Characterization of a hepatitis A virus strain suitable for vaccine production

.?ournnlofHepatoZogy,1991; 13(Supgf. 4): S146-s151 @ 1991Elsevier Science Publishers B.V. AII rights reserved.016%8278/91/$03.50

5146 HEPAT 01039

Characterization of a he

is 44 virus strai roductio

Nicoletta Fineschi’ , Filippo Cavalier?, Hemda Garelick2, Anna Brugnda’, Vittoria Pellegrini’ and Arie J. Zuckerman3 ‘Sclavo Research Center

and RID Vaccines, Siena, Italy, ‘London School of Hygiene and Tropical Medicine, London and “The Royal Free Hospital School ofMedicine, London, UnitedKingdom

A novel isolate of hepatitis A virus, obtained from a clinical sample, has been adapted to grow on cultured human

dipI& cells. Growth and purification parameters have been optimized to obtain conditions suitable for the development of an inactivated vaccine. The entire viral genome was molecularly cloned, and the gene encoding the VP3 capsid protein was expressed in Escherichia co& The resulting recombinant VP3 was used to obtain rabbit antisera which recognize the denatured protein in purified virion preparations. Nucleotide sequencing data are presented and compared to known sequences of different strains.

Hepatitis A virus is a Picornavirus belonging to the genus Enterovirus (1). Attempts to characterize the virus have been hampered by difficulties in growing the virus in cell culture, which usually result in long-term incubation, low virus recovery, absence of cyopathic effect and persistent infection (2). In this study, we analyze the LSH/S strain of hepatitis A virus, a candidate for the production of an inactivated vaccine which was isolated from a clinical case and adapted to grow in human diploid MRCS cells at the London School of Hygiene and Tropical Medicine (3). The genome of the LSH/S strain, at the twelfth passage on MRC-5 cells, was completely cloned by reverse PCR and partially sequenced. The gene encoding the capsid protein VP3, has been expressed as coliphage MS2 RNA polymeraseNP3 fusion protein, and employed to obtain antibodies that recognize the viral VP3 from purified virion protein in Western blot.

MRC-5 cells were grown at 3PC using Dulbecco’s modified medium (D-MEM) containing 4 mM L-glut-

amine, 0.37% NaHCO,, 1 mM piruvate, 100 &ml neomycine sulphate and 10% foetal calf serum (FCS). Maintenance medium used after infection was supplemented with 3% FCS. Virus seed

The hepatitis A virus strain LSWS, used in this study, was isolated in MRC-5 cells from a sonicated fecal extract derived from a documented case of hepatitis A at the London School of Hygiene and Tropical Medicine. The virus grows at 32”C, mostly cell associated, with an incubation time of 3 weeks. It gives a persistent infection on MRC-5 cells and is not cytopathic. After the isolation, the virus was serially passaged in MRCJ cells. The seed used in this study was obtained after 12 passages in MRC-5 cells. Virus production and harvest

MRC-5 confluent monolayers were used for HAV production at a doubling population level of 30. Roller bottles (850 cm2) were inoculated with seed virus diluted in serum-free D-MEM at a MOI of 0.5. After adsorption (2 h; 32°C) maintenance medium was added to a final volume of 200 ml. Infected cultures were incubated at 32°C for 21 days (rotation 0.75 rpm). Ten days post-infection

Correspondent@: VittoriaPeilegrini,SCLAVO IUD Vaccines. Via Fiorentina 1, 53100 Siena, Italy.

maintenance mediu ml/roller.

was added to a final volume of 360

E

sl s2 s3 s4

Molecular cloning of HAV cBNA A battery of eight synthetic oligos were syrrthetized on an Applied Biosystems DNA Synthesizer. This battery comprised four sense oligos (sequence identical to the coding, +, viral RNA) = s; and four antisense oligos (sequences complementary to the coding, +, viral RNA) = a; ordered in four couples as follows: oligo sequences and map coordinates were inferred from Cohen et al. (4), since we had indications that our strain shared significant sequence homology with the one described (not shown). These indications were amply confirmed by sequencing data obtained from our cDNA clones.

s oligos were preceded by the recognition sequence for restriction nuclease Kpp2I and oligos by that for .SmaI. Total A was prepared from infected weeks post-infection as described (5). ‘I!vo micrograms of RNA were used for each cDNA synthesis reaction. cDNA was synthetized with ringer cDNA kits, uader the conditions recom by the manufacturer, except for the substituti e a-series oligos for oligo-dT primer. The re ture was diluted 10fold in water and one pa polymeraze chain reaction ( actions were carried out directly on the heteroduplex, with reagents of the PerkinGene-Amp kit, under the conditions recommended by the manufacturer, in the presence of the a~~r~~riate couple of oligos. The amplimers were gel purified, digested with @I and SnzaI and cloned in ~~lues~ri~t (Stratagene), by standard techniques (6). Restriction maps of the construct were obtained and compared with published sequences. Whenever possible, single cutter restriction enzymes, whose recognition sequences lie in the overlap, were used to fuse two subclones. Recourse to partial digestion was unavoi able in other cases. full-length clone was thus obtained by fusing amplimers

assay antigen was detected by a doub ELISA assay (3). Wells of microti plates (Nunc) were coated with 30 nglmg of the anti monoclonal antibody 14I-ULSI-IT purified by protein A chromatography. After washing, serial dilutions of each sample were added. uman anti-I-IAV IgG, purified from a convalescent serum, was used as first antibody. The second antibody was a peroxidase-conjugated mouse anti-human IgG (Jackson). For quantitative assay, serial dilutions of an FIAV internal standard were tested. The MAV content of each sample, expressed in EU/ml, was determined on the standard curve.

VP4

= = = =

nt 1 to 27, ali = rat 2127 nt 1900 to 1927, a2 = nt nt 3300 to nt 3327, a3 = rat 4900 to nt 4927, a4 =

to 2100 3400 to 3573 nt 551BOto 5473 nt 74% to 7461.

38

38

Fig. 1. a: map of HAV genome. b: regions of HAV LSHfS strain that has been sequenced. c: DNA clones of HAV LSWS strain with the location of restriction enzyme sites used for the constructiou ot fuii-ien& douc.

1

S143

59

G~~GG~CA,;IT?C?GGRG~~?~T~W.C~ACAG?T~C?~CAGGACAGR~

11~111 11111111111111111111111ll11ll1111111 G77GG,.GAtGA?7C7GCAGG?T?TtCMCMCAG?t?C7AC,.GMCA0AA . 51 TGTTCCAGA7CCCCMG?TGCtAtMCM~CA?GMGG~~TT......~ ll1111~l11~111111~111llllllllII11111 It111 2259 ~G~TCCAGA~CCCCMG~~GG~ATMCMCCA~GAAAGA~~TGAAAC~

III11

2304

LSH/S

1

mu75

1

50

7fCMWGG~GTCWCCGGiM~TtCCCGG~C~~CttC7t~CGMGTCCAli IIIIIIIllill1 Illllll11ll1IIIIIllllII IIIIIllllllI ??CM~GGCGtC.?CCGGGMtttCCGGAGtCCC?C.ttGtMGIC~t

48

51 ~G?CAGCGG;C~GA?ACC;CICCGCCC~,GCCTA0GCTA,~GCC,~ 1111IIIIIIIIIII11111l11111111111111111111111111111 49 GG?~0GG~C~GA,A~C?~~~~G~~~7~G~~,AG0~7A7~~0~7~ LO1 99 142 1*9 192

77fTCCcrrkCcCtTcC&7.......:.CCt,C???.;tA,?Gt& I IIIIIIII 111lIIlllIIII II lllll T??tccctttccct~Tccc7T?ccTAt~ccctt~G?tttGc~tG~~T

141 11111111 I40

231

20

249

2156

I

3d9

5'

non

coding':Q6.?%

295

29d

2306

. vE5

100

I II

631

15, tCttCAGT?i,,7AC,GC?G~GGt,CGA?C~CA,QAGG?T&ACC?T7GA~

200

IS1

IIII1111111111 lll1llllll1lllllI lllllIIlllllllll 655 ?C~CAGttCAtACACC7GAOG~GGAtCh~CCAGCttCMCC~OA9

934

3162

150

10,

98d

3212

IIIIIl1llllll

266

LSWS HM175

I 1470

3312

A7GA7GAG~tGM?7TLiGG7~AG?AC~AC?G~T~?GGtGM?~

III11111111111111111111111111 IIIII IIIIIIIIIlll ~?G~?G~~T~?~?~GGO?CAC?~?AC?~GM?G?GG?GM?C?

51 GTC~?,~~C~GA?GC~GhGC~GA~G7CT???GC~,,AGA?CAG~ 1610

101 1570 151 1520 201 1670

IIIlllllfIIIIIIIflIlIIIII~IIIlll1lrlillIII G~C~t7AtGMGAtGCMGhGCAAA~TGtCttttCCTttGGAtCAGG

tIlllIt

100

1569

1619

1669

IIIItlIIIIllIIIIIII

II II

17lO ATtTtttCCMATGACAMthCGM7CCtGhCCLMAAtGtAThACtGCt

I1lll11111II111111111111

301 T7GGC??Ct~t77G?CAG~~GT??TG?tt~?GGAGACGA~A ll1l1lllllllllll1llllllllllllllllllllllll ?TGGCTtC?AZTtCTChGG~Zf?GT?tt?GGAGAGGAGA

VP3

:95.3%

94.3%

9311

3347

identity

1119

lll1IIIllllllIIIIIlllll!l IIIIIlllllI IllI ~TGGhCA,~TG777CAT,GA7~GAt77GCTCAG7TC?f?hGTCAt~~CA

55 4950 105 5000

IS0 1111

3261 300

336

?, CITGGACA~~~G?7~C~T7iA~~~~CtTC~TCACT~CCC~4G~~AtGA~~ 4900

a00

.

ORTAC,AG,&TT,UCGA~G~t7GGATT~?C???G llllIllllIILIIlll1lllllllllll llllll GAtAC,AGAGC?,TMFGAGG?T,GGA??CTCtt?G

155 5050

300

105

1769

9100

341

101 4999

204

tCTGt7hGTkACAkG7~GG7tGC7G7~GGAGClGCAk’tGGtGt7C~ tIllIll lIlllllllIIIIlll1lIIIIIIIIIIl1ll1III ?C?GtTACTMTCACMGtGGGT?G~797GGGAGCTGCAG?~GCAT?C?

254

2C/3A:

94.5%

5099

III1 297 5392

identity

50

ATCCA7GTlGttGGIGGMAtTCM71C7tG77GCAAMTTGGT7AC7C*

51 AG~tGtt~CMMTAff;rA,MG-;,t~G,Cl;ihGMTCL,C~ IIIIIIIIIllllllllllIIIIIIIIIIIll1lllllllllllI 5909 AG-7GTTCCMMThT7Cn7MG~7?G~ClCAG~G~tlhTCA

5901

100 Illi

Iltllll lllllllllllllllllIIIIII lIIIl~llllllllIlII 5959 MGtCCAGt7tACtCAGtGTTCP.ATG~lGTGG7CTCC~CGCtT7TT 151

301 6059 151 6109

hShMGhG*ccCri,?CA,C;~cA~~,,G~i~~~~~~G~,~~,TT~c~ 1111IIIIIIIIIII IIIIIIIIIIIlIIlIIIIIIl~lI AGRMGhGTCCCAt7thtC~~C~Ch77Ght~CCA7C~tT~TTttC~ ?GChGC7ht~CCt7t?tC7;I7FCCGUA;TCLtC~~l~G~7~7G~lG~ IlIIIl~lllll l1llllll II IIIIIIIIIIIlllllll ?GCAGCtA7GCCC7?TTC7AMGCTGhMf7GMCCMTGGCfGTGAtGT

6004 200 llllllll

(OS9 350

llllll

6106

TITC7~GC~7tCA??~~CC;~T,GtACM~IGCC~G~CC~T,~?hMGA6 IIIlII!I 1111111111 1~111111111 IIIIllll1ll1llllII

300

?h7CTMG7AT7CATPACC7ATtG~AGMG~EEA.TbGChTTAT~GAG

6150

301 AC 6159 6:

302 6160 3C/3D

of the HAV LSWS strain genome compared

5956 150

101 MGTCC~77~1AC7C~G,G;?C~,G~?~,~G7C,CC~A~GC7~77~

CO09

5149

-

1 ATTCAlC7l~C~CCACG~TT~M?,~~~G7,C~~l~~tTl~~?~~ II IIIIIIIllllllllllllll1~llllllllllIIIIIIIIIIIIIII S4SP

154

$049

Ct7tICCAt~tGGTG~CC~tCGMT~CC~~TATC~G~C~~t~TCC~ III1111111111111111111111 II 1IIIIIIIIIIIIIIllllII C????C~?C?GC?~CCATC0MCTC~?tAtC7GGCTTT??C~

identity

$H/S

(949

GGGMTT~.C~GATGA7Ch~~~G~~~G~G;AG~GGt~t~~T~,7~CCAG~ IIIlllllllllllllll1lllllllllIIIII II IIIIIllllllll GGGM?T?CAtA?GATGhtAA7G?.1AC1GCAG7AGC?GAGtf?77CCAG?

lllllllll1l1lll IIIIlllllllIIIIIlllllllllII 51.50 TGGAGtGCICGT7GGAGGAtGGTtTGTG7AfMGCA77?CTCC

1810

sd Illtll

Ot7G1U,C;GGhMC~CATMCTG~lT7ATGGAC~~GTGGfCTC~ Iill~lll II 11111!III1! llll~lll IIllllllIIll11lII GT7GAAA7tAG-ChCA7G~C,GAATTCATGG~G7~G?GG?C?CA

255 ?GtA07G~,~G77GGGCCh~~CGit?~TGf~,~G~A7~7~TCC

HM175

Fig. 2. Sequences

3211 350

IIIIIIIIIIIIIIIIII~IlllIIIllll IIIItllll1lllll~II CCCtAG~tGMCGGACTC~t~7CACMECC-~TtCTC~7~Tt~

vpl/a:

150

?GCTtCAGhZ~tAt??CG?C*~C~~t~G7TAT?C~r;Gt?GA~CCA~ llllll1ll I11l1ltllll lIIIIIIllllllllIllllll1 TGt??CAGhC?CAG??GG?CAACAAAt?AAAG?tA77CC~G??tACCCA2

I lllll

1161 200

1519

AACAWCG~~C~GhtCC~tCCCMGC~iGCGGnA~~AiM?tA~~CA~

t~tAC?ACt;G0ACAtC~A~CCCAAC7~~&3C~GCtCAGittCCA?tT~ IIIIItltlltlllllllll lllllllll1llllllllI1lllIIIIlI tTtACTACtTC0ACATC7A?WXAAC7?TG0C7GCtCAGtttCCA7tTAA

CAAMAC7CMG?h,GCt&G.MGM,t~?CMhTGUi?CG?CCCAC~ lllllI~IIIIIIIIlIIIIIIIIII1llllllllllIIII ~GACTCM07,.3GC?CAGGMGMT?ETCW?GAhGZICrTeCAC??CCACC

SO

I11I1/I1I11I1111111 ffI(I II(lI II II 111111111111 MCA?TGGAAAtCTGAtCC0tCCCAtGG7GGtGCGAtCMMTtACTCA7

251 ATTt?TtCC~tGAC~5AC~CC~?~ACC~t~T~T~C,tC)

1770

I

lllll 111111111111 1111111 IIl1IlIIIIII &ll~lllli A?~~AG~?GC&GGAAGCCA,A,M~~AACTGA~A?TAGMGT,CG~AM

~~ACTGAGG~GCA,GMAT~TCUGTTT~CC,CCAGAC~AG?GAC?CC~ I IIIIIIIIIIIIIIIIIIIIlIIIIIIIIIlllllIlI llllll A1AC,GAGGACCATGAUtAA?GW.GtT?,CC,GGhGA0GTG?GAC?CCT

301

identity

3111 150

C~ct*G~Tt~cCaG~~cTT7Tc~c~~~c~7~~c*~~~r~~

3262

VP4/VP2:93.6%

50

~)Zht~9M+~tlGG-CC~~At~CM~~G~G~TTGC~G~TGGG~

251

1000

identity

3061

,111

985

2546

llllllll11lIlllllllllllllll ~GAG,AtACltttCCTh,~~~t,G

1OD

DBd

tCCtGOTtC~ttCTGC II II l1llllllll tCttGh7tCATtCtGC

lllllflI III AAttCMh,Mt......

2507

5L 07C,,tCAG,~GATGA?CC?kA?ChGA0G~GGAT-t;l77tGAGAGT~ IIIlIIlllll1llllIIIIllllll1IlI IIIIIIIllllllIIIIII 3062 07CAtCAG?GGAtGA?CC?AGA?CAGA0GMGA?-GATTTGAGAGTC lG1

251

119

I n70t?A,~~;~,G~?C~iG*?GAGCAG~G,,GCGGC?iChGAC7TGG~ Itl111111111111111111111111111 IllI IlIIlllllll1ll AtGTtA~CCAC?GMTCMtGh7GAGCACiM~~CCAGC~CGAGhCttGGA

150

935 MCCtCtGt~GAt~CCCGG7tt~WAGAC~CAtGG~GAG~~Tt2

MTTC-CMT-GIG~t~GTLC*C~t+fCCThT~cCTTG

3012

LO6 tTGh~AGGA~tGtAG~G~C~GGAGC~~C~~ATT~tACtt~~C~GG~~C~ lIIIIlllllll lllllllll tIIIIIIIIIllllllIIlIlIIIIr~ 635 TtGh+AG~CtGChC~CACtG0tGCttC~tA~t~~ACttC~G~G~~C~~

201 MCCTC?G7;CtC~CCt~GAt~~G~GA~TC~GG~OAG~~~~ lllll II lIIIIIIIIIIIII IIII111111~

II

~~RTCt#.CMt?,TA?GGGMGGTCtCAT?tC?TGTGC~Cf??TACAttC

VPli 94.6%

CChCAtCC~G~ttCtGGCA~~~AtTOACG~G~CC~~~At~CA~~~~~ IIIIIIIIIIIII 1111111 11111111111111111111111 CCACATCC?G,Ct77GGCAGAC,.77GACGAAGAGC~TGA??CAA7CAC

2d57 29,

~?A~?~AC~G~+TII~GGG~GG~C~CA~~~?~~GT~CA~?~T~AC~~~~ .I II IlIIiltllllllllll1lllllllI lIIIIIIlllllII

identity

50

51

1dd

,,Od

27 2

704

3101

195 A~,cCC~G~~TGMLCC~~GAGAGT~~A~G~~~A~A~~~GA~~A~ATG~ Ill IlIIIIIIIIIIIIIIIIII lllll IlIIIIIIllIIIlll1II ~~~~cc~GMT~GMAc~TGG~G~~~C~GA~~T~~A~C~GA~C~~~~G~

C~CM~~T~~ACGC~~?C~~~CT~C?~~C~~CCA.GGCT~~CCC...~~~ Iltl 11111111111111111111111111111 l1lllllll

191)

194

I,6 ~~+~,~A~~CM,?~ACG;,C~7G777T~tC~G~~7~C~,G~GA~ IIIIIllIIIIIllIIIIIIIII IIIIIIll11llllIlll1lllllll 2356 Gc~A,cACMCMT?GACG~?CCAGTT??AGCAMG~UAGTACC,GAGAC

191

.199 C?Ch?t7TtCACGC???CtGTCtTC7?tCttCCACG‘X?C?CCCCW.iCC 234

1dd

9E

~t?AA~fCCiCGhGG?TCAGGtt7C7t~tC~G~?tCTC~At~~CA Illlllltll II1111111111111111111111i1111I11111111 *Z+M77CClb~~G??tAOG~?7~*7~t~7G77?~7~7*~~~~~

~thGGCt&GCC~ttGCGtCCGGC.GGCkMCtC 1111111111111111111111111 llllllllll CtAGGC?C?GtCCG??GCGCC~GGCGCGG,CF.AC,C

94 2307

TiiGATG7t7CA~GAGTGCMC~ACCTG7GtGA 95 MGCTMT*~GCGumA 'I II IIIIlIIllllIIIIlIIII IIIIlllllllIi1l

100

339.3

with the sequence

:95-d%

identity

of the wild type HM175 strain (4);

1 and 2 (HkdiIT% digestion an

Bigatisn) to obtain clone l-2 (nt O-3600). Avd partial digestion was necessary to fuse clone l-2 to amplimer 3 originating clone l-3 (nt O-5500), which was in turn fused to a partial digestion followed by ligati length recombinant. Partial nucleotide sequence was determined by standard dideosy chain termination techniques (6).

of HAY VP3 capsid protein %norder to obtain a VP3 cDNA in a frame suitable for expression, the VP3 coding sequence was amphfied by PCR from the full length e synthetic oligos employed were VP3-s = nt 1470-1497, preceded Cloning and expression

1

51 101

the procaryotic expression ve the N-terminus of coliphage under the control of the t The construct was transformed into E. coli K-12dehaHBdeltatrp (7,8). The fusion protein was purified and used to immunize rabbits as described (8).

les containing purified virions were subjected to

MlSRQCIPl?

TVGSGLDHl[L

SLADIEEEQH

IQ(jL"DRTbfTGASYFTSVDQ

SSVHTAEVGS

H&LRTSV

\KPGSKKTQG

EKFFL&u3

WLTT~~~LFHE

VAKLDV

1

MMRNEFRVST

TENVVNLSNlt EDARAKMSFA

LDQEDWKSDP

SQGGGIXJTH

51

FTTWTSIPTL

AAQFPFNASD

VDPYFFQHTN

TNPDQKCITA

SVGQQIKVIP

101

LASICQHFCF WRGDLLDP’

1

VGHDSGC;FST TVSTGQSVPD

PQVGITTflKD L

TTXEDPVLAK

KVPETFPELR

PG&SRHTSDH

NNME_YTFP

ITL

1

MLSTESMMSR

VAAGDLESSV

DDPRSEEDKR

FESHIE:RKP YKELRLEVGR

51

QRLKYAQEEL

SNE&PPRK

MKGLFSQAKI

SLFYTEEH'EI HKFSWRGVT:

IRKQN:TEFH

ELWSQGISDD

DNDSAVAEFF

L PI

D

51 101

RGKHDV

SGVQAPVGAI

flSIYKFl?GRSHFLCTFTFNS

I

101

1

DTRALRRFGF SL

MDGHNVSL,MD LLSSLVRTVE

51

QSFPSGEPSN

SKLSGFFQSV :N"KWVA"GA

AVG b LGVLVG

GWFVYKHPS

1

IHVAGGNSIL

VAKLVTQEflF

QNIDKKIESQ

RIMKV;FTQC

SMNWSKTLF

RKSPII:HHID

KTflINFPAAn

PFS:AEIDP”

Ai$lLSK:SLP

IVEEPEDYKE

51

101

TR

@$. 3. Peptide sequences of the HAV LSWS strain. The sequences were compared with the sequence of the wild type X-Ml75 strain (4); differences are shown above the line.

N. FINESCHI et al.

s150 kD

--l;-iii ,--++I

90K67 K--,

43K--c

,,

,

5 . !!

(,I ! 1

!

I

-VP3

fig, 4. a: VP3 expression vector. b: immunoblot with rabbit’s antibodies to the VP3 fusion protein. Lane A,B, SDS-PAGE of purified HAV transferred to nitrocellulose and stained with colloidal gold; lane C. immunoblot of the sample A with the anti-VP3 antibodies.

SDS-PAGE with discontinuous buffers (9) in 12% acrylamide gel and transferred to nitrocellulose as described by Tobin et al, (10). Rabbit anti-VP3 antibodies were used as first antibody. The second antibody was a peroxidase conjugated anti-rabbit antibody.

Results and Comments

The HAV LSH/S isolate has been adapted to grow on human fibroblasts (MRC-5), and parameters important for vaccine production (viral yield, purification procedures, quantitative assays, inactivation protocols) have been optimized. Alongside this work, we initiated the molecular characterization of the isolate with a double purpose: (a) establishing the uniqueness of our isolate and saving the information in a stable storage form; (b) obtaining highly specific reagents necessary for strict quality controls during purification. To achieve these aims, a full-length cDNA clone of the viral genome was obtained by reverse polymerase chain reactions followed by standard molecular cloning. The cloning strategy is outlined in Materials and Methods and a graphic representation is shown in Fig. 1. Experiments aimed at assessing the infectivity of the cDNA clone are underway. Data obtained from partial nucleotide sequencing of the clone are shown in Fig. 2. Sequences shown are compared to known sequences of the same regions of wild-type HAV isolate HM175 (4), the results of which showed it to be the most closely related of all isolates whose sequences are available, The degree of identity at the nucleotide level is more than 90% differences including deletions, substitutions and insertions, and slightly higher at the amino acid level. VP2 and VP4 are. apparently, the least homologous of

the analyzed sequences, showing 93.6% identity to their HM175 equivalent, while the 5’ non-coding region is the most homologous rating 96.7% identity. Nevertheless, four deletions of 9, 1, 3 and 1 nucleotides, respectively, at positions 121-129,233,243-245 and 274 were mapped together with two insertions of one nucleotide at positions 14 and 38. Deletions and insertions (of six bases each, at positions 2300-2305 and 2520-2525, respectively) were detected in VP1 coding sequences as well. Remarkably, two ‘hot spots’ in which a mutation occurs in most strains adapted to propagate in tissue culture, were found in our isolate as well: nt 203 in the 5’ noncoding region, (nt 203-207) ant nt 1742 (G to A) in the VP3 ORF (11-13). Sera from patients or experimental animals mostly recognize conformational epitopes which are lost during virion denaturation. We needed antisera capable of reacting specifically with denatured virion components in order to test our purified virion preparations by easily reproducible Western blot procedures. Fig. 3 shows how we obtained the first such reagent. The VP3 coding region was subcloned into the procaryotic expression vector pEX34B, and expressed in E. coli as a coliphage MS2 RNA polymeraseNP3 fusion protein (MS2NP3). The fusion protein was identified and purified thanks to the reactivity of its MS2 moiety with anti-MS2 sera. The purified protein was then used to immunize rabbits and the antiserum tested against disrupted, denatured purified virions. Bands of the expected apparent molecular weight were detected in viral samples but not in preparations from mock-infected cultures (Fig. 4). The antiserum is not reactive in either ELISA tests or virus neutralization tests (14). We are presently employing the same procedure to obtain anti-VP1 antisera.

MOLECULAR CHARA~~R~ZAT~GN

The authors wish

OF A NOVEL HEPATITIS A VI

80 t an& G. Cmsi for artwork.

8 Nicosia A, Bartoloni A, Ferugini M, Rappuoli R. Expression 1 Gust IDI Coulepis AG, Feinstone SM et al. Taxonomic classi-

fication of hepatitis A virus. Intervir 1983; 20: 1-7. 2 Gust ID, Locarnini AL, Coulepis A@, von der Helm K. The biology of hepatitis A virus. In: Deinhart P, Deinhardt J, eds. Viral Hepatitis: Laboratory and Clinical Science. New York: Marcel Dekker, 1983; 35-55. 3 Garelick H, Mann GF, Harrison TJ, Zuckerman AJ. Defective interfering particles in hepatitis A. In Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R Liss. 1988; 12-5. 4 Cohen JI, Ticehurst JR, Purcell RH. Buckler-White A, Baroudy BM. Complete nucleotide sequence of wild type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses J Viral 1987; 61: 50-9. 5 Sharp PA, Gallimore P , Flint SJ. Mapping adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines. Cold Spr Harb Symp Quant Biol 1974; 39: 457-74. 6 Maniatis T. Fritsch EE Sambrook J. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 7 Klinkert M. Herrmann R, Shaller H. Surface proteins of mycoplasma hyopneumoniae identified from an Eschericlria co/i expression plasmid library. Infect Immun 1985; 49: 329-35.

9 10

11

12

13

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