Complete Nucleotide Sequence of Wild-Type Hepatitis A Virus: Comparison with Different Strains of Hepatitis A Virus and Other Picornaviruses

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1 JOURNAL OF VIROLOGY, Jan. 1987, p Vol. 61, No X/87/ $02.00/0 Copyright 1987, American Society for Microbiology Complete Nucleotide Sequence of Wild-Type Hepatitis A Virus: Comparison with Different Strains of Hepatitis A Virus and Other Picornaviruses JEFFREY I. COHEN,* JOHN R. TICEHURST, ROBERT H. PURCELL, ALICIA BUCKLER-WHITE, AND BAHIGE M. BAROUDYt Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Received 27 June 1986/Accepted 1 October 1986 The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82: , 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(a) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; >99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation. Hepatitis A virus (HAV) is an RNA virus belonging to the picornavirus family. In 1983, about 21,500 cases of hepatitis A were reported in the United States, accounting for about 38% of all reported hepatitis cases (4). The true incidence of hepatitis A is thought to be much higher because most cases are not reported. Hepatitis A is endemic in developing countries, where virtually entire populations are infected during childhood (13). HAV also causes hepatitis in chimpanzees and certain new world monkeys. The virus has been propagated in several human and primate cell lines, including Alexander hepatoma cells, human diploid fibroblasts, and monkey kidney cells (34). Wild-type virus generally grows poorly in cell culture, but after several passages, the virus adapts to growth in vitro, resulting in higher titers of progeny virus and shorter replicative cycles. It is unknown what changes occur in the HAV genome during adaptation to cell culture. However, after only 10 passages of wild-type HAV HM-175 virus in monkey kidney cells, the virus becomes partially attenuated for chimpanzees (9). Recently, several groups have reported the nucleotide sequences of different strains of HAV (2, 23, 28, 30, 42, 44). These strains were isolated from hepatitis outbreaks of diverse geographic origin. Four of the HAV strains (23, 28, 30, 44) were adapted to growth in cell culture before molecular cloning. Strain HM-175 was isolated from an outbreak in Australia (14) and subsequently passaged three times in marmosets. The HM-175 virus used for cdna cloning was * Corresponding author. t Present address: Division of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, MD purified from the livers of marmosets with acute hepatitis and had never been in cell culture (42). We determined the complete nucleotide sequence of wild-type HM-175 HAV, portions of which were previously reported (2, 42). We compared this sequence to those from cell culture-adapted HAV strains as well as to sequences from other picornaviruses. MATERIALS AND METHODS Cloning. Molecular cloning of cdna representing greater than 99% of HAV HM-175 was described previously (42). The 5' end of the genome was cloned by primer extension (20, 35). Virion RNA template was extracted from HAV purified from marmoset liver. A primer was prepared from cdna clone phavlb113 (42) by successive digestion with restriction enzymes NciI and NcoI. A 223-nucleotide fragment (HAV nucleotides 45 to 268) was isolated and annealed to HAV RNA; cdna was synthesized with reverse transcriptase (160 U/ml), and the RNA was subsequently hydrolyzed (42). A homopolymer tail of dcmp was added to the cdna by terminal deoxynucleotidyl transferase (42), and the second strand of cdna was synthesized by using the large fragment of Escherichia coli DNA polymerase I with oligo(dg) as the primer (20, 35). The double-stranded DNA was separated on a 2% low-melting-point agarose gel, and DNA fragments from 200 to 500 nucleotides in length were isolated. The selected DNA was tailed with dcmp by deoxynucleotidyl transferase, annealed to oligo(dg)-tailed pbr322, and used to transform E. coli HB101 (42). Recombinant plasmids were screened for the presence of Hinfl (base 28) and NcoI (base 45) restriction sites. Plasmids containing both sites were characterized with additional

2 VOL. 61, 1987 NUCLEOTIDE SEQUENCE OF WILD-TYPE HEPATITIS A VIRUS 51 restriction enzymes, and nucleotide sequences were determined for several clones thought to contain the 5' terminus. One clone (of more than 300 evaluated), phavl5375, extended to the 5' end of the genome, and its 5' terminal sequence is similar to that of another strain of HAV (28). Sequence determination. A portion of the nucleotide sequence (4,886 nucleotides) has been previously published (2). Additional sequence was determined both from labeled DNA fragments by the procedure of Maxam and Gilbert (26) and directly from plasmid DNA by the method of Zagursky et al. using reverse transcriptase with oligonucleotide primers and dideoxynucleotide triphosphates (48). The entire genomic sequence of HAV HM-175 was determined by the method of Zagursky et al., and 95% was also determined by the procedure of Maxam and Gilbert. A total of 95% of the sequence was obtained on both strands, and the remainder was obtained from multiple determinations on one strand. Computer analysis. Sequences were analyzed by using a VAX 11/750 computer. Comparisons of HAV nucleotide sequences were made with the SEQH program (11). Secondary structures and free energies for the 5' terminus (bases 1 to 750) of HAV RNAs were predicted with the folding programs of Zuker and Steigler (49). Putative peptide cleavage sites for HAV were identified by the alignment of amino acid sequences from HAV with other picornaviruses. The SEQHP program (11) was used to align sequences with standard parameters (deletion penalty-8) or by reducing the deletion penalty to 6. Graphic matrix analysis (25) was performed with a window size of 25 and a minimum score of 15. Amino acid sequences surrounding putative dipeptide cleavage sites were compared by using the RELATE program (6), and regions with the highest scores from the mutation data matrix (37) were identified. The two picornaviruses with the highest homology to HAV near each cleavage site were determined. Sequences of 100 amino acids from each of the three viruses were aligned at each site by using a program for comparing the amino acid sequences (27) with a gap penalty of 8. RESULTS Sequence of HAV HM-175. The complete nucleotide sequence of HAV HM-175 is shown in Fig. 1. The genome is 7,478 nucleotides long followed by a poly(a) tail and encodes a polyprotein of 2,227 amino acids. A 5' noncoding region (734 bases) precedes a single long open reading frame (6,681 bases), which is followed by a 3' noncoding region (63 bases). Either methionine codon (bases 735 to 737, 741 to 743) may initiate translation; both are surrounded by several of the consensus nucleotides preferred by eucaryotic ribosomes for initiation of protein synthesis (19). The longest open reading frame initiated by an AUG codon in the 5' noncoding region is only 60 nucleotides long (bases 673 to 732). The 5' noncoding region contains two pyrimidine-rich tracts. The first region, near the 5' terminus (bases 99 to 138), has a 95% pyrimidine content. The second tract (bases 712 to 720) lies immediately before the initiation codon. On the basis of limited amino acid homology with other picornaviruses, we identified putative posttranslational cleavage sites for the polyprotein encoded by HAV (Fig. 1). The exact location of the VP3/VP1 site and approximate location of the VP2/VP3 site have been determined by direct amino acid sequence data from VP1 and VP3 (23). Locations of HAV VP4/VP2, VP3/VP1, 3A/3B, and 3B/3C cleavage sites have been mapped previously by using amino acid homology with other picornaviruses (2, 45). Amino acid alignments for HAV with other picornaviruses that were used to locate the remaining putative cleavage sites (VP1/2A, 2A/2B, 2B/2C, 2C/3A, and 3C/3D) are shown in Fig. 2A through E. Assignment of the VP1/2A cleavage site is also supported by data from antibodies to synthetic peptides representing sequences surrounding the site. Antibody to a peptide located proximal to the proposed site reacted with VP1; antibody to a peptide located distal to the site failed to react with VP1 by Western blot analysis (C. Wheeler, personal communication). Although a few of the peptide cleavage sites that we predicted differ markedly from those of Najarian et al. (28), other groups have assigned many cleavage sites which are similar to ours (7; A. C. Palmenberg, in D. J. Rowlands, B. W. J. Mahy, and M. Mayo, ed., Molecular Biology of Positive-Strand RNA Viruses, in press). The molecular weights of VP1, VP2, and VP3 predicted from the sequence data (33,200, 24,800, and 27,800) are similar to those determined by Western blot analysis with antibodies to synthetic peptides (33,000, 27,000, and 29,000 [47]). However, the predicted molecular weight of VP4 (2,500) differs from that determined by biophysical methods (14,000 [5]). The nucleotide composition of HAV HM-175 has a very low G+C content (38%), less than that of any of the picornaviruses that have been sequenced (Palmenberg, in press). The 5' noncoding region of HAV has a much higher G+C content (47%) than does the rest of the genome. The low G+C content of HAV RNA is also reflected in the codon selection pattern used for translation (data not shown). The C-G dinucleotide content of HAV (0.52%) is lower than that expected on a random basis (3.5%) considering its base composition. A lower than expected C-G content is also seen in polioviruses (43) and in eucaryotic mrna (12). Comparison with other strains of HAV. (i) Overall genome. The complete nucleotide sequence of one other HAV strain has been previously reported (designated here as strain LA; isolated in Los Angeles, Calif. [28]). Unlike HAV HM-175, strain LA was isolated from an outbreak in the Western hemisphere and was adapted to growth in cell culture before cdna cloning. A comparison of the nucleotide and predicted amino acid sequences of the two strains is shown in Table 1. Both strains have a genome length of 7,478 nucleotides; compared with strain LA, HM-175 has one additional nucleotide in the 5' noncoding region but one less nucleotide in the 3' noncoding region. Sequence comparison shows 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). Of the 589 nucleotide differences in the coding region, 511 (87%) are in the third position of codons, 61 (10%) are in the first position, 9 (2%) are in the second position, and 8 are due to insertions or deletions of nucleotides. The small number of changes in the first and second codon positions reflects the low number of amino acid differences between the two strains. When the two strains were compared for the presence of 100 restriction endonuclease sites present in HM-175 cdna, only 56 of the 100 sites were present in strain LA. (ii) 5' Noncoding region. The complete 5' noncoding region sequence was determined for strains HM-175 and LA. The nucleotide sequence of this region is the most conserved portion of the two genomes (96% identity). Most of the changes (79%) are nucleotide transitions. A possible secondary structure for the 5' terminus of the HAV RNA for both strains is shown in Fig. 3. The nucleotide differences be-

3 52 COHEN ET AL. J. VIROL. rtc^gagggctcgggattccgggtcctctgg^gtcatgtgaggggacttgatacctcaccgccgtttgcctaggctataggctaaatttrtccctttcccttttcccctttc CTATTCCCTTTGTTTTGCTTGTAAATATTAATTCCTGCAGGTTCAGGGTTCTTAAATCTGTTTCTCTATAAACACTCATTTTTCACGCTTTCTGTCTTCTTTCTTCCAGGGCTCTCCC CTTGCCCTAGGCTCTGGCCGTTGCGCCCGGCCGGGGTCAACTCCATGATTAGCATGGAGCTGTAGGAGTCTAAATTGGGGACACAGATGTTTGGAACGTCACCTTGCAGTGTTAACTTGGC TTTCATGAATCTCTTTGATCTTCCACAAGGGGTAGGCTACGGGTGAAACCTCTTAGGCTAMATACTTCTATGAMAGAGATGCCTTGGATAGGGTAACAGCGGCGGATATTGGTGAGTTGTTA AGACAAAAACCATTCAACGCCGGAGGACTGACTCTCATCCAGTGGATGCATTGAGTGGATTGACTGTCAGGGCTGTCTTTAGGCTTAMATTCCAGACCTCTCTGTGCTTAGGGCAAACATC ATTTGGCCTTAAMATGGGATTCTGTGAGAGGGGATCCCTCCATTGACAGCTGGACTGTTCTTTGGGGCCTTATGTGGTGTTTGCCTCTGAGGTACTCAGGGGCATTTAGGTTTTTCCTCAT 730 < VP4 - IA (nt / oa 1-23 / 23 oa) VP4 >< VP2 * 18 (nt / aa / TCTTAAATAATAATGAACATGTCTAGACAAGGTATTTTCCAGACTGTTGGGAGTGGTCTTGACCACATCCTGTCTTTGGCAGACATTGAGGAAGAGCAAATGATTCAATCAGTTGATAGG M N M S R O G I F Q T V G S G L D H I LSLA D I E E E M I S V D R 222 aa) ACTGCAGTGACTGGTGCTTCTTATTTTACTTCTGTGGATCAMATCTTCAGTTCATACAGCTGAGGTTGGATCACACCAGGTTGAACCTTTGAGAACCTCTGTTGATAAACCCGGTTCAAAG T A V T G A S Y F T S V D Q S S V H T A E V G S H Q V E P L R T S V D K P G S K AAGACTCAGGGAGAGAAATTTTTCTTGATTCATTCTGCAGATTGGCTTACTACACATGCTCTTTTCCATGAMAGTTGCAAAATTGGATGTGGTGAAATTATTATACAMATGAGCAGTTTGCT K t O G E K F F L I H S A D W L T T H A L F H.E V A K L D V V K L L Y N E O F A GTTCAAGGGTTGTTGAGATACCATACATATGCAAGATTTGGCATTGAAATTCAAGTTCAGATAAACCCTACACCTTTCCAACAGGGGGGATTGATCTGTGCTATGGTTCCTGGTGACCAG V O G L L R Y H T Y A R F G I E I Q V O I N P T P F Q Q G G L I C A M V P G D O AGCTATGGTTCTATAGCATCATTGACTGTTTATCCTCATGGTTTGTTAAATTGCAMATATTAACAATGTGGTTAGAMAT MAGGTTCCATTTATTTACACAMAGAGGTGCTTACCACTTTAAA S Y G S I A S L T V Y P H G L L N C N I N N V V R I K V P F I Y T R G A Y H F K GATCCACAATACCCAGTTTGGGAATTGACAMATTAGAGTTTGGTCAGAMATTAAATATTGGGACAGG,I\CTTCAGCTTATACTTCACTCAMATGTTTTAGCTAGATTTACAGATTTGGAGTTG D P O Y P V W E L T I R V W S E L N I G T G T S A Y T S L N V L A R F T D L E L 1450 VP2 >< VP3 - IC (nt / a / 246 aa) 1550 CATGGATTAACTCCTCTTTCTACACAAATGATGAGAMAATGAATTTAGGGTCAGTACTACTGAGAATGTGGTGAATCTGTCAAATTATGAAGATGCAAGAGCAAAGATGTCTTTTGCTTTG H G L T P L S T O M M R N E F R V S T T E N V V N L S N Y E D A R A K M S F A L GATCAGGAAGATTGGAATCTGATCCGTCCCAGGGTGGTGGGATCAATTACTCATTTTACTACTTGGACATCTATTCCAACTTTGGCTGCTCAGT TTCCAtTTTATGCTTCAGACTCA D O E D W K S D P S G GG I K I T H F T T W T S I P T L A A O F P F N A S D S GT TGGTCAACAATTAAGTTATTCCAGTTGACCCATATTTTTTCCAAATGACAATACGAATCCTGACCAAATGTAT AACTGCTTTGGCTTCTATTTGTCAGATGTTTTGTT TTTGG V G OQOI K V I P V D P Y F F O M T N T N P D O K C I T A L A S I C O M F C F W AGAGGAGATC TTGTCTTTGATT TTCAAGTTTT TCCCACCAATATCATTCAGGTAGATTACTGTTTTGTTTTGTTCCTGGCAATGAGCTAATAGATGTTTCTGGAATCACATTAAGCAA R G D L V F D F O V f P T K Y H S G R L L f C f V P G N E L I D V S G I t L K "0 GCAACTACTGCTCC TTGTGCAGTAATGGATATTACAGGAGTGCAGTCAACTTTGAGATTTCGTGTTCCCTGGATT TCTGACACTCCT TACAGAGTGAACAGGTATACAAAGTCAGCACAT A T T A P C A V M D I T G V O S T L R F R V P W I S D T P Y R V N R Y T K S A H CAGAAAGGTGAGTACACTGCCATTGGGAAGCTTATTGTGTATTGTTATAACAGATTGACCTCTCCTTCTAACGT TGCTTCCCATGTCAGAGTGAATGTTTATCTTTCAGCAATTAACTTG O K G E Y T A I G K L I V Y C Y N R L T S P S N V A S H V R V N V Y L S A I N L FIG. 1. Complete nucleotide and predicted amino acid sequence of cdna from wild-type HAV HM-175. Nucleotides at positions 6208, 6282, 6299, and 6301 have been corrected from the previously published sequence of this region (2). The nucleotides were sequenced from multiple determinations on complementary strands. The nucleotide at position 871 is C in phavlb39 and T in phavlb113 (changes to Ile codon); the nucleotide at position 2196 is G in phavlb39 and A in phavlb1307 (changes to Ile codon); the nucleotide at position 6216 is T in phavlb93 and C in phavlb24 (no change in agnino acid). Asterisks indicate stop codons. tween the strains reside in the loops, resulting in a similar reported to cause cytopathic changes in cell culture (44). The free energy for both structures. Compared with other sequence of the latter strain resembles the other three strains picornaviruses, the 5' terminal hairpin of HAV is much for the first 159 nucleotides; however, there is no detectable longer, resulting in a higher free energy (V. Rivera, personal homology for the remaining 29 nucleotides. communication). (iii) Coding region. The nucleotide sequence of the entire A large portion (498 nucleotides) of the 5' noncoding capsid region of three strains (HM-175, LA, and CR-326) and region sequence has been determined for HAV CR-326 (23). 95% of the capsid region of a fourth strain (HAS-15 [30]) has When sequences representing the two cell culture-adapted been determined. The amino acid differences for the capsid strains are compared, there is over 99% identity in the 5' region of the four strains are shown in Fig. 4. Strain HM-175 noncoding region sequences. Furthermore, both strains have most closely resembles the consensus sequence for the identical sequences in the last 280 nucleotides of this region. capsid region (five different amino acids; 99.4% identity). A small portion (188 nucleotides) of the 5' noncoding region The two most closely related strains in the capsid region are sequence has also been determined for a strain of HAV HM-175 and CR-326 (98.6% identity). The two most diver-

4 VOL. 61, 1987 NUCLEOTIDE SEQUENCE OF WILD-TYPE HEPATITIS A VIRUS VP3 >< VP1 1D (nt / oo / 300 oo) GAATGTTrTTGCTCCTCTTTATCATGCTATGGATGTTACTACACAAGTTGGAGATGATTCTGGAGGTTTTTCAACAACAGTTTCTACAGAACAGAATGTTCCAGATCCCCAAGTTGGTATA E C F A P L Y H A M D V T TT V G D D S G G F S T T V S T E O N V P D P O V G ACAACCATGAAAGATTTGAAAGGAAAAGCTAACAGAGGGAAAATGGATGTTTCAGGAGTACAAGCACCTGTGGGAGCTATCACAACAATTGAGGATCCAGTTTTAGC T T M K D L K G K A N R G K M D V S G V O A P V G A I T T I E D P V L A K K V P GAGACATTTCCTGAATTGAAACCTGGAGAATCCAGACATACATCAGATCATATGTCCATCTACAAGTTTATGGGAAGGTCTCATTTCTTGTGCACTTTTACATTCAATTCAATAATAAA E T F P E L K P G E S R H T S D H M S I Y K F M G R S H F L C T F T F N S N N K GAGTACACATTTCCTATAACCTTGTCTTCAACCTCTAATCCTCCTCATGGTTTGCCATCAACACTGAGGTGGTTTTTCAACTTGTTTCAGTTGTATAGAGGGCCTTTAGATCTGACAATT E Y T F P I T L S S T S N P P H G L P S T L R W F F N L F O L Y R G P L D L T I TCT I I T G A T D V D G M A W F T P V G L A V D T P W V E K E S A L S I D Y K T A L ATTATTACAGGAGCAACTGATGTAGATGGCATGGCCTGGTTCACTCCAGTAGGTCTTGCCGTTGATACTCCTTGGGTAGAGAAGGAGTCAGCTTTrGTCTATTGACT GGAGCTGTCAGATTTAACACAAGGAGAACAGGGAACATTCAGATTAGATTACCATGGTATTCTTATTTATATGCTGTGTCTGGAGCACTGGATGGTTTGGGTGACAAGACAGATTCTACA G A V R F N T R R T G N I Q I R L P W Y S Y L Y A V S G A L D G L G D K T D S T TTTGGATTGGTTTCTATTCAGATTGCAATTACAATCATTCTGATGAATACTTGTCTTTTAGTTGTTATTTGTCTGTCAICAGAACAATCAGAGTTTTATTTTCCCAGAGCTCCATTG0A0 fg L V S I O I A N Y N H S D E Y L S F S C Y L S V T E O S E F Y F P R A P L N VPI >< 2A TCAAATGCCATGTTATCCACTGAATCAATGATGAGCAGAATTGCAGCTGGAGACTTGGAGTCATCAGTGGATGATCCTAGATCAGAGGAAGATAAGATTTGAGAGTCATATAGAATGC S N A M L S T E S M M S R I A A G D L E S S V D D P R S E E D K R F E S H I E C (nt / oa / 189 ao) 3200 AGGAAGCCATATAAAGAACTGAGATTAGAAGTTGGGAACAAGACTCAAGTATGCTCAGGAAGAATTGTCAATGAAGTACTTCCACCCCCTAGGAATGAAGGGACTGTTTTCACAA R K P Y K E L R L E V G K O R L K Y A O E E L S N E V L P P P R K M K G L F S O GCCAAAATTTCTCTTTTTTATACTGAGGAGCATGAATAATGAAGTTTTCCTGGAGAGGTGTGACTGCTGATACTAGAGCTTTAAGGAGGTTTGGATTCTCTTTGGCCGCAGGCAGAAGT A K I S L F Y T E E H E I M K F S W R G V T A D T R A L R R F G F S L A A G R S GTGTGGACTCTTGAATGGATGCTGGGGTTCTTACTGGGAGACTGATTAGATTGAATGATGAGAATGGACAGAATGAAGGATGACAAGATTGTTTCATTGATTGAAGTTTACAAGT V W T L E M D A G V L T G R L I R L N D E K W T E M K D D K I V S L I E K F T S AACAAATATTGGTCCAAGTGAATTTCCCACATGGGATGTTGGATCTTGAAGAATTGCTGCCAATTCTAAGGATTTTCCTAACATGTCTGAACGGATTTGTGTTTCTTGCTGCATTGG N K Y W S K V N F P H G M L D L E E I A A N S K D F P N M S E T D L C F L L H W A >< 28 (nt / 0a / 107 oo) TTAAATCCAAGAATTA ATTTAGCAGATAG AATGCTTGGATTGTCTGGAGTTCAGGAAATTAAAGACAGGTGTTGGATTAATAGCAGAGTGTAGAACTTTCTTAGATTCTATTGCT L N P K K I N L A D R M L G L S G V Q E I K E Q G V G L I A E C R T F L D S I A GGAACTTTAAAATCTATGATGTTTGGATTTCATCATTCTGTGACTGTTGAAATTATAAACACTGTGCTCTGTTTTGTTAAGAGTGGAATTTTGCTTTATGTAATACAACAATTGAATCAG G T L K S M M F G F H H S V T V E I I N T V L C F V K S G I L L Y V I O O L N O GATGAACATTCTCACATAATTGGTTTGTTGAGAGTCATGAATTATGCAGATATTGGTTGTTCAGTTATTTCATGTGGCAAAGTTTTTTCCAAAATGCTGGAAACAGTCTTTAATTGGCAA D E H S H I I G L L R V M N Y A D I G C S V I S C G K V F S K M L E T V F N W O 28 >< 2C (nt / o / 335 oo) 4050 ATGGACTCCAGAATGATGGAGTTAAGGACTCAGAGTTTTTCCAACTGGTTAAGAGATATTTGTTCTGGGATCACCATTTTTAAAAACTTCAAGGATGCAATTTATTGGCTTTATACAAAA M D S R M M E L R T O S F S N W L R D I C S G I T I F K N F K D A I Y W L Y T K TTAAAGGACTTTTATGAAGTGAATTATGGCAAGAAGAAGGACATTTTAAATATTCTTAAAGATAACCAACAAxAAAATAGAGAAAGCCATTGAGGAAGCCGATGAATTTTGCATTTTGCAA L K D F Y E V N Y G K K K D I L N I L K D N O O K I E K A I E E A D E F C I L O FIG. 1.-Continued. 2400GTCC gent strains, LA and HAS-15 (97.3% identity), have prominent differences from the consensus sequence. HAS-15 has a deletion of six amino acids in VP1 (positions 26 to 31); this area was confirmed by nucleotide sequence from two different plasmids (30). When strain LA is compared with the sequences of the three other strains, it apparently has three clusters of frameshift mutations (VP1 amino acid positions 49 to 56, 168, and 229 to 232) resulting in the addition of an amino acid (between positions 54 and 55) and subsequent deletion of an amino acid (position 168). VP1 has the most amino acid diversity of the capsid proteins. In the capsid region of the four HAV strains, there are 10 amino acid differences from the consensus sequence that result in amino acids of different charges; 8 of the 10 amino acids are located in VP1. All of the amino acid insertions and deletions occur in VP1. Proteins 2A, 3B, and 3C have identical amino acid sequences in HAV HM-175 and LA. When the complete amino acid sequences of strains HM-175 and LA are compared, there are six amino acid differences resulting in amino acids of different charge in the P1 region; however, there are only two with different charges in the P2 region and two with different charges in the P3 region. Although the P1 region is the site of several amino acid insertions or deletions among different strains, there are no apparent amino acid insertions or deletions in the P2 or P3 region. Comparison of the amino acid sequences of strains HM-175 and LA shows that all of the dipeptide cleavage

5 54 COHEN ET AL. J. VIROL ATCCAAGATGTGGAAAAATTTGAACAGTATCAGAAAGGGGTTGACTTGATACAAAAATTGAGAACTGTTCATTCAATGGr,TCAGGTTGATCCAAATTTAATGGTTCATTTGTCACCTTTG I O D V E K F E O Y O K G V D L I O K L R T V H S M A O V D P N L M V H L S P L AGAGATTGTATAGCAAGAGTTCATCAGAAACTTAAAAACCTTGGATCTATAAATCAGGCAATGGTAACGAGATGTGAGCCAGTTGTTTGTTATTTATATGGCAAAGAGGGGGAGGAAAG R D C I A R V H O K L K N L G S I N O A M V T R C E P V V C Y L Y G K R G G G K AGCTTAACATCAATTGCATTGGCAACCAAAATTTGTAAACATTATGGTGTTGAGCCTGAAAAGAATATCTATACT^AACCTGTGGCTTCAGATTACTGGGATGGATATAGTGGACAATTA S L T S I A L A T K I C K H Y G V E P E K N I Y T K P V A S D Y W D G Y S G Q L GTTTGCATCATTGATGATATTGGCCAAAACACAACAGATGAGGATTGGTCAGATTTTTGTCAGTTAGTGTCAGGATGTCCAATGAGATTAAACATGGCCTCTCTTGAGGAGAAGGGTAGG V C I I D D I G O N T T D E D W S D F C O L V S G C P M R L N M A S L E E K G R CATTTTTCTTCTCCTTTTATAATAGCAACTTCAAATTGGTCAAATCCAAGTCCAAAAACAGTTTATGTTAAGGAAGCAATTGACCGCAGACTCCATTTCAAGGTTGAAGTTAAACCTGCT H F S S P F I I A T S N W S N P S P K T V Y V K E A I D R R L H F K V E V K P A TCATTTTTCAAAAATCCTCACAATGATATGTTGAATGTTAATTTAGCTAAAACAAATGATGCAMATCAAAGATATGTCTTGTGTTGATTTGATAATGGATGGACATAATGTTTCATTGATG S F F K N P H N D M L N V N L A K T N D A I K D M S C V D L I 'M D G H N V S L M C >< 3A (nt / / 74 aa) GATTTGCTCAGTTCTTTAGTCATGACAGTTGAAATTAGAACAACATGACTG AATTCATGGAGTTGTGGTCTCAGGGAATTTCAGATGATGATAATGATAGTGCAGTAGCTGAGTTT D L L S S L V M T V E I R K O N M T E F M E L W S O G I S D D D N D S A V A E F TTCCAGTCTTTTCCATCTGGTGAACCATCGAACTCTAATTATCTGGCTTTTTCCAATCTGTTACTAATCACAAGTGGGTTGCTGTGGGAGCTGCAGTTGGCATTCTTGGAGTGCTCGTT F O S F P S G E P S N S K L S G F F O S V T N H K W V A V G A A V G I L G V L V A >< 38 - VPg (nt / / 23 oc) GGAGGATGGTTTGTGTATAAGCATTTCTCCCGCAAGAGGAGGAACCAATCCCAGCTGAAGGGGTATATCATGGTGTAACTAAGCCCAAGCAAGTGATTAATTAGATGCAGATCCAGTA G G W F V Y K H F S R K E E E P I P A E G V Y H G V T K P K O V I K L D A D P V 36 >< 3C (nt / aa / 219 co) GAATCTCAGTCAACTTTGGAAATAGCAGGACTGGTTAGGAAGAACTTGGTTCAGTTTGGAGTTGGAGAGAGAATGGATGTGTGAGATGGGTTATGAATGCCTTGGGAGTGAMAGATGAT E S O S T L E I A G L V R K N L V O f G V G E K N G C V R W V M N A L G V K D D TGGCTGCTTGTGCCTTCCCATGCTTATAAATTTGAGAAAGATTATGAAATGATGGAGTTTTATTTTAATAGAGGTGGAACTTACTATTCAATTTCAGCTGGTAATGTTGTTATTCAATCT W" L L V P S H A Y K F E K D Y E M M E F Y F N R G G T Y Y S I S A G N V V I O S TTGGATGTGGGATTCCAGGATGTTGTTCTGATGAAGGTTCCTACAATTCCTAAGTTTAGAGATATTACTCAGCATTTTATTAAGAAAGGGGATGTGCCTAGAGCTTTGAATCGCCTGGCA L D V G F O D V V L M K V P T I P K F R D I T Q H F I K K G D V P R A L N R L A ACATTAGTGACACTGTVNATGGLCCCCTATGTTATTTCTGAGGGCCCACTLMAGATGGAGAGAGCTACTTATGTTCATYGVAAATGAGGTAC VCAGTTGATTT VCTGTG T L V T T V N G T P MI L I S E G P L K M E E K A T Y V H K K N D G T T V D L T V GATCAGGCATGGAGAGGAAGGCGAAGGTCTTCCTGGAATGTGTGGTGGGCCTTGGTTTCATCGAATCAATCTATACAGAATGCAATCTTGGATCCATGTTGCTGGAGGAAATCA D O A W R G K G E G L P G MC G G A L V S S N O S I O N A I L G I H V A G G N S C >< 3D (nt / 0o / 489 ca) 6000 AT TCTTGTTGCAAATTGGT TACTCAAGAAATGTTCCAAAATATTGATAAGAAAATTGAAGTCAGAGAATTATGAAAGTGGAGTTTACTCAGTGTTCAATGAATGTGGTCTCCAAAACG I L V A K L V T O E M F O N I D K K I E S O R I M K V E F T O C S M N V V S K T CTGAAATTGATCCAATGGCTGTGATGTTTTAArGTATTCA L F R K S P I Y H H I D K T M I N F P A A M P F S K A E I D P M A V M L S K Y S CTTTTTAGAAGAGTCCCATTTATCATCACATTGATAACCATGGATTATTTrCCTGCAGCTATGCCCTTTCTAArc TTACCTATTGTAGAAGAACCAGAGGATTATAAAGAGGCTTCAATTTTTTATCA^AAATbAAAAAGTGGGTAAGACTCAGTTAGTTGATGATTrTTTAGATCTTGATATGGCCATTACAGGG L P I V E E P E D Y K E A S I F Y O N K I V G K T O L V D D F L D L D M A I T G FIG. 1.-Continued. sites proposed above are conserved. The areas surrounding these cleavage sites are also identical, except for one amino acid at the 1D/2A junction (Fig. 4, VP1 amino acid 297). (iv) 3' Noncoding region. The complete sequence of the 3' noncoding region was determined for strains HM-175 and LA. The nucleotide sequences of the two strains are most divergent in the 3' noncoding region (88.9% identity). Both strains have a stop codon (UGA) followed six nucleotides later by a second stop codon (UAA). Comparison with other picornaviruses. HAV shows less homology with other picornaviruses than the non-hav picornaviruses show with each other. HAV is most homologous with encephalomyocarditis virus (EMCV); proteins 2C and 3C have 28 and 25% amino aci-d identity between the two picornaviruses. Part of the amino acid sequence from the carboxy portion of protein 2C for HAV and other picornaviruses is shown in Fig. 2G. If analogous with poliovirus protein 2C (41), HAV protein 2C may be involved in transcription. The carboxy portion of 2C is highly conserved among different picornaviruses (1). The amino acid at position 198 (Fig. 2G, arrow) has been shown to correlate with guanidine sensitivity for several picornaviruses (33). Poliovirus and rhinovirus type 14 (asparagine at position 198) are inhibited by guanidine; however, EMCV, foot-and-mouth disease virus, and HAV (glycine at position 198) are guanidine resistant. Protein 3C is a protease in poliovirus (15) and in EMCV (32). Two amino acids in 3C, cysteine and histidine (Fig. 2E, asterisks), are thought to be reactive residues of the functional site for the protease (1). These two amino acids

6 VOL. 61, 1987 NUCLEOTIDE SEQUENCE OF WILD-TYPE HEPATITIS A VIRUS GCCCCAGGAATTGATGCTATCAACATGGATTCATCTCCTGGATTTCCTTATGTCCAGGAGAAG TrGACCAAGAGArTTTAATTGGTTGGATGAAAGGTTTATTGCTGGGAGTTCAT A P G I D A I N M D S S P G F P Y V Q E K L T K R D L I W L D E N G L L L G V H CCAAGAT TGGCTCAGAGAATCT TAT TCAATACTGTCATGATGGAATTGTTCTGATT TGGATGTTGTT TTTACAACCTGTCCAAGATGAATTGAGACCATTAGAGAAGTGTTGGAA P R L A O R I L F N T V M M E N C S D L D V V F T T C P K D E L R P L E K V L E TCAAAAACAAGAGCTAT TGATGCTTGTCCTCTGGATTACTCAATTTTGTGCCGAATGTATTGGGGTCCAGCTATTAGTTAT TTTCATTTGAATCCAGGTTTCCATACAGGTGTTGCTATT S K T R A I D A C P L D Y S I L C R M Y W G P A I S Y F H L N P G F H T G V A I 665e 6700 GGCATAGATCCTGATAGACAGTGGGATGAATTATTTAAACAATGATuAAGATCGGAGATGTTGGTCTTGATTTAGATTTCTCTGCTTTTGATGCTAGTCTTAGTCCAT TTATGATTAGA G I D P D R O W D E L F K T M I R F G D V G L D L D F S A F D A S L S P F M I R GAAGCAGGTAGAATCATGAGTGAACTATCTGGAACTCCATCCCATTTTGGCACAGCTCTTATCAATACTATCATTTATTCCAAGCATTTGCTGTATAACTGTTGTTACCATGTCTGTGGT E A G R I M S E L S G T P S H F G T A L I N T I I Y S K H L L Y N C C Y H V C G TCAATGCCCTCTGGGTCTCCT TGTACAGCTTTGCTAAATTCAATTATTAATAATGTCAATTTGTATTATGTGTTTTCCAAGATATTTGGAAGTCTCCAGTTTTCTTTTGTCAGGCTTTG S M P S G S P C T A L L N S I I N N V N L Y Y V F S K I F G K S P V F F C O A L AAGATTCTCTGTTATGGAGATGATGTTTTAATAGTTTTCTCTCGAGATGTTCAGATTGATAATCTTGATTTGATTGGACAAAAAATTGTAGATGAGTTTAAGAAACTTGGCATGACAGCT K I L C Y G D D V L I V F S R D V O I D N L D L I G Q K I V D E F K K L G M T A ACTTCTGCTGACAAGAATGTACCTCAGCTGAAACCAGTTTCGGAATTGACTTTTCTCAAAAGATCTTTC MATTTGGTAGAGGATAGAMATTAGACCTGCAMATTTCGGAAAAAACAMATTTGG T S A D K N V P Q L K P V S E L T F L K R S F N L V E D R I R P A I S E K T I W TCTTTAATAGCATGGCAGAGAAGTAACGCTGAGTTTGAGCAGAATTTAGAAAATGCTCAGTGGTTTGCTTTTATGCATGGCTATGAGTTTTATCAGAAATTTTATTATTTTGTTCAGTCC S L I A W O R S N A E F E O N L E N A O W F A F M H G Y E F Y Q K F Y Y F V Q S D > TGTTTGGAGAAAGAGATGATAGAATACAGACTTAAATCTTATGATTGGTGGAGAATGAGATTTTATGACCAGTGTTTCATTTGTGACCTTTCATGATTTGTTTAAACAAATTTTCTTAAA C L E K E M I E Y R L K S Y D W W R M R F Y D Q C F I C D L S ATTTCTGAGGTTTGTTTATTTCTTTTATCAGTAAATAAAAAAAAAAAAAAA FIG. l.-continued. are conserved among several picornaviruses, including HAV. Protein 3D of HAV has 29% amino acid identity with 3D of poliovirus type 1 (Mahoney). Protein 3D has been identified as an RNA-dependent polymerase in poliovirus (24). HAV, like other picornaviruses, has a 14-amino-acid region (Fig. 2F) consisting of two aspartate residues surrounded by hydrophobic amino acids. This region is thought to be an active site or recognition site of RNA polymerases (17). Of the 10 proposed dipeptide cleavage sites used by HAV, 4 are shared with EMCV (VP4/VP2, 2A/2B, 2B/2C, and 2C/3A). Other picornaviruses have two or fewer dipeptide cleavage sites shared with HAV. DISCUSSION Sequence variation among different HAV strains has been examined previously by using ribonuclease T1 oligonucleotide mapping. With this procedure, the estimated variation in nucleotide sequences ranges from 0.8 to 10% among different strains (46). In contrast, comparison of cdnas from different HAV strains shows only 50 to 60% conservation of restriction endonuclease sites. When the complete nucleotide sequences of two strains (HM-175 and LA) are compared, there are 624 nucleotide differences (91.7% identity) and 34 amino acid differences (98.5% identity). Thus, restriction site mapping overestimated nucleotide variation, whereas oligonucleotide mapping more closely reflected actual sequence data. Nucleotide sequences of poliovirus types 1 and 3 have been determined for both vaccine and parent wild-type strains (18, 29, 35, 40, 43). For poliovirus type 2, sequences have been reported for a cell culture-adapted (vaccine) strain and a mouse-adapted (neurovirulent) strain (21, 43). The numbers of nucleotide differences between wild-type and vaccine strains of types 1, 2, and 3 are 57, 1308, and 10, respectively. The numbers of amino acid differences between the wild-type and vaccine strains of poliovirus types 1, 2, and 3 are 21, 83, and 3, respectively. Thus, the number of amino acid differences between the two HAV strains studied is similar to the number of amino acid differences between wild-type and attenuated variants of the same poliovirus strains. The majority of amino acid differences between HAV HM-175 and LA occur in the capsid region. Within this region, the highest variation is seen in VP1. When the capsid regions from all four HAV strains are compared, about half (15 of 31) of the amino acid differences are located in the N terminus of VP1. A similar pattern of variation is seen for poliovirus types 1 and 2 (21, 29). When the wild-type and vaccine strains of these viruses are compared, most of the amino acid differences are located in the capsid region, and about half of the amino acid differences in the capsid region are clustered in the N terminus of VP1 (5 of 12 amino acids for poliovirus type 1 and 15 of 22 for type 2). Presumably, different environmental influences (including adaptation to cell culture) may select for amino acid changes in VP1, the immunodominant protein of the virion. Two groups have attempted to locate antigenic sites in the capsid region of HAV. Emini et al. (8) proposed the location of three HAV antigenic sites by comparing surface probability profiles of HAV with those of poliovirus. A synthetic peptide corresponding to one of three sites induces anti- HAV neutralizing antibody. All four of the HAV strains described above have identical amino acid sequences in the three sites. Palmenberg (personal communication) has

7 56 COHEN ET AL. J. VIROL. A VP1 / 2A B 2A / 2B EMCV PRPT VFFPWPTSGDKIDMTPR AGVLMLESPNALDISRTGTL HVLIOFNHRGLEV HAV PRAP LNSNAMLSTE SMMSR IAAGDLESSVDDPRSEEDKRFZHIECRKPYKELRLEV RV2 PRPPRALEYTRAHRTN FKIEDRSIOTAIVTRPIITTAGPSDhM HVG NLIYRNLHL EMCV RP RKO VFO TZ:AA VSSMAOTLL PNDLAS K HAV AD RMLGLSGVOEIKEZ G LIAECRTFL DSIAGTLK RV14 ADIROLEC IAE E*LSDYITGLGRAFGVGFTDQIST K C 28/ 2C D 2C / 3A EMCV PTISLFQ SLPLKQVNDIFSLAKNLDW HAV RMMIEL R T'FSNWLRDICSGITIFKN RV14 PYIER &NOGWFRKFNDACNAAKGLEW EMCV HAV RV2 ISLVDVIERAVARIERKKKVLTTVOTLVA<PVDEVSFHS VSLMDLLSSLVMTVEIRKQNMTEFMELW * tisddndsa YSLAQVYNIMIEE DRRRROVVDVMTAIF WIDWKNPPP E 3C ACTIVE SITE AND 3C / 3D * 6 EMCV KGWCGSALLA DLGGSKKILGIHSAGSMGIAAASIVSOEMIRAVVNAFEPsALER LPDGP RIHVPRKTALRPT HAV PGMCGGALVSSNOSIONAILGIHVAGGNSILVAKLVTOEMFQNIDKKIESEIKW VEFTOC SMNWSKTLFRKS FMDV AGYCGGAVLA KDGADTFIVGTHSAGGNGVGYCSCVSRSMLLKWAHI DPEPH*tLIVDTRDVEERVHVMRKTKLAPT F EMCV HAV RV2 3D ACTIVE SITE 6*6 VKVLSYGDDLLVAT LKILCYGDDVLIVF LKIIAYGDDVIFSY G 2C: REGION OF GUANIDINE RESISTANCE MARKER V EMCV RCEPW I VLRGDAGOGKSLSSOV I AOAVSKT I F GROSVYSLPPDSDFFDGYENOFAA IMDDLGNPOGSDFTTFCOMVSTTNFLPNMASLERKGTPFTSQ HAV RCEPVVCYLYGKRGGGKSLTS I ALATK ICKHYGVEPEKN I YTKPVASDYWDGYSGOLVC II DD IGQNTTDEDWSDFCOLVSGCPMRLNMASLEEKGRHFS PV1 RI EPVCLLVHGSPGTGKSVATNLIARAIAER ENTSTYSLPPDPSHFDGYKOGWVIMDDLNONPOGADMKLFCOMVSTVEFIPPMASLEEKGI LFTSNYV FIG. 2. Selected amino acid alignments for HAV with other picornaviruses. Alignments were made as described in Materials and Methods and then truncated for this figure. Abbreviations: EMCV, encephalomyocarditis virus (31); RV2, rhinovirus type 2 (38), RV14, rhinovirus type 14 (3, 39), FMDV, foot-and-mouth disease virus type 01K (10). Amino acids conserved with HAV are indicated by colons, cleavage sites are indicated by reverse-type letters, putative active sites are indicated by asterisks, and the guanidine resistance site is indicated by an arrow. A to E, Amino acid alignments surrounding cleavage sites; E to G, alignments containing proposed functional sites. aligned the amino acid sequences of HAV capsid proteins with those of other picornaviruses, including two viruses (poliovirus type 1 [Mahoney] and rhinovirus type 14) for which the three-dimensional structures have been deter- TABLE 1. Amino acid and nucleotide differences between HAV HM-175 and LA No. of amino acid No. of nucleotide Region differences/total differences/total (% identity) (% identity) 5' Noncoding 28/734a (96.2) VP4 1/23 (96) 5/69 (93) VP2 2/222 (99.1) 62/666 (90.7) VP3 1/246 (99.6) 61/738 (91.7) VP1 15/300 (95.0) 89/900 (90.1) 2A 0/189 (100) 50/567 (91.2) 2B 0/107 (100) 24/321 (92.5) 2C 6/335 (98.2) 107/1,005 (89.4) 3A 2/74 (97) 15/222 (93.2) 3B 0/23 (100) 7/69 (90) 3C 0/219 (100) 59/657 (91.0) 3D 7/489 (98.5) 110/1,467 (92.5) 3' Noncoding 7/63b (89) Total 34/2,227 (98.5) 624/7,478c (91.7) a In the 5' noncoding region, HAV HM-175 has 734 nucleotides; strain LA has 733. b In the 3' noncoding region, HAV HM-175 has 63 nucleotides; strain LA has 64. c Of the 624 nucleotide differences, 616 are point mutations, 4 are insertions, and 4 are deletions. The latter insertions and deletions are all in VP1. mined (16, 36). Strain LA differs from the other three strains in one region (amino acids 229 to 232 of VP1) which aligns with a portion of cluster I on VP1 of poliovirus type 1 (residues 245 to 251 [16]). Cluster I is one of four discrete groups of amino acid residues which form external loops known to represent antigenic sites on poliovirus (16). The G G 'AU A..G A A U GIC G C C G U A uc G G GCGU{: G C G C G C A U G C A A U C G U G 1-U A-41 FIG. 3. Predicted secondary structure of RNA at 5' terminus of HAV HM-175. Bases surrounding the primary structure are those which differ in strain LA. The stem and loop structure has a AG of kcal/mol (1 cal = J) for strain HM-175 and a AG of kcal/mol for strain LA.

8 VOL. 61, 1987 NUCLEOTIDE SEQUENCE OF WILD-TYPE HEPATITIS A VIRUS 57 HM-175 LA CR-326 HAS-15 TTMKDL TTM r DL TTMKDL T T M K D L *-I--***-1I V AITTIE r s v q qql n V AITTIE V AIT TI E I I 11 V V a VI ' HM-175 LA CR-326 HAS-15 r DSv rg K DSIKG K v I IKs - -SIKG J.V'/ S T T T T E * E T E T k 11 VSIQE k f I f ee R VSIQq R V SIOE R \/1 L 'I N I ' l li l VP G G S G I VP2 222 aa VP3 246 aa FIG. 4. Location of amino acid differences between capsid proteins of HAV HM-175, LA, CR-326, and HAS-15. Letters indicate amino acids and numbers refer to the locations of amino acids within each capsid protein. Upper case letters indicate amino acids of consensus sequence. -, Amino acid not determined; 0, amino acid deleted. VPI 300 aa remaining amino acid differences in the capsid regions of the four HAV strains (Fig. 4) do not align with known antigenic sites on picornaviruses. Thus, the four different HAV strains may have similar antigenic sites. Similar findings have been obtained by virus cross-neutralization studies; using this procedure, no antigenic differences were found among HAV strains of different geographic origins (22). Comparison of HAV with other picornaviruses shows that HAV proteins are less homologous with other picornaviruses than the latter proteins are with each other. The proteins of HAV most homologous with their analogs in other picornaviruses are 2C and 3D. These two proteins are known to be involved in picornavirus RNA transcription, and one might expect conservation of these proteins among the different picornaviruses (Palmenberg, in press). Thus, although the genomes of HAV and other picornaviruses are organized quite similarly, their amino acid sequences are quite divergent. Adaptation of viruses to cell culture often results in attenuation; this phenomenon has been exploited to develop viral vaccines. After 10 passages of wild-type HM-175 HAV in monkey kidney cells, the virus becomes partially attenuated for chimpanzees (9). When the sequences of wild-type and different cell culture-adapted strains of HAV are compared, the nucleotide differences in the 5' noncoding region and amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation. Using recombinants of poliovirus cdna clones, the capsid region has been shown to be responsible for mouse neurovirulence of poliovirus 2 (Lansing) (21). Exact determination of HAV attenuation markers, however, will need to await sequence comparison and recombination experiments between wild-type and attenuated strains. We are currently pursuing these goals in an attempt to learn more about attenuation of HAV for development and evaluation of candidate live vaccines. ACKNOWLEDGMENTS The authors are indebted to Melanie Spriggs and Peter Collins for assistance with dideoxynucleotide sequence determination; to Ann Palmenberg, Victor Rivera, and Jacob Maizel for sequence alignment and RNA folding information; to Cosette Wheeler for useful discussions; to Stephen Feinstone for critical review of the manuscript; and to Linda Jordan for editorial assistance. We thank the Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, for the use of their VAX 11/750 computer. ADDENDUM Since submission of our paper, Ross et al. (B. C. Ross, B. N. Anderson, A. G. Coupelis, M. P. Chenoweth, and I. D. Gust, J. Gen. Virol. 67: , 1986) have reported the sequence of nucleotides 29 to 1002 from the 5'end of a cell culture-adapted isolate of HAV HM-175. There are eight nucleotide changes from wild-type HAV HM-175, all of which are in the 5' noncoding region. LITERATURE CITED 1. Argos, P., G. Kamer, M. J. H. Nicklin, and E. Wimmer Similarity in gene organization and homology between proteins of animal picomaviruses and a plant comovirus suggest common ancestry of these virus families. Nucleic Acids Res. 12:725

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