was de-tected with similar sensitivity by each HAV probe, as othershave demonstrated with strains HM-175 and MS-1 (17).Thus, the portions of these genomes detected by the probes(0.75 to 7.0 kb) did not contain major sequence differences

FIG. 1. Estimation of HAV RNA mass by densitometry. (A) Denatured pHAV/J was diluted, applied to nitrocellulose paper, and

hybridized with HAV probe Pi. The tracing, colinear with the autoradiogram, and area (AD) measurement were produced by thedensitometer. (B) Extracts of feces containing HAV HM-175, SD-11, or MS-1 were applied in volumes corresponding to 0.2 or 0.5 x log1oID5oof HAV. (C) Control extracts of marmoset feces collected 1 day prior to (negative) and 9 days after (positive) inoculation with HAV (appliedas 0-2, 10-3, and 1O-4 dilutions) (Fig. 4). As, Area measurement of signal by densitometer. For calculation, LD = 8,900; %C± = 19; %C-= 21 (5).

A Feces

Pt Day

r FiAV Probe

RNA P1/opBR Pl pBR322pg

7 3

8 2

9 ND

7

9

B Marrroset lIver

C pBR322 DNA

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1826 TICEHURST ET AL.

that were restricted to discrete regions. Calculated ratios ofHAV genome equivalents to ID50 approximated particle/ID50ratios determined for other picornaviruses (50 to 103) (28),except that of strain HM-175 in feces [(2 x 104 genomes)/ID50)]. HAV RNA was detected in '-1 ng and thereforerepresented approximately 0.01% of cytoplasmic RNA fromcells infected with strain HM-175 (data not shown).RIA was 3- to 20-fold less sensitive than EH was in

detecting HAV (Table 1). Other comparisons of hybridiza-tion results with those from RIA and IEM are describedbelow.

Specificity of EH and effect of contamination with pBR322.Hybridization with HAV probes was abolished by boilingfecal suspensions to release RNA from virions (9) and bytreating them with RNase A or NaOH before extraction.However, before concluding that a specimen containedHAV RNA, it was necessary to exclude any effect ofhybridization between residual pBR322 in probes and pro-caryotic nucleic acid in extracts. HAV probes were 200- to1,000-fold more sensitive for homologous DNA than forpBR322, but 10 to 100 pg of pBR322 was still detected (Fig.2C). Although <5% of extracts hybridized with pBR322 atthis level of sensitivity, some produced signals equivalent to200 pg of pBR322, demonstrating that lowered specificitywas due to reaction with contaminating vector DNA in theprobe preparations which hybridized with homologous se-quences extracted from fecal samples (Fig. 2A, patients 1and 2). Signals produced by positive samples were measur-ably greater by using HAV probes than with pBR322 (Fig.2A, patients 1, 2, 8, and 9). Ratios of signal produced byHAV probe Pi versus that produced by pBR322 ranged from7.3 to 27.We also attempted to reduce nonspecificity by using the

technique of Jansen et al. (13), who partially purified HAVby immunoadsorption to monoclonal anti-HAV prior tohybridization. In our experiments, HAV strains SD-11 andHM-175 were bound by both monoclonal and polyclonalanti-HAV, but the latter was not as efficient. The yield ofHAV HM-175 RNA following immunoadsorption was two-to threefold less than that obtained by EH (Fig. 3). Althoughthe recovery ofHAV RNA after immunoadsorption ofHAVSD-11 from one stool sample was only 4% (data not shown),

TABLE 1. Detection of different HAV strains: sensitivityof RIA and EH

Minimum logo ID"o"HAV strain Specimen detected by:

RIAb EH"

HM-175 Fecesd 2.0 1.0Cell cultures 4.0 2.7

SD-11 Fecesf 4.0 3.5MS-1Fecesi 3.5 3.0

Serum"l NA 3.0

aApproximate titers were determined in chimpanzees by intravenousinoculation of serial 10-fold dilutions and in cell culture (ID50, 50% tissueculture infective dose) by immunofluorescence (25).hSamples (25 PI) were diluted to contain 0.5 x loglo ID50. NA, Not

assayed.c A 160-,ul sample of each stock was extracted, and volumes were applied

to nitrocellulose paper corresponding to 1 x logl0 ID50 (strain HM-175, cellculture) or 0.5 x logo ID50.

d Suspensions of 5 or 0.02% (wt/vol) (10).e Harvests at 48 and 72 h of rapidly growing variant (25).f Suspensions of 2 or 0.2% (wt/vol) (6).I Suspensions of 0.2% (wt/vol) (2).h Serum-PBS (50% [vol/vol]) (15).

Feces

A Extraction

B InTrunoadsorpt ion

-2n,%Ab 10

-4ch irpanzee 10

400 40 4 ug

0.66 pg HAV RNA

33%

1p14%

oe32% 43%

34% 22%

FIG. 3. Comparison of HAV RNA detection by EH and byimmunoaffinity-hybridization. Samples, assayed in duplicate, werehybridized with HAV probe P2-P3 after extraction (A) or immunoaf-finity purification (B) (13). Sera coating immunoaffinity wells weremonoclonal antibody K3-2F2 (mAb) (19) and hyperimmune serumobtained from a chimpanzee (21). Indicated dilutions were deter-mined to be optimal. Marmoset stool was collected 10 days afterinoculation with HAV HM-175 (Fig. 4), and serial 10-fold dilutionsof a 5% (wt/vol) suspension were made in PBS-1% (wt/vol) bovineserum albumin prior to extraction or immunoadsorption. An 80-,ulsample of a 0.005% (wt/vol) suspension (4 ,ug of feces) contained0.66 pg of HAV RNA, quantified by densitometry. The signal islisted below the autoradiographic data as the percentage of thatobtained with extracted sample.

approximately 50 pg of pBR322-like nucleic acid was elimi-nated.Other attempts to eliminate hybridization between probes

and pBR322-like nucleic acids in the fecal extracts wereunsatisfactory. The addition of unlabeled pBR322 to thehybridization mixture resulted in an unacceptable level ofbinding of the probe. HAV cDNA selectively labeled atrestriction sites or RNA (23) was a less specific probe forHAV RNA. An adaptation of a method for RNA isolation (4)resulted in partial loss of HAV RNA without completelyremoving pBR322-like nucleic acid. Jiang et al. (14) showedthat DNase hydrolyzed pBR322, while leaving purified HAVRNA intact. However, when we incubated fecal suspensionswith DNase I under conditions that hydrolyzed at least 100ng of exogenous pBR322, endogenous procaryotic nucleicacid was not eliminated from all specimens. Furthermore,the yield of HAV RNA from some stool suspensions wasdiminished.

Application of hybridization analysis to HAV infections.The course of hepatitis A in an experimentally infectedmarmoset is shown in Fig. 4. The onset of the most severehepatitis coincided with the appearance of serum anti-HAV.Although immunofluorescence analysis demonstrated HAVantigen in biopsied liver from early in the infection, HAVwas maximally shed during the first 20 days after inoculationand was detected by RIA during this period only. EH was atleast 10-fold more sensitive than RIA was, and the ratio ofEH to RIA increased after day 16. After day 20, the smallamounts of HAV excreted were detected only by EH.

In addition, samples from a large outbreak of hepatitis A innaval recruits at San Diego, Calif., were assayed by EH andcompared with results of IEM (6, 21, 27) and RIA. Weanalyzed the sample collected earliest from each individualand every fecal suspension from individuals who had detect-able HAV RNA or serum bilirubin of '8 mg/dl. One extractthat hybridized with pBR322 was excluded.Of 134 fecal samples, 116 were collected from 57 individ-

uals with acute type A hepatitis, and 53 of these had

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EXTRACTION-HYBRIDIZATION 1827

Anti-HAV (eum) EHAV Ag (hRoer) E

Path <h«u E

Ea

o*.~0

10.7

Jz

DAYS AFTER INOCULATION

FIG. 4. Course of hepatitis A in an experimentally infected marmoset. A marmoset was inoculated intravenously with a human fecalsuspension containing 2 x 106 chimpanzee ID50s of HAV HM-175. Samples of serum, liver, and feces were collected until the animal died40 days after inoculation. ICD, Serum isocitric dehydrogenase (A). The average of four ICD determinations before inoculation was 372 + 84(standard deviation) 30°C Sigma units/ml. RIA (0), Results from RIA of 20% (wt/vol) fecal suspensions; HAV RNA (O), molecules per gramof stool, calculated from densitometry data. RIA and RNA values are plotted as log1o. Anti-HAV, Results of HAVAB RIA of serum; HAVAg, results of immunofluorescence analysis of liver tissue, scored - to 4+; Path, histopathologic changes in liver tissue, scored - to 4+.

detectable HAV (Table 2 and Fig. 2A and 5). HAV was morefrequently detected by EH than by IEM or RIA, especiallyafter the onset ofjaundice. The efficiency of detecting HAVin the 53 positive specimens was 98% for EH, 66% for RIA,and 53% for IEM. HAV RNA was detected in 22 of 57specimens (39%) collected during the second week of symp-toms, compared with 7 (12%) positive by IEM and 12 (21%)by RIA, indicating that the relative sensitivity of EH in-creased with time from the onset of illness. There was nocorrelation between the level of serum aminotransferases or

TABLE 2. HAV in fecal suspensions from the San Diegooutbreak: comparison of IEM, RIA, and EH'

Detection method No. of samplespositive (%)b

IEM, RIA, and EH .................................. 23 (43)IEM and EH only .................................... 4 (7.5)RIA and EH only ................................... 12 (23)IEM only........................................ 1 (1.9)EH only........................................ 13 (25)

a IEM was performed in 1974 and 1975 (6). For RIA, some samples were

previously analyzed with blocking immunoassays to determine specificity(21), and some were not included in Fig. 5 because collection dates were notrecorded.

b Percentages are the percent of 53 positive by any assay.c Two not assayed by IEM.d Five virus particles in three grid squares (Fig. 2A, patient 5).

bilirubin and the presence of HAV by IEM, RIA, or EH.Four extracts, prepared from stool collected after 3 monthsof disease from four patients with prolonged hepatitis A (27),did not hybridize with HAV probes. Viral RNA was notdetected in extracts of acute-phase sera that contained '800ID50s of HAV. The remaining 14 fecal samples were from 13patients who did not have a diagnosis of hepatitis A. Ex-tracts from two normal uninfected individuals, three withpresumed chronic non-A, non-B hepatitis, five with hepatitisB virus infection, two with alcoholic hepatitis, and one withinfectious mononucleosis did not hybridize with clonedHAV cDNA. Specificity of the probe applied to thesesamples was 100% (14 of 14).

DISCUSSION

We adapted a protocol for the extraction of cytoplasmicRNA from HAV-infected cells (20, 31) to fecal suspensions,sera, and liver homogenates. Stools were used to evaluatethis protocol because they may contain factors which lowerspecificity and sensitivity, including RNases, other picorna-viral RNAs (12), or procaryotic nucleic acids. High concen-trations of buffer and salt with EDTA, proteinase K, andSDS minimized sample-to-sample variations in digestionconditions, while inhibiting RNases. This protocol was sub-sequently used to extract other picornaviral RNAs that didnot hybridize with HAV probes under these or less stringent

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22 -

20

Co> 18

a.16

<14-

U.

12-

10

z..

6

4-

2

-9 -7 -5 -3 -1 1 3 5 7 9 11

DAYS FROM ONSET OF JAUNDICE

FIG. 5. HAV in fecal suspensions from an outbreak of hepatitisA and relationship to onset ofjaundice. Samples from the San Diegoepidemic (6, 27) were tested for HAV by RIA and EH. Each squareindicates a single sample (totaled along the ordinate) collected on a

day relative to the onset of jaundice (abscissa). These data are asubset of those in Table 2. C, Negative by RIA and EH; O, negativeby RIA and positive by EH; O, positive by RIA and EH.

conditions (Ticehurst et al., submitted). On the other hand,some fecal samples contained nucleic acid that hybridizedwith residual pBR322 DNA in HAV probes. AU but one ofour possible false-positive results were excluded by compar-ing autoradiograms produced by sequential probing withHAV cDNA and pBR322.Endogenous pBR322-like nucleic acid was eliminated

from a specimen by immunoadsorption of HAV, consistentwith results obtained after seeding fecal suspensions withplasmid-containing bacteria (13). While immunoaffinity-hybridization was easier to perform and more rapid for largenumbers of specimens, the autoradiographic signals were

stronger following EH. More experiments are needed toresolve this discrepancy between our results and those ofJansen et al. (13), who obtained substantially less HAV RNAfrom a similar extraction method (13).Sequence differences probably do not explain why the

homologous wild-type strain HM-175 was detected withgreater apparent sensitivity by EH and RIA. Geographicallydistinct HAV isolates have >90% sequence identity (5, 33).However, infectivity titers of wild-type viruses were deter-mined in small numbers of animals, and this imprecision mayexplain the observed differences in sensitivity.The diagnosis of acute hepatitis A is reliably made by

detecting anti-HAV immunoglobulin M in serum. Althoughwe detected HAV RNA in one serum, viremia generallyprecedes illness, and many viremic sera will not havedemonstrable HAV RNA. Hybridization is more sensitivethan IEM or RIA for detecting HAV in feces but morecumbersome than are assays for anti-HAV immunoglobulinM. In addition, HAV is usually excreted for only a few daysafter symptoms appear (for a review, see reference 16), a

period that may end before a patient seeks medical care. Onthe other hand, some patients continue to excrete HAV afterhospitalization. We detected HAV late in disease more

readily by EH, consistent with the finding that virus particles

excreted early are often empty (3). In addition, coproanti-body might interfere with immunoassays, resulting in arelative advantage for EH. HAV RNA was also frequentlydetected in RIA-negative feces collected during the secondweek of illness from children with sporadic hepatitis A (30).

If a genome-to-ID50 ratio of 100 is assumed, HAV excre-tion from patients in San Diego (Fig. 1 and 2) or from amarmoset (Fig. 4) was as high as 108 to 10i ID50s/g of stool.These estimates agree with titrations of HAV in humanstools by inoculation of chimpanzees (25). The risk ofexposure to such HAV concentrations must be assessedfrom epidemiologic data, but current guidelines do notrecommend isolation (7). It might be important, for diagnosisand control of infections, to quantitate viral excretion byhybridization, particularly when a patient has already devel-oped anti-HAV or when hepatitis occurs after the acutephase (29).

Hybridization is a sensitive screening technique for labo-ratories that study HAV. Qualitative and quantitative anal-yses of the small amounts of HAV RNA produced byinfected cells can be readily performed, during virus purifi-cation (18), for example, or during studies of viral macromo-lecular synthesis (1, 32). Thus, hybridization is especiallyuseful for the characterization of HAV because of its rela-tively poor growth in cell culture and the limited range oflaboratory animal hosts.

ACKNOWLEDGMENTS

The contributions of Diane Blackmore, Ronald Engle, KathleenMihalik, and Doris Wong to laboratory studies and Linda Jordanand Debra Brunelle to manuscript preparation are greatly appreci-ated. Lucid records, prepared by Jules Dienstag and John Routen-berg, facilitated our analysis of the San Diego outbreak. Theexperience of Jorge Flores was very helpful for extraction, as wasthat of Thomas Sargent for DNase, Bahige Baroudy and MarkBerninger for probe preparation, Stanley Lemon for immunoaffinity-hybridization, and Brent Korba and George Yoakum for densitom-etry. We thank the Laboratory of Carcinogenesis, National CancerInstitute, for the use of their densitometer, Anthony Coulepis andIan Gust for monoclonal anti-HAV, and Mary Estes for sharing dataprior to publication.

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