AU639209B2 - Enterically transmitted non-a/non-b hepatitis viral agent - Google Patents

Description

i 11 OPI DATE 12/01/90 AOJP DATE 15/02/90 APPLN- ID 38574 89 PCT NUMBER PCT/US89/02648

PCTI

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/12462 A61K 39/29, 39/42, C07H 17/00 C07K 7/10, 15/04, C12N 7/02 Al C12Q 1/68, 1/70, G01N 33/531 (43) International Publication Date: 28 December 1989 (28.12.89) GOIN 33/536, 33/543 (21) International Application Number: PCT/US89/02648 (74) Agent: NEELEY, Richard, Leydig, Voit Mayer, 350 Cambridge Avenue, Suite 200, Palo Alto, CA 94306 (22) International Filing Date: 16 June 1989 (16.06.89) (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 208,997 17 June 1988 (17.06.88) US pean patent), CH (European patent), DE (European pa- 336,672 11 April 1989 (11.04.89) US tent), DK, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European pa- -a1c.no @o a g r tent), NL (European patent), SE (European patent).

(71) Applicants: GENELABS, £&BQ9 [US/US]; 505 Penobscot Drive, Redwood City, CA 94063 (US).

THE GOVERNMENT OF THE UNITED STATES OF Published AMERICA as represented by THE SECRETARY OF With international search report.

THE DEPARTMENT OF HEALTH AND HUMAN Before the expiration of the time limit for amending the SERVICES AND HIS SUCCESSORS [US/US]; Wash- claims and to be republished in the event of the receipt of ington, DC 20231 amendments.

(72) Inventors: REYES, Gregory, R. 2112 St. Frances Drive, M Palo Alto, CA 94303 BRADLEY, Daniel, W. .r A 2938 Kelly Court, Lawrenceville, GA 30244 AuslIAuAN EC. 0

AUSTRAUAN

A A 104.. 12 JAN1990 6 3 9 2 09 PATENT OFFICE 639 U20 3 (54) Title: ENTERICALLY TRANSMITTED NON-A/NON-B HEPATITIS VIRAL AGENT BILE BILE (INFECTED) (NON-NFECTED) (57) Abstract Viral proteins derived from an enterically transmitted non-A/ EtoRI %RNA ERNA ion-B viral hepatitis agent are disclosed. In one embodiment, the pro- I 'tein is immunologically reactive with antibodies present in individuals t EcoR cDNA cDNA infected with the viral hepatitis agent. This protein is useful in a diag- LINKER nostic method for detecting infection by the enterically transmitted EcaRI RAIOLABEL agent. Also disclosed are DNA probes derivtd from a cloned se- cDNA cDNA quence of the viral agent. These probes are uset'ul for identifying and PROBE PROBE sequencing the entire viral agent and for assaying the presence of the LIGAT viral agent in an infected sample, using probe-specific amplification ECR-I E"oRI of virus-derived DNA fragments. Ao/ HINDI II EcoRI SELECT CLONES pZ T I EcR I. ISOLATE cDNA FRAGMENT EcoRIl

LIGATE

B

A pTZKFI

T

ISOLATE 1.1 \(ETI.1) EcrrI. ISOLATE FRAGMENT/

FRAGMENT

LAE/ PRIMER \ONta--I 100-300 bp ET-NANB SEQUENCE CLONE INTO it I PROBE

RECOMBINANT

PROTEINS

WO 89/12462 PC/US89/02648 ENTERICALLY TRANSMITTED NON-A/NON-B HEPATITIS VIRAL AGENT CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. Application Serial No. 336,672, filed April 11, 1989, which is a continuation-in-part of U.S.

Application Serial No. 208,997, filed June 17, 1988, both of which are herein incorporated by reference.

INTRODUCTION

Field of Invention This invention relates to recombinant proteins, genes, and gene probes and more specifically to such proteins and probes derived from an enterically transmitted nonA/nonB hepatitis viral agent, and to diagnostic methods and vaccine applications which employ the proteins and probes.

Background Enterically transmitted non-A/non-B (ET-NANB) hepatitis viral agent is the reported cause of hepatitis in several epidemics and sporadic cases in Asia, Africa, and the Indian subcontinent. Infection is usually by water contaminated with feces, although the virus may also spread by close physical contact.

The virus does not seem to cause chronic infection.

The viral etiology in ET-NANB has been demonstrated by infection of volunteers with pooled fecal isolates; Immune electron microscopy (IEM) studies have shown virus particles with 27-34 nm diameters in stools from infected individuals. The virus particles reacted with WO 89/12462 PCT/US89/0264? 2 antibodies in serum from infected individuals from geographically distinct regions, suggesting that a single viral agent or class is responsible for the majority of ET-NANB hepatitis seen worldwide. No antibody reaction was seen in serum from individuals infected with blood-transmitted NANB virus, indicating a different specificity between the two NANB types.

In addition to serological differences, the two types of NANB infection show distinct clinical differences. ET-NANB is characteristically an acute infection, often associated with fever and arthralgia, and with portal inflammation and associated bile stasis in liver biopsy specimens (Arankalle). Symptoms are usually resolved within six weeks. Blood-transmitted NANB, by contrast, produces a chronic infection in about 50% of the cases. Fever and arthralgia are rarely seen, and inflammation has a predominantly parenchymal distribution (Khuroo, 1980). The two viral agents can also be distinguished on the basis of primate host susceptibility. ET-NANB, but not the blood-transmitted agent, can be transmitted to cynomolgus monkeys. The blood-transmitted agent is more readily transmitted to chimpanzees than is ET-NANB (Bradley, 1987).

There have been major efforts worldwide to identify and clone viral genomic sequences associated with ET-NANB hepatitis. One goal of this effort, requiring virus-specific genomic sequences, is to identify and characterize the nature of the virus and its protein products. Another goal is to produce recombinant viral proteins which can be used in antibody-based diagnostic procedures and for a vaccine. Despite these efforts, viral sequences associated with ET-NANB hepatitis have not been successfully identified or cloned heretofore, nor have any virus-specific proteins been identified or produced.

WO 89/12462 PCI'/US89/02648 3 Relevant Literature Arankalle, et al., The Lancet, 550 (March 12, 1988).

Bradley, et al., J Gen. Virol., 69:1 (1988).

Bradley, D.W. et al., Proc. Nat. Acad. Sci., USA, 84:6277 (1987).

Gravelle, C.R. et al., J. Infect. Diseases, 131:167 (1975).

Kane, et al., JAMA, 252:3140 (1984).

Khuroo, Am. J. Med., 48:818 (1980).

Khuroo, et al., Am. J. Med., 68:818 (1983).

Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982).

Seto, et al., Lancet, 11:941 (1984).

Sreenivasan, M.A.m et al., J. Gen. Virol., 65:1005 (1984).

Tabor, et al., J. Infect. Dis., 140:789 (1979).

SUMMARY OF THE INVENTION Novel compositions, as well as methods of preparation and use of the compositions are provided, where the compositions comprise viral proteins and fragments thereof derived from the viral agent for ET- NANB. Methods for preparation of ET-NANB viral proteins include isolating ET-NANB genomic sequences which are then cloned and expressed in a host cell.

The resultant recombinant viral proteins find use as diagnostic agents and as vaccines. The genomic sequences and fragments thereof find use in preparing ET-NANB viral proteins and as probes for virus detection.

WO 89/12462 PCT/US89/02648 4 4 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows vector constructions and manipulations used in obtaining and sequencing cloned ET-NANB fragment; and Figures 2A-2B are representations of Southern blots in which a radiolabeled ET-NANB probe was hybridized with amplified cDNA fragments prepared from RNA isolated from infected and non-infected (N) bile sources and from infected and noninfected stool-sample sources (2B).

DESCRIPTION OF SPECIFIC EMBODIMENTS' Novel compositions comprising generic sequences and fragments thereof derived from the viral agent for ET-NANB are provided, together with recombinant viral proteins produced using the genomic sequences and methods of using these compositions.

The genome of the ET-NANB viral agent is identified as containing a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KF1 (ET1.1) carried in E. coli strain BB4 and having ATCC deposit no. 67717. Initial studies sequenced the two terminal regions of the insert and an intermediate region. The 5'-end region of this insert contains the sequence: 1 42 GAT GGA AGG CAC TAA TCT GGC AAG ACC TGT CCC TGT TGC AGC 43 84 TGT TCT ACC ACC CTG CCC CGA GCT CGA ACA GGG CCT TCT CTA 126 CCT GCC CCA GGA GCT CAC ACA CCC TGT GAT AGT GTC GTA ACA 127 168 TTT GAA TTA ACA GAC ATT GTG CAC TGC CGC ATG GCC GCC CCG WO 89/12462 PCT/US89/02648 169 210 AGC CAG CGC A-AG GCC GTG CTG TCC ACA CTC GTG GGC CGC TAC 211

GGC.

An intermediate region has the sequence: a 691 731 CTA GAG TGT GCT ATT ATG GAG GAG TGT GGG ATG CCG CAG TGG 733 774 CTC ATC CGC CTG TAT CAC CTT ATA AGG TCT GCG TGG ATC TTG 775 816 CAG CCC CCG AAG GAG TCT CTG CGA GGG ITT TGG AAG AAA CAC 817 858 TCC GGT GAG CCC GGC ACT CTT CTA TGG AAT ACT GTC TGG AAT 859 900 ATG GCC GTT ATT ACC CAC TGT TAT GAC TTC CGC GAT TTT GAG 90. 942 GTG GCT CCC TTT AAA GGT GAT GAT TCG ATA GTG CTT TGC AGT 943 984 GAG TAT CGT GAG ACT CCA GGA GCT GCT GTC CTG ATC CCC GGC 985 1026 TGT GGC TTG AAG TTG AAG GTA GAT TTC CCC CCG ATC GGT TTG 1027

TAT.

WO 89/12462 PCT/US89/0264~ 6 The 3'-end region contains the sequence: 1191 1232 TGA GTA GAG GAT GTT GTT TCC CGT GTT TAT GGG GTT TCC CCT 1233 1274 GGA CTC GTT CAT AAC CTG ATT GGC ATG CTA CAG GCT GTT GCT 1275 1316 GAT GGC AAG GCA CAT TTC ACT GAG TCA GTA AAA CCA GTG CTC 1317 1327 GAC CGG AAT TC.

Additional work has provided the entire sequence, in both directions, as set forth below. The sequence of both strands is provided, since it is not known in which strand the gene is located. However, the sequence in one direction has been designated as the "forward" sequence because of statistical similarities to known proteins. This sequence is set forth below along with the three possible translation sequences.

There is one long open reading frame that starts at nucleotide 145 with an isoleucine and extends to the end of the sequence. The two other reading frames have many termination codons. Standard abbreviations for nucleotides and amino acids are used here and elsewhere in this specification.

Forward Seauence R I P P T D G R H Z S G K T C P C C S C E F R Q L M E G T N L A R P V P V A A V N S A N Z W K A LI WQ DL S L L Q L F 1 11 21 31 41 51

CGAATTCCGCCAACTGATGGAAGGCACTAATCTGGCAAGACCTGTCCCTGTTGCAGCTGT

WO 89/12462PC7U8/24 PCT/US89/02648 7 S T T L P R A R T G P S L P AP G A H H L P P C FE L E Q G L L Y L P Q E L T T Y H PA P S S N R A F S T C P R S S P P 61. 71 82. 91 101 il1 TCTACCACCCTGCCCCGAG CTCGAACAGGG CCTTCTCTACCTG CC CCAGGAC CTCACCAC L Z Z C R N I Z I N R. H C A L P H C R P C D S V V T F E LT D IV H C R M A A P V I V S Z H L N Z Q T L C T A A W P P R 121 131 141 151 161 171 CTGTGATAGTGTCGTA.ACATTTGAATTAACAGACATTGTGCACTGC CCCATC C CCCCCC EP A Q GCR A V HT R G P L R R R T K L S Q R K A V L S T L V C RY G. VA Q S S A S A R P C C P H S W A A T A S H K A L 181 191 201 211 221 231

GAGCCAGCGCAAGGCCCTCCTCTCCACACTCGTGGGCCGCTACGGCGTCGCACAAAGCTC

Y N A S H S D V R D S L A R F IFP A I G T M L P T L M F A T L S P V L S R F L A Q C F P L Z C S R L S R P F Y P G H W P 241 251 261 271 281 291

TACAATGCTTCCCACTCTGATCTTCGCGACTCTCTCGCCCGTTTTATCCCGGCCATTGGC

P V QV T T C EL YE L VEA MV E KG P Y R L QL V N C T S Z W RP W S R RA P. T G Y N L Z I V RA SCG G H G R 2 G P 301 311 321 331 341 351

CCCGTACAGGTTACAACTTGTGAATTGTACGAGCTACTGGAGGCCATGGTCGAGAAGGC

WO 89/12462 WO 8912462PCT/US89/02648 8 Q D G S A V L E L D L C N R D V S R I T R M A P P S L S L I F A T V T C P G S P G W L R R P Z A Z S L Q P Z R V Q D HL 362. 371. 381 391 401 412.

CAGGATGGCTCCGCCGTCCTTGAGCTTGATCTTTGCAACCGTGACGTGTCCAGGATCACC

F F Q K D C N K F T T G ET I A H G K V S S R K I VT S S P Q V RP L PM VK W L PE R L Z Q V H HR Z D H C P W Z S G 4221 4321 442. 451 461 471

TTCTTCCAGAAAGATTGTAACAAGTTCACCACAGGTGAGACCATTGCCCATGGTAAAGTG

G Q G I S A WS K T F C AL F G P W F R AR A S R P GA R P S A PS L AL GS A P G H LG L E QDL LR P L W P L V P R 481 491. 501 511. 521 531

GGCCAGGGCATCTCGGCCTGGAGCAAGACCTTCTGCGCCCTCTTTGGCCCTTGGTTCCGC

AlI EK AlI LA L L P Q G V F Y G D A F L L R R L F W P C S L R V C F T V M P L Y Z E G Y SG PA P S G C VL R Z C L Z 541 551 561 571 581 591

GCTATTGAGAAGGCTATTCTGGCCCTGCTCCCTCAGGGTGTGTTTTACGGTGATGCCTTT

D D T V F S AA V AA A KAS M V F E N M TP S S RR L WP Q QR HP WC L R M Z H R L L G GC G R S KG I HG V Z E Z 601 611 621 631 641 651

GATGACACCGTCTTCTCGGCGGCTGTGGCCGCAGCAAAGGCATCCATGGTGTTTGAGA.AT

WO 89/1 2462 PTU8/24 PCY/US89/02648 9 D F S E F D S T Q N N F S L G L E C A I T F L S L T P P R I T F LW V Z S V L L L F Z V Z L H P E Z L F S G S R V C Y Y 661 671 681 691 701 711.

GACTTTTCTGAGTTTGACTCCACCCAGAATAACTTTTCTCTGGGTCTAGAGTGTGCTATT

M4 E EC G M P Q W L I R L Y H L I R S A W R S V G CR S G S S A C I T L Z G L R G G '71 W D A A V A H P P V S P Y K V C V 721 731 741 751 761 771

ATGGAGGAGTGTGGGATGCCGCAGTGGCTCATCCGCCTGTATCACCTTATAAGGTCTGCG

W I L QA P K E S L RG FW KK H S G E G S CR P R R S L CEG F GR N T P V S D L A G P E G V SA R VL E E T L R Z A 781 791 801 811 821 831

TGGATCTTGCAGGCCCCGAAGGAGTCTCTGCGAGGGTTTTGGAAGAAACACTCCGGTGAG

P G T L L W N T V WN M AV I T H C Y D P AL F Y G IL S G I W P L L P T V M T R H S S M E YC L EY G RY Y P L L Z L 841 851 861 871 881 891

CCCGGCACTCTTCTATGGAATACTGTCTGGAATATGGCCGTTATTACCCACTGTTATGAC

F R D F Q V -A A F K G D D S I V L C S E S A I F R WL PL KV M IR Z C F AV S P R F S G G C L Z R Z Z FOD S AL Q Z V 901 911 921 931 941 951

TTCCGCGATTTTCAGGTGGCTGCCTTTAAAGGTGATGATTCGATAGTGCTTTGCAGTGAG

WO 89/12462 WO 8912462PCf/US89/0264 Y R Q S P G A A V L I A G C G L K L K V I V R V Q E L L S Z S PA V A Z S Z R Z S S E S R S C C P D R R L W L E VE G R 961 971 981. 991 1001 1011 TATCGTCAkGAGTCCAGGAGCTGCTGTCCTGATCGCCGGCTGTGGCTTGAAGTTGAAGGTA D F R P I G L Y A G V V V A P G L GA L I SA R S V C M QV L W W P P AL A R S F PP DR F V CRCC GGPR P WRAP 1021 1031 1041 1051. 1061 1071.

GATTTCCGCCCGATCGGTTTGTATGCAGGTGTTGTGGTGGCCCCCGGCCTTGGCGCGCTC

P D V V R F A G R L T E K N W G P G P E L M L C A SP A G L P R R IG A LA L S Z C CA L R R P A Y R EE L G P W P Z A 1081 1091 1101 1111 1121 1131

CCTGATGTTGTGCGCTTCGCCGGCCGGCTTACCGAGAAGAATTGGGGCCCTGGCCCTGAG

R A E Q L R L A V S D F L R K L T N VA G R S SS A SL LV I S S A SS R M Z L G G AA P P R C Z Z F P P QARH E C S S 1141 1151 11.61 1171 1181 1191

CGGGCGGAGCAGCTCCGCCTCGCTGTTAGTGATTTCCTCCGCAAGCTCACGAATGTAGCT

Q M C V D V VS RV YG V S P G L V HN R C V W M L F P V F MG F P L D SF1I T D V C G C C -F P C L W G F P W T R S Z P 1201 1211 1221 1231 1241 12531

CAGATGTGTGTGGATGTTGTTTCCCGTGTTTATGGGGTTTCCCCTGGACTCGTTCATAAC

WO 89/12462 WO 8912462PCT/US89/02648 L I G M L Q A VA DG K A H F T E S V K Z L A CY R L L L MA R H I S L S Q Z N D W HA T G C C Z W Q G T F H Z V S K T 1261 1271 1281 1291 1301 1311

CTGATTGGCATGCTACAGGCTGTTGCTGATGGCAAGGCACATTTCACTGAGTCAGTAAAA

P V L D R N S S Q C S T G I R S A R P E F E 1321 1331 1341

CCAGTGCTCGACCGGAATTCGAGC

The complimentary strand, referred to here as the "reverse sequence," is set forth below in the same manner as the forward.sequence set forth above.

Several open reading frames, shorter than the long open' reading frame found in the forward sequence, can be seen in this reverse sequence.

Reverse Seauence A R I P V EH WF Y Z L S E M C LAI S L E F R S S T G F T D S V K C A L-P S A S N S G R A LV L LTQ Z NV PC HQ Q 1 11. 21 31 41 51

GCTCGAATTCCGGTCGAGCACTGGTTTTACTGACTCAGTGAAATGTGCCTTGCCATCAGC

N S L Z H A N Q VMN E S R G N P IN T T A C S M P I R L Z T SP GE T P Z T R Q P V A C Q S G Y E R V 'Q G K P H K H G 61 71. 81 91 101 ill

AACAGCCTGTAGCATGCCA.ATCAGGTTATGAACGAGTCCAGGGGAAACCCCATAAACACG

WO 89/12462 PTU8/24 PCT/US89/026-0 12 G N N I H T H 1, S Y I R E L A E E I T N E T T S T H. I Z A T F V S L R R K S L T K Q H P H T S E L H S Z A C G G N H Z Q 121 131 141 151 161 171

GGAAACAACATCCACACACATCTGAGCTACATTCGTGAGCTTGCGGAGGAAA.TCACTAAC

S EALE L L R P L R A R AP I LL G K P A R R S C S A R S G P G P Q F F S V S R R G G A A PP A Q G Q G P N S S R ZA G 181 191 201 211 221 231

AGCGAGGCGGAGCTGCTCCGCCCGCTCAGGGCCAGGGCCCCAATTCTTCTCGGTAAGCCG

AG E A H N IR E R A K A G G H H N T C P A K R T T S G S AP R P CA T T T P A R R S A Q HQ GA R Q GRCG P P QH L H 241 251 261 271 281 291

GCCGGCGAAGCGCACAACATCAGGGAGCGCGCCAAGGCCGGGGCCACCACAACACCTGC

I Q T D AA E I Y L Q L QA T AG D Q D Y K P IG R K S T F N F K P Q P A IR T T N R S G GN L P S T S S HS R R S G Q 301 311 321 331 341 351

ATACAAACCGATCGGGCGGAAATCTACCTTCAAC!TTCAAGCCACAGCCGGCGATCAGGAC

S S S W T L T I L TA KHY R I I T F K A A P G L Z R Y S L S ST I E S S P L K Q L L D S D D T H C K A L S N H H L Z R 361 371 381 391 401 -111

AGCAGCTCCTGGACTCTGACGATACTCACTGCAAAGCACTATCGAATCATCACCTTTAAA

WO 89/12462 WO 89/1242ICT/US89/02649 13 G S H L K I A E V I T V G N N G H I P D A A 'T Z K S R K S Z Q W V I T A I F Q T Q P P E N R. G S H N S G Z Z R P Y S R Q 421 4321 4421 4521 4621 471

GGCAGCCACCTGAAAATCGCGGAAGTCATAACAGTGGGTAATAACGGCCATATTCCAGAC

SI P Z K SA G L T G V F L P K P S Q R V F H R R V P G S PE C F F Q N P R R D Y S I E EC R A H R S V S S K T L A E T 481 4921 501 5121 521 531

AGTATTCCATAGA.AGAGTGCCGGGCTCACCGGAGTGTTTCTTCCAAAACCCTCGCAGAGA

L L RG L Q D P R RP Y KV I QA D E P S F G A C K I H A D L I R Z Y R. P.M S H P S G P AR S T Q T L Z G D T G G Z A T 541 551 561 571 581 591

CTCCTTCGGGGCCTGCAAGATCCACG--CAGACCTTATA-AGGTGATACAGGCGGATGAGCCA

L R HP T L L H N T L Z T QR K V I L C G I P H S S I I A HS R P R E K L F W A A S H T P P Z H T L D P E K S Y S G 6021 611 621 631 641 651 CTG CGG CAT CC CACACT CCTC CATAATAGCACACTCTAGAC CCAGAG AAAAGTTATT CTG G G V K L P. K V I L K H H. G C L C C G H V E S N S E K S F S NT MD A F A A A T W S QT Q K S F S QTP W MP L L R. P Q 661 671 681 691 701 711 GGTGGAGTCA.AACTCAGAAAAGTCATTCTCAAACACCATGG,GCCTTTGCTGCGG CCAC WO 89/12462 WO 8912462PCTIUS89/0264,8 14 S P. RE D G V I K(G I T V K H T L P. E Q A A E K T V S S K A SP Z N T P Z G S R P PRPR C HQRHHR KT HP E GAG 721 731 741 751 761 771 AGCCGC CGAGAAGACGGTGTCATCAA.AGGCATCACCGTAAAACACACCCTGAGGGAGCAG G Q N S L L N S A E P R A KE G A E G L A P. I A F S I A P. N Q G P K P. A Q K V L P E Z P S Q Z R G T K G Q R G P.R R S C 781 791 801 811 821 831

GGCCAGAATAGCCTTCTCATAGCGCGGAACCA.AGGGCCAAAGAGGGCGCAGAAGGTCTT

A PGR RDALAHF TMG NG L TC GE L Q A E M P W P TL P W AM V S P V V N S R P R C P G P L Y HG Q W S H L W Z T 841 851 861 871 881 891

GCTCCAGGCCGAGATGCCCTGGCCCACTTTACCATGGGCAATGGTCTCACCTGTGGTGAA

T V T I F L E E GD P G H V T V A K I K L L Q S F W K KVI L DT S R L Q R S S C Y N L S G P.R Z S W T R H G C K D Q A 901. 911 921 931 941 951

CTTGTTACAATCTTTCTGGAAGAAGGTGATCCTGGACACGTCACGGTTGCAAAGATCAAG

L K DG G A I LA L LD HG L H Z L V Q S P.T A E P S WP F ST M AS T SS Y N Q G R P. S H P G P S P. P W P P L P. T I 961 971 981 991 1001 1011

CTCAAGGACGGCGGAGCCATCCTGGCCCTTCTCGACCATGGCCTCCACTAGCTCGTACA

Wo 89/12462 WO $9/12462 IJ/S89/O2648 F T S C N L Y G A NG R D K T G E R V A S Q V V T C T G P M AG I1K R AR E S R H K L Z P V RG Q W PG ZN G RE S R E 10221 1031. 10421 1051 1061 1071

TTCACAAGTTGTAACCTGTACGGGGCCAATGGCCGGGATAAAACGGGCGAGAGAGTCGCG

N I R V G S I V E L CA T P Z R P T S V T S EW E A L ZS F V R R R S G P R V W H Q S G K H C R A L C D A V A A H E C G 1.081 1091 1101 1111 1121 1131

AACATCAGAGTGGGAAGCATTGTAGAGCTTTGTGCGACGCCGTAGCGGCCCACGAGTGTG

D S T A L RW L G A A MR Q C T M S V N T AR P C A G -S G RP C G S AQ C L L I Q H G L A L A R G G H A AV H N V C Z F 1141 1151 1161 1171 1181 1191

GACAGCACGGCCTTGCGCTGGCTCGGGGCGGCCATGCGGCAGTGCACAATGTCTGTTAAT

S N V T T L S Q VVS S WG R Z R RP C Q ML RHY H RW ZA PG A GR E G PV K C Y D T I T G GE L L GQ V E K A L F 1201 1211 1221 1231 1241 1251 TCAAATGTTACGACACTATCACAGGTGGTGAGCTCCTGGGGCAGGTAGAGA-aGGCCCTGT G ~G G RT AA TG T G LA RL V R A R G R V V E Q L Q Q G Q V L P D Z C E L G A G W Z N S C N R DR S C Q I S A 1261 1271 1281 1291 1301 1311

TCGAGCTCGGGGCAGGGTGGTAGAACAGCTGCAACAGGGACAGGTCTTGCCAGATTAGTG

WO 89/12462 WO 89/12462 PM/US$9 I02W4 16 P SI S W R N S L P S V G G. I FEHQ L A E F 1321 1331 1341

CCTTCCATCAGTTGGCGGAATTCG

Identity of this sequence with sequences in etiologic agents has been confirmed by locating a corresponding sequence in a viral strain isolated in Burma. The Burmese isolate contains the following sequence of nucleotides.

Non-A Non-B .ET: Burmese strain 1 CGGTTGTTCA GTACCAGTTT ACTGCAGGTG TGCCTGGATC CGGCAAGTCC 51 CGCTCTATCA 101 GTTGCGTAAT 151 CTGCCGCCAG 201 TCCCTCCCCC 251 CCACCTTCTT 301 CTGGGCTCGT' 351. ATGTTACCCA 401 TACCCCATGA 451 TGAGCCTGCC 501 CCAACCCCGG 551 GAGACCACTA 601 TCGGGCTCAT 651 TCATTGACGC 701 GTTA.ATAACT 751 AGTTATTCCC 801 TCCCGCCGTC 851 GGCCACAGAC 901 CGAACAGGGC 951 TCGTAACATI

CCCAAGCCGA

GCCTGGCGCC

AGTCACCCAG

CTCACCTGCT

GGCGCCCCGA

CCC CG CCAT C

TCGCTGCCCT

TCCAGACCAC

GTCGGGCAGA

CT CAG TGAC G

TTATTGCCAC

GCCATTGTTG

ACCAGGCCTG

TTTTCCTCGC

CGTGGCAACC

TTGCCAGATT

CTGTCCCTGT

CTTCTCTACC

TGAATTAACA

rGTGGACGTT

GTCGCGGCTT

GGGCGCCGGG

GCTGCTCCAC

PCCAGATCCC

AGGC CCGACT

GCGGATGTAT

TAGCCGGGTT

AACTAGTGTT

GTCCACGAGG

AGCAGATGCC

CTCTGACGCG

CTTCGCGAGG

TGGTCGCGAA

CTGACGCCAA

AGTGCCTTCC

TGCAGCTGTT

TGCCCCAGGA

GACATTGTGC

GTCGTGGTCC

TGCTGCTTTT

TTGTCATTGA

PTGCAGCGGG

AGCCATCGAC

TAG CC CCACC

GCGAGCTCAT

CTCCGTTCGT

CACCCAGGCG

CGCAGGGCGC

CGGGGCCTTA

CCACACTGAG

TGGGCATCTC

ATTGGTCACC

TGTTGACACC

ATCAGTTGGC

CTACCACCCT

GCTCACCACC

ACTGCCGCAT

CGACGCGTGA

PCCCCGCATA

TGAGGCTCCA

CCGCCACCGT

TTTGAGCACG

TCCTGGTGGC

CCGTGGTGCA

TGTTCTGGGG

GCCAAGGCCG

TACCTACACG

TTCAGTCGTC

AAGTGCGTCA

CGATGCAATC

AGCGCCCATC

CTGGCTGCCT

TGAGGAGCTT

GCCCCGAGCT

TGTGATAGTG

GGCCGCCCCG

WO 89/12462PC/U9064 13CT/US89/02648 1001 AGCCAGCGCA AGGCCGTGCT 1051 ACAA AGCTCT ACAATGCTTC 1101 1153.

1201 1251.

1301 1351 1401.

1451 1503.

1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2102 2153 220] 225] 2301 235:.

240: 245: 250.

TTTTATCCCG

AGCTAGTGGA

GAG CTTGATC AG AT TGCTAA C

GCCAGGGCAT

TGGTTCCGCG

GTTTTACGGT

CAGCAAAGGC

ACCCAGAATA

TGGGATGCCG

GGATQTTGCA

TCCGGTGAGC

TATTACCCAC

GTGATGATTC

GCTGTCCTGA

GATCGGTTTG

CTGATGTTGT

GGCCCTGAGC

CAAGCTCACG

ATGGGGTTTC

*GTTG3CTGATC

*CTTGACAAA'I

*TGCGCCCATC

*CCTCATGTT9 L GCCGCCGTCC L GACCGGGTT( L. CCCCTTCGCC I. GCCAACCCG( 3. CCCGCCGTT(

GCCATTGGCC

GGCCATGGTC

TTTGCAACCG

AAGTTCACC.

CTCGGCCTGG

CTATTGAGAA

GATGCCTTTG

ATC CATGGTG

ACTTTTCTCT

CAGTGGCTCA

GGCCCCGAAG

CCGGCACTCT

TGTTATGACT

GATAGTGCTT

TCGCCGGCTG

TATG CAGGTG

GCGCTTCGCC

GGGCGGAGCA

M.TGTAGCTC

CCCTGGACTC

GCAAGGCACP,

TCAATCTTGI

GGTTCGCGAC

'TTGCCTATGC

TGGGCGGCGC

SATTCTCAGC(

2CCCGATGTCI 2CCGACCACT( I CCTCACGTC( GTCCACACTC G CCACTCTGAT G CCGTACAGGT TI GAGAAGGGCC P~ TGACGTGTCC P~ CAGGTGAGAC C AGCAAGA.CCT t

GGCTATTCTG

ATGACACCGT

TTTGAGAATG

GGGTCTAGAG

TCCGCCTGTA

GAGTCTCTGC

TCTATGGAAT

TCCGCGATTT

TGCAGTGAGT

TGGCTTGAAG

TTGTGGTGGC

GGCCGGCTTA

GCTC CCCCTC

AGATGTGTGT

GTTCATAACC

TTTCACTGAC

GTCCGGTGGA

'2"ATGCGCCCT

TGCCCGCGCC

AGCGGCGGTT

SCTTCGCAATC

k~ CCGCTGCGGC

:GCGTCCGCTT

3 TAGLCCTACC TGCGCCGCT A ~TTCCCGACT C 'ACAACTTCT G ~GGATGGCTC C ~GGATCACCT 93 :ATTCCCCAT C

CTGCGCCCT

;CCCTGCTCC

:TTCTCGGCG

%CTTTTCTGA

rGTGCTATTA flCACCTTATA

GAGGGTTTTG

ACTGTCTGCA

T CAGCT C CCT

ATCGTCAGAG

TTGAAGGTAG

CCCCGGCCTT

CCGAGAAGAA

GCTGTTAGTG

GGATGTTGTT

TCATTGG CAT

TCAGTAAAAC

ATGAATAACA

CCC CCTATTT

ACCGCCCGGT

CCGGCGGTGG

CCCTATATTC

CGGGGCTGGA

GGCGTGACCA

ACAGCTGGGG

CGGCGTCC

:TCTCGCCCC

AATTGTACG

GCCGTCCTT

'CTTCCAGAA

GTA.AAGTGG

:TTTGCCCCT

:TCAGCCTGT

3CTGTGGCCG

GTTTGACTCC

TGGAGGAGTC

.GGTCTGCGT

GAAGAAACAC

ATATGGCCCT

GCCTTTAAAG

TCCAGGACCT

ATTTC CG CC C

GGCGCGCTCC

TTGGGGCCCT

ATTTCCTCCG

TCCCGTGTTT

GCTACAGGCT

CAGTCCTCGA

TGTCTTTTGC

TGTTGCTGCT

CAGCCGTCTG

TTTCTGGGGT

ATCCAACCAA

CCTCGTGTTC

GGCCCAGCGC

CCGCGCCGCT

2551 AACCGCGGTC CCTCCGGCCC CG The ability of the methods described herein to isolate and identify genetic material from other NANB hepatitis strains has been confirmed by identifying genetic material from an isolate obtained in Menico.

WO 89/12462 WO 8912462PCT/US89/02648, 18 The sequence of this isolate was about 75% identical to the ET1.1 sequence set forth above. The sequence was identified by hybridization using the conditions set forth in Section II.B below. A partial cDNA sequence consisting of 1661 nucleotides is set forth below.

Non-A Non-B ET: Mexican Strain 1 51 101 151 201 250 301 350 401 451 501 551 601 651 701 752 802 901 100: 105:.

110, 115 120 125 130 135

GCGGCCGCTC

TAAGCAAGGA

CAGATG CCAT

CAGAGACCAT

CTTGCGGCGT

TGAGGAGCTG

GAGCTTGAGC

CAGTGTTGTG

CCCCTAGCCA

AGACGCACAA

TGCGCGCTTT

TCTTTGAGCT

GTCCTCGAGT

C CAGAAGGAT

*AAGTCGGTCA

*GGCCCCTGG

t

I

*AAGCTGTGT'I

*GTGGCTGGC.

LTGACTCGAC9 L AAGAGTGTGC L TCGGCGTGGI I. GAAGCATTC' 1 TGGCAATCtkr 1 TTCAAGGGC( 1 AGGCGCCGG' 1 TCCGGCCGA' 1 GCCCTACCC 1 GGGGCCTGA

GGTGGGGTTC

ATTA.ATTCGC

TGTTAATAAT

CGGTCATTCC

TTCCACCTTC

GGCCACCGGC

AGGGCCTTCT

ACATTTGAG C

AAGGAAAGCT

GGCTTTATGA

ATTCCCACTC

TGTAGAGGCG

TGGATTTGTG

TGTAACAAGT

GGGTATCTCC

TCCGTTGCGA

CTACGGGGA7

CCAGCCATGC

CAGAATAAC'I

TATGCCCCAC

k. TCCTGCAGGC P' GGTGAGCCGC r' TGCCCATTG( "i ACGACTCGG' P' TCGCTTATA( T TGGGCTGTA! G ATGTCGTTC T CCGGAGCGG

CACCTCTCTT

GGCCGCTCGT

TTCTTCCTTT

GCGAGCAAC C

ATGCCA.AATA

GGCGCCGGTG

CTATCTGCCA

TAACTGACAT

GTTTTGTCCA

TGCGGGTCAC

TCGGGCGGGT

ATGGTGGA.GA

CAGCCGAGAT

TCACGACCGG

GCCTGGAGTA

TTGAGAAGGC

GCTTATGACG

CATGGTGTTT

TTTCCCTAGG

TGGCTTGTCP

CCCAAAAGAC

SGCACGTTGC I

TATGAGTTCC

r~ CGTCCTCTG' 3 CAGGCTGTGC r GCCGGGGTT( 37 ATTCGCCGGj G CAGAGCAGC'

CCCATTCCGA

GTTGCGTGAG

CGGGTGGCGA

CTGACCGCAA

AGCGCCTTCC

CTGTGCTACC

CAG GAG CTAG

TGTGCACTGC

CGCTGGTAGG

ACCGATGTCC

TACTGCCACC

AGGGCCAAGA

GTCTCCCGCA

CGAGACAATT

AGACCTTTTG

TATTCTATCC

ACTCAGTATI

GAAAATGAT'I

iTCTTGAGTGC

GGTTGTACCI

TCTTTGAGAC

CTGGAATAC(

GGGACCTCC)

LAGTGAATAC(

3 TTTGAAGCCI STCGTCGCCCi ~CGGCTTTCGi r' CCGCCTCGC

ACAGAGAAGT

GTGGGTATCT

GGTTGGTCAC

TGTTGACGTG

ATCAG CTTG C

TCCCTGCCCT

CCTCCTGTGA

CGCATGGCGG

CCGGTATGGC

GCGCCTCCCT

ACCTGTGAAC

CGGTTCAGCC

TAACCTTTTT

GCGCATGGCA

TG CCCTGTTT

CTTTTACCAC

CTCTGCTGCC

TTTCTGAGTT

GCCATTATGG

TGCCGTCCGG

GGTTCTGGAA

GTGTGGAACA

k. GGTTGCCGCC 2GCC,7LGAGCCC 3AAGGCTGACT

CGGGGCTCGGG

G AGAAGAACTG C GTGCA,3"GATT 1401 TCCTCCGTAG GTTAACGAAT GTGGCCCAGA TTTGTGTTGA GGTGGTGTCT WO 89/12462 PCr/ US8902648 19 1451 AGAGTTTACG GGGTTTCCCC GGGTCTGGTT CATAACCTGA TAGGCATGCT 1501 CCAGACTATT GGTGATGGTA AGGCGCATTT TACAGAGTCT GTTAAGCCTA 1551 TACTTGACCT TACACACTCA ATTATGCACC GGTCTGAATG AATAACATGT 1601 GGTTTCCTGC GCCCATGGGT TCGCCACCAT GCGCCCTAGG CCTCTTTTGC 1651 CGAGCGGCCG C When comparing the Burmese and Mexican strains, 76.7% identity is seen in a 1372 nucleotide overlap beginning at nucleotide 13 of the Mexican strain and nucleotide 331 of the Burmese strain.

It will be recognized by one skilled in the art of molecular genetics that each of the last two sequences teaches a corresponding complementary DNA sequence as well as RNA sequences corresponding to both the principal sequence shown and the complementary DNA sequence. Additionally, open reading frames encoding peptides are present, and expressible peptides are disclosed by the nucleotide sequences without setting forth the amino acid sequences explicitly, in the same manner as if the amino acid sequences were explicitly set forth as in the ET1.1 sequence above.

DETAILED DESCRIPTION OF THE INVENTION I. Defini'tions The terms defined below have the following meaning herein: 1. "Enterically transmitted non-A/non-B (ET-NANB) hepatitis viral agent" means a virus, virus type, or virus class which causes water-borne, infectious hepatitis, (ii) is transmissible in cynomolgus monkeys, (iii) is serologically distinct from hepatitis A virus (HAV) and hepatitis B virus (HAB), and (iv) includes a genomic region which is homologous to the 1.33 kb cDNA WO 8;9/12462 PC-r/US89/0264P, insert in plasmid pTZ-KFl(ET1.1) carried in E. coli strain BB4 identified by ATCC deposit number 67717.1 2. Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions in which hybridized strands contain at most about 25-30% basepair mismatches. In general, two single-strand nucleic acid species will be homologous if they hybridize under the conditions described in Maniatis et al., op. cit., pp. 320-323, but using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50 0 C once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes each. More preferably, homologous nucleic acid strands contain 25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material), as is well known in the art.

3. A DNA fragment is "derived from" an ET-NANB viral agent if it has the same or substantially the same basepair sequence as a region of the viral agent genome.

4. A protein is "derived from" an ET-NANB viral agent if it is encoded by an open reading frame of a DNA or RNA fragment derived from an ET-NANB viral agent.

II. Obtaining Cloned ET-NANB Fragments According to one aspect of the invention, it has been found that a virus-specific DNA clone can be produced by isolating RNA from the bile of a cynomolgus monkey having a known ET-NANB infection, (b) cloning the cDNA fragments to form a fragment library, and screening the library by differential hybridization to radiolabeled cDNAs from infected and non-infected bile -sources.

WO 89/12462 PCT/US89/02648 21 A. cDNA Fragment Mixture ET-NANB infection in cynomolgus monkeys is initiated by inoculating the animals intravenously with a 10% w/v suspension from human case stools positive for 27-34 nm ET-NANB particles (mean diameter 32 nm).

An infected animal is monitored for elevated levels of alanine aminotransferase, indicating hepatitis infection. ET-NANB infection is confirmed by immunospecific binding of seropositive antibodies to virus-like particles (VLPs), according to published methods (Gravelle). Briefly, a stool (or bile) specimen taken from the infected animal 3-4 weeks after infection is diluted 1:10 with phosphate-buffered saline, and the 10% suspension is clarified by lowspeed centrifugation and filtration successively through 1.2 and 0.45 micron filters. The material may be further purified by pelleting through a 30% sucrose cushion (Bradley). The resulting preparation of VLPs is mixed with diluted serum from human patients with known ET-NANB infection. After incubation overnight, the mixture is centrifuged overnight to pellet immune aggregates, and these are stained and examined by electron microscopy for antibody binding to the VLPs.

ET-NANB infection can also be confirmed by seroconversion to VLP-positive serum. Here the serum of the infected animal is mixed as above with 27-34 nm VLPs isolated form the stool specimens of infected human cases and examined by immune electron microscopy for antibody binding to the VLPs.

Bile can be collected from ET-NANB positive animals by either cannulating the bile duct and collecting the bile fluid or by draining the bile duct during necropsy. Total RNA is extracted from the bile by hot phenol extraction, as outlined in Example 1A.

The RNA fragments are used to synthesize corresponding duplex cDNA fragments by random priming, also as WO 89/12462 PCT/US89/02648 referenced in Example 1A. The cDNA fragments may be fractionated .by gel electrophoresis or density gradient centrifugation to obtain a desired size class of fragments, 500-4,000 basepair fragments.

Although alternative sources of viral material, such as VLPs obtained from stool samples (as described in Example may be used for producing a cDNA fraction, the bile source is preferred. According to one aspect of the invention, it has been found that 0 bile from ET-NANB-infected monkeys shows a greater number of intact viral particles than material obtained from stool samples, as evidenced by immune electron microscopy. Bile obtained from an ET-NANB infected human or cynomolgus monkey, for use as a source of ET- NANB viral protein or genomic material, or intact virus, forms part of the present invention.

B. cDNA Library and Screening The cDNA fragments from above are cloned into a suitable cloning vector to form a cDNA library. This may be done by equipping blunt-ended fragments with a suitable end linker, such as an EcoRI sequence, and inserting the fragments into a suitable insertion site of a cloning vector, such as at a unique EcoRI site.

After initial cloning, the library may be recloned, if desired, to increase the percentage of vectors containing a fragment insert. The library construction described in Example 1B is illustrative. Here cDNA fragments were blunt-ended, equipped with EcoRI ends, and inserted into the EcoRI site of the lambda phage vector gtlO. The library phage, which showed less than fragment inserts, was isolated, and the fragment inserts recloned into the lambda gtlO vector, yielding more than 95% insert-containing phage.

The cDNA library is screened for sequences specific for ET-NANB by differential hybridization to cDNA probes derived from infected and non-infected WO 89/12462 PCr/US89/02648 23 sources. cDNA fragments from infected and non-infected source bile or stool viral isolates can be prepared as above. Radiolabeling the fragments is by random labeling, nick translation, or end labeling, according to conventional methods (Maniatis, p. 109). The cDNA library from above is screened by transfer to duplicate nitrocellulose filters, and hybridization with both infected-source and non-infected-source (control) radiolabeled probes, as detailed in Example 2. In order to recover sequences that hybridize at the preferred outer limit of 25-30% basepair mismatches, clones can be selected if they hybridize underthe conditions described in Maniatis et al., op. cit., pp.

320-323, but using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50 0 C once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes each. These.conditions allowed identification of the Mexican isolate discussed above using the ET1.1 sequence as a probe. Plaques which show selective hybridization- to the infected-source probes are preferably replated at low plating density and rescreened as above, to isolate single clones which are specific for ET-NANB sequences. As indicated in Example 2, sixteen clones which hybridized specifically with infected-source probes were indentified by these procedures. One of the clones, designated lambda gtl0- 1.1, contained a 1.33 kilobase fragment insert.

C. ET-NANB Sequences The basepair sequence of cloned regions of the ET-NANB fragments from Part B are determined by standard sequencing methods. In one illustrative method, described in Example 3, the fragment insert from the selected cloning vector is excised, isolated by gel electrophoresis, and inserted into a cloning vector whose basepair sequence on either side of the insertion site is known. The particular vector employed in Example 3 is a pTZ-KFI vector shown at the left in Figure 1. The ET-NANB fragment from the gtlO-1.l phage was inserted at the unique EcoRI site of the pTZ-KF1 plasmid. Recombinants carrying the desired Insert were identified by hybridization with the isolated 1.33 kilobase fragment, as described in Example 3. One selected plasmid, identified as pTZ-KFl(ETI.1), gave the expected 1.33 kb fragment after vector digestion with F.coRI. E. coli strain BB4 infected with the pTZ-KFI(ET1.1) plasmid was deposited with the American Type Culture Collection, Rockville, MD, on 27 May 1988 and is J identified by ATCC deppsit number 67717.

The pTZ-KF1(ET1.1) plasmid is illustrated at the bottom in Figure 1.

The fragment insert has 5' and 3' end regions denoted at A and C, respectively, and an intermediate region, denoted at B. The sequences In these regions were determined by standard dideoxy sequencing and are set forth above. The three short sequences B, and C) are from the same insert strand. As will be seen in Example 3, the B-reglon sequence was actually determined from the opposite strand, so that the R-region sequence shown above represent the complement of the sequence in the sequenced strand. The base numbers of the partial sequences are approximate.

Later work in the laboratory of the inventors identified the full sequence, also set forth above. Fragments of this total sequence can readily be prepared using restriction endonucleases. Computer analysis of both the forward and reverse sequence has identified a number of cleavage sites. The specific cleavage sites are summarized (for the forward direction) in the following tables.

WO 89/12462Pr/S8024 PC'T/LJS89/02648 Pattern identifier cleavage site) Pattern matched Base number matched in "forward" strand CC*WGG(BstNI) CC*SGG(NciI) GAAGANNNNNNNN* (mboii) TCTTC( <-7-MboII) GCGTC(<-10-HgaI) GCATCNNNNN* (Sf aNI) GCAGCNNNNNNNN* (BbvI) GCTGC(<-12-BbvI) GGATGNNNNNNNNN* (FokI) CATCC(<-13-FokI) GGTGANNNNNNNN*(Hp hl) TCACC (<-7-HphI) GP*CGYC (AhaII) GDGCH*C (Bsp11286) GPGCY*C (BanII) C*YCGPG(AvaI) Y*GGCCP (EaeI) GWGCq* C (Gg iAI C*CTTGG (Styl) P*GATCY (XholI) CAG"Y,.'TG(PvuII) C*CATGG (NcoI) CGAT*CG(PvuI) C*GGCCG ag I) G*AATTC (EcoRl) GAGCT*C (SacI) GCATG*C (SphI) GCC*GGC(NaeI) G*CGCGC (BssHII) GGGCC*C(ApaI)

CCWGG

CCSGG

GAAGA

TCTTC

GCGTC

GCATC

GCAGC

GCTGC

GGATG

CATCC

GGTGA

TCACC

GPCGYC

GDGCHC

GPGCYC

CYCGPG

YGGCCP

GWGCWC

CCTTGG

PGATCY

CAGCTG

CCATGG

CGATCG

CGG CCG

GAATTC

GAGCTC

GCATGC

GCCGGC

GCGCGC

GGGCCC

106, 360, 497, 973, 288, 841, 822, 1116 422, 611, 225 488, 640 53, 631, 919, 979 363, 734, 641, 750 454, 589, 114, 416, 224 77, 110, 1125, 1324 77, 110, 74, 178 171, 290, 1101 77, 110, 529, 1068 782 54 344, 468, 1031 1101 2, 1335 77, 110 1268 994, 1099, 1073 .1125 410, 1129, 1063 849 1149 1212 835, 446, 931 762 158, 838, 838, 1125 626, 875, 158, 1324 483, 1243 644 1103 WO 89/12462 PCT/US89/02648 26 TCG*CGA(NruI) TCGCGA 264 T*CTAGA(XbaI) TCTAGA 705 TTT*AAA(DraI) TTTAAA 925 G*TGCAC(ApaLI) GTGCAC 158 ACCTGCNNNN*(BpsMI) ACCTGC 99 GCAGGT(<-8-BspMI) GCAGGT 1045 GACN*NNGTC(TthlllI) GACNNNGTC 604 CCANNNN*NTGG(PflMI) CCANNNNNTGG CC*TNAGG(MstII) CCTNAGG 571 GCCNNNN*NGGC(BglI) GCCNNNNNGGC 216, 359, 738 CCANNNNN*NTGG(BstXI) CCANNNNNNTGG 204 III. ET-NANB Fragments According to another aspect, the invention includes ET-NANB-specific fragments or probes which hybridize with ET-NANB genomic sequences or cDNA fragments derived therefrom. The fragments may include full-length cDNA fragments such as described in Section II, or may be derived from shorter sequence regions within cloned cDNA fragments. Shorter fragments can be prepared by enzymatic digestion of full-length fragments under conditions which yield desired-sized fragments, as will be described in Section IV.

Alternatively, the fragments can be produced by oligonucleotide synthetic methods, using sequences derived from the cDNA fragments. Methods or services for producing selected-sequence oligonucleotide fragments are available.

To confirm that a given ET-NANB fragment is in fact derived from the ET-NANB viral agent, the fragment can be shown to hybridize selectively with cDNA from infected sources. By way of illustration, to confirm that the 1.33 kb fragment in the pTZ-KFl(El.1) plasmid is ET-NANB in origin, the fragment was excised from the pTZ-KFl(ETl.1) plasmid, purified, and radiolabeled by random labeling. The radiolabeled fragment was WO 89/12462 PCT/US89/02648 27 hybridized with fractionated cDNAs from infected and non-infected sources to confirm that the probe reacts only with infected-source cDNAs. This method is illustrated in Example 4, where the above radiolabeled 1.33 kb fragment from pTZ-KFl(ET1.l) plasmid was examined for binding to cDNAs prepared from infected and non-infected sources. The infected sources are (1) bile from a cynomolgus monkey infected with a strain of virus derived from stool samples from human patients from Burma with known ET-NANB infections and a viral agent derived from the stool sample of a human ET-NANB patient from Mexico. The cDNAs in each fragment' mixture were first amplified by a linker/primer amplification method described in Example 4. Fragment separation was on agarose gel, followed by Southern blotting and then hybridization to bind the radiolabeled 1.33 kb fragment to the fractionated cDNAs. The lane containing cDNAs from the infected sources showed a smeared band of bound probe, as expected (cDNAs amplified by the linker/primer amplification method would be expected to have a broad range of sizes). No probe binding to the amplified cDNAs from the non-infected sources was observed. The results indicate that the 1.33 kb probe is specific for cDNA fragments associated with ET-NANB infection. This same type of study, using ET 1.1 as the probe, has demonstrated hybridization to ET-NANB samples collected from Tashkent, Somalia, Borneo and Pakistan. Secondly, the fact that the probe is specific for ET-NANB related sequences derived from different continents (Asia, Africa and North America) indicates the cloned ET-NANB Africa sequence is derived from a common ET-NANB virus or virus class responsible for ET-NANB hepatitis infection worldwide.

In a related confirmatory study, probe binding to fractionated genomic fragments prepared from human or cynomolgus monkey genomic DNA was examined. No WO 89/12462 PCT!US89/02640 28 probe binding was observed to either genomic fraction, demonstrating that the ET-NANB fragment is not an endogenous human or cynomologus fragment.

Another confirmation of ET-NANB specific sequences in the fragments is the ability to express ET-NANB proteins from coding regions in the fragments. Section IV below discusses methods of protein expression using the fragments.

One important use of the ET-NANB-spe"ific fragments is for identifying ET-NANB-derived cDNAs which contain additional sequence information. The newly identified cDNAs, in turn, yield new fragment probes, allowing further iterations until the entire viral genome is identified and sequenced. Procedures for identifying additional ET-NANB library clones and generating new probes therefrom generally follow the cloning and selection procedures described in Section

.II.

The fragments (and oligonucleotides prepared based on the sequences given above) are also useful as primers for a polymerase chain reaction method of detecting ET-NANB viral genomic material in a patient sample. This diagnostic method will be described in Section V below.

IV. ET-NANB Proteins As indicated above, ET-NANB proteins can be prepared by expressing open reading-frame coding regions in ET-NANB fragments. In one preferred approach, the ET-NANB fragments used for protein expression are derived from cloned cDNAs which have been treated to produce desired-size fragments, and preferably random fragments with sizes predominantly between about 100 to about 300 base pairs. Example describes the preparation of such fragments by DNAs digestion. Because it is desired to obtain peptide WO 89/12462 PCT/US89/02648 29 antigens of between about 30 to about 100 amino acids, the digest fragments are preferably size fractionated, for example by gel electrophoresis, to select those in the approximately 100-300 basepair size range.

A. Expression Vector The ET-NANB fragments are inserted into a suitable expression vector. One exemplary expression vector is lambda gtll, which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the beta-galactosidase gene. Thus, the inserted sequence will be expressed as a beta-galactosidase fusion protein which contains the N-terminal portion of the beta-galactosidase gene, the heterologous peptide, and optionally the C-terminal region of the beta-galactosidase peptide (the Cterminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon). This vector also produces a temperature-sensitive repressor (c1857) which causes viral lysogeny at permissive temperatures, 32 0

C,

and leads to viral lysis at elevated temperatures, 42 0 C. Advantages of this vector include: (1) highly efficient recombinant generation, ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and high levels of recombinant fusion protein production. Further, since phage containing a heterologous insert produces an inactive betagalactosidase enzyme, phage with inserts can be readily identified by a beta-galactosidase colored-substrate reaction.

For insertion into the expression vector, the viral digest fragments may be modified, if needed, to contain selected restriction-site linkers, such as EcoRI linkers, according to conventional procedures.

Example 1 illustrates methods for cloning the digest WO 89/12462 PCT/ US589/02648 fragments into lambda gtll, which includes the steps of blunt-ending the fragments, ligating with EcoRI linkers, and introducing the fragments into EcoRI-cut lambda gtll. The resulting viral genomic library may be checked to confirm that a relatively large (representative) library has been produced. This can be done, in the case of the lambda gtll vector, by infecting a suitable bacterial host, plating the bacteria, and examining the plaques for loss of betagalactosidase activity. Using the procedures described in Example 1, about 50% of the plaques showed loss of enzyme activity.

B. Peptide Antigen Expression The viral genomic library formed above is screened for production of peptide antigen (expressed as a fusion protein) which is immunoreactive with antiserum from ET-NANB seropositive individuals. In a preferred screening method, host cells infected with phage library vectors are plated, as above, and the plate is blotted with a nitrocellulose filter to transfer recombinant protein antigens produced by the cells onto the filter. The filter is then reacted with the ET-NANB antiserum, washed to remove unbound antibody, and reacted with reporter-labeled, anti-human antibody, which becomes bound to the filter, in sandwich fashion, through the anti-ET-NANB antibody.

Typically phage plaques which are identified by virtue of their production of recombinant antigen of interest are re-examined at a relatively low density for production of antibody-reactive fusion protein.

Several recombinant phage clones which produced immunoreactive recombinant antigen were identified in the procedure.

The selected expression vectors may be used for scale-up production, for purposes of recombinant protein purification. Scale-up production is carried WO 89/12462 PCT/US89/02648 31 out using one of a variety of reported methods for (a) lysogenizing a suitable host, such as E. coli, with a selected lambda gtll recombinant culturing the transduced cells under conditions that yield high levels of the heterologous peptide, and purifying the recombinant antigen from the lysed cells.

In one preferred method involving the above lambda gtll cloning vector, a high-producer E. coli host, BNN103, is infected with the selected library phage and replica plated on two plates. One of the plates is grown at 32 0 C, at which viral lysogeny can occur, and the other at 42 0 C, at which the infecting phage is in a lytic stage and therefore prevents cell growth. Cells which grow at the lower but not the higher temperature are therefore assumed to be successfully lysogenized.

The lysogenized host cells are then grown under liquid culture conditions which favor high production of the fused protein containing the viral insert, and lysed by rapid freezing to release the desired fusion protein.

C. PeDtide Purification The recombinant peptide can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. In the case of a fused protein, such as the beta-galactosidase fused protein prepared as above, the protein isolation techniques which are used can be adapted from those used in isolation of the native protein. Thus, for isolation of a betaglactosidase fusion protein, the protein can be isolated readily by simple affinity chromatography, by passing the cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody.

WO 89/12462 PCT/US89/02648 32 D. Viral Proteins The ET-NANB protein of the invention may also be derived directly from the ET-NANB viral agent. VLPs isolated from a stoo" sample from an infected individual, as above, are one suitable source of viral protein material. The VLPs isolated from the stool sample may be further purified by affinity chromatography prior to protein isolation (see below). The viral agent may also be raised in cell culture, which provides a convenient and potentially concentrated source of viral protein. Co-owned U.S.

Patent Application Serial No. 846,757, filed April 1, 1986, describes an immortalized trioma liver cell which supports NANB infection in cell calture. The trioma cell line is prepared by fusing human liver cells with a mouse/human fusion partner selected for human chromosome stability. Cells containing the desired NANB viral agent can be identified by immunofluorescence methods, employing anti-ET-NANB human antibodies.

The viral agent is disrupted, prior to protein isolation, by conventional methods, which can include sonication, high- or low-salt conditions, or use of detergents.

Purification of ET-NANB viral protein can be carried out by affinity chromatography, using a purified anti-ET-NANB antibody attached according to standard methods to a suitable solid support. The antibody itself may be purified by affinity chromatography, where an immunoreactive recombinant ETNANB protein, such as described above, is attached to a solid support, for isolation of anti-ET-NANB antibodies from an immune serum source. The bound antibody is released from the support by standard methods.

\VO 89/12462 PCT/US89/02648 33 Alternatively, the anti-ET-NANB antibody may be a monoclonal antibody (Mab) prepared by immunizing a mouse or other animal with recombinant ET-NANB protein, isolating lymphocytes from the animal and immortalizing the cells with a suitable fusion partner, and selecting successful fusion products which react with the recombinant protein immunogen. These in turn may be used in affinity purification procedures, described above, to obtain native ET-NANB antigen.

V. Utility A. Diagnostic Methods The particles and antigens of the invention, as well as the genetic material, can be used in diagnostic assays. Methods for detecting the presence of ET-NANB hepatitis comprise analyzing a biological sample such as a blood sample, stool sample or liver biopsy specimen for the presence of an analyte associated with ET-NANB hepatitis virus.

The analyte can be a nucleotide sequence which hybridizes with a probe comprising a sequence of at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences). The analyte can be RNA or cDNA. The analyte is typically a virus particle suspected of being ET-NANB or a particle for which this classification is being ruled out. The virus particle can be further characterized as having an RNA viral genome comprising a sequence at least about 80% homologous to a sequence of at least 12 consecutive nucleotides of the "forward" and "reverse" sequences given above, usually at least about homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially homologous to the full-length sequences. In order to detect an analyte, where the WO 89/12462 PCT/US89/02648 34 analyte hybridizes to a probe, the probe may contain a detectable label.

The analyte can also comprise an antibody which recognizes an antigen, such as a cell surface antigen, on a ET-NANB virus particle. The analyte can also be a ET-NANB viral antigen. Where the analyte is an antibody or an antigen, either a labelled antigen or antibody, respectively, can be used to bind to the analyte to form an immunological complex, which can then be detected by means of the label.

Typically, methods for detecting analytes such as surface antigens and/or whole particles are based on immunoassays. Immunoassays can be conducted either to determine the presence of antibodies in the host that have arisen from infection by ET-NANB hepatitis virus or by assays that directly determine the presence of virus particles or antigens. Such techniques are well known and need not be described here in detail. Examples include both heterogeneous and homogeneous immunoassay techniques. Both techniques are based on the formation of an immunological complex between the virus particle or its antigen and a corresponding specific antibody. Heterogeneous assays for viral antigens typically use a specific monoclonal or polyclonal antibody bound to a solid surface. Sandwich assays are becoming increasingly popular. Homogeneous assays, which are carried out in solution without the presence of a solid phase, can also be used, for example by determining the difference in enzyme activity brought on by binding of free antibody to an enzyme-antigpn conjugate. A number of suitable assays are disclosed in U.S. Patent Nos. 3,817,837, 4,006,360, 3,996,345.

When assaying for the presence of antibodies induced by ET-NANB viruses, the viruses and antigens of the invention can be used as specific binding aqnts to detect either IgG or IgM antibodies. Since IgM antibodies are typically the first antibodies that appear \VO 89/12462 PCT/US89/02648 during the course of an infection, when IgG synthesis may not yet have been initiated, specifically distinguishing between IgM and IgG antibodies present in the blood stream of a host will enable a physician or other investigator to determine whether the infection is recent or chronic.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having surface-bound ET-NANB protein antigen. After binding anti-ET-NANB antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-ET-NANB antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.

The solid surface reagent in the above assay prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activate carboxyl, hydroxyl, or aldehyde group.

In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium. Known general types of homogeneous assays proposed heretofore include spin-labeled reporters, where antibody binding to the antigen is detected by a change in WO 89/12462 PCT/US89/02648 36 reported mobility (broadening of the spin splitting peaks), fluorescent reporters,.where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents.

In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody. The examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent phase), and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.

Also forming part of the invention is an assay system or kit for carrying out the assay method just described. The kit generally includes a support with surface-bound recombinant protein antigen which is (a) immunoreactive with antibodies present in individuals infected with enterically transmitted nonA/nonB viral agent and derived from a viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETl.1) carried in E. Coli strain BB4, and having ATCC deposit no. 67717. A reporter-labeled anti-human antibody in the kit is used for detecting surface-bound anti-ET-NANB antibody.

O 89/12462 PCT/US89/02648 37 B. Viral Genome Diagnostic Applications The genetic material of the invention can itself be used in numerous assays as probes for genetic material present in naturally occurring infections.

One method for amplification of target nucleic acids, for later analysis by hybridization assays, is known as the polymerase chain reaction or PCR technique. The PCR technique can be applied to detecting virus particles of the invention in suspected pathological samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth above. The primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt). This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain- DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer. The degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2 n where n is the number of cycles. Given that the average efficiency per cycle ranges from about to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence. The PCR method is described in a number of publications, including Saiki et al., Science (1985) 230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf et al., Science (1986) 233:1076-1078. Also see U.S. Patent Nos. 4,683,194; 4,683,195; and 4,683,202.

The invention includes a specific diagnostic method for determination of ET-NANB viral agent, based on selective'amplification of ET-NANB fragments. This method employs a pair of single-strand primers derived WO 89/12462 PCT/US89/02648 38 from non-homologous regions of opposite strands of a DNA duplex fragment, which in turn is derived from an enterically transmitted viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETl.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717. These "primer fragments," which form one aspect of the invention, are prepared from ET-NANB fragments such as described in Section III above. The method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S.

Patent No. 4,683,202, as discussed above.

C. Peotide Vaccine Any of the antigens of the invention can be used in preparation of a vaccine. A preferred stazting material for preparation of a vaccine is the particle antigen isolated from bile. The antigens are preferably initially recovered as intact particles as described above. However, it is also possible to prepare a suitable vaccine from particles isolated from other sources or non-particle recombinant antigens.

When non-particle antigens are used (typically soluble antigens), proteins derived from the viral envelope or viral capsid are preferred for use in preparing vaccines. These proteins can be purified by affinity chromatography, also described above.

If the purified protein is not immunogenic per se, it can be bound to a carrier to make the protein immunogenic. Carriers include bovine serum albumin, keyhole limpet hemocyanin and the like. It is desirable, but not necessary, to purify antigens to be substantially free of human protein. However, it is more important that the antigens be free of proteins, viruses, and other substances not of human origin that may have been introduced by way of, or contamination of, the nutrient medium, cell lines, tissues, or patho- WO 89/12462 PCT/ US89/02648 39 logical fluids from which the virus is cultured or obtained.

Vaccination can be conducted in conventional fashion. For example, the antigen, whether a viral particle or a protein, can be used in a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like. The immunogen is administered using standard techniques for antibody induction, such as by subcutaneous administration of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens. An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less.

A specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant protein or protein mixture derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETI.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717. The vaccine is administered at periodic intervals until a significant titer of anti-, ET-NANB antibody is detected in the serum. The vaccine is intended to protect against ET-NANB infection.

D. Prophylactic and Therapeutic Antibodies and Antisera In addition to use as a vaccine, the compositions can be used to prepare antibodies to ET-NANB virus particles. The antibodies can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines. The host serum or plasma is collected WO 89/12462 PPcr/ US8/02648 following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-vial agents such as drugs.

The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas.

The antibodies can also be used as a means of enhancing the immune response since antibody-virus complexes are recognized by macrophages. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the ET-NANB virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an ET-NINB virus to enhance the immune response and/or the effectiveness of an antiviral drug.

Alternatively, anti-ET-NANB-virus antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified antiET- NANB-virus antibody preparation prepared as descibed above is used 'n induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following VO 89/12462 PM'//US8/026,t8 41 administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-ET-NANBvirus antibodies, or by affinity chromatography using anti-ET-NANB-virus antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic ET-NANB antigen and may be used to prepare an ET-NANB vaccine rather than using a ET-NANB particle antigen.

When used as a means of inducing anti-ET-NANBvirus antibodies in a patient, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable.

The anti-idiotype method of induction of anti-ET-NANBvirus antibodies can alleviate problems which may be caused by.passive administration of anti-ET-NANB-virus antibodies, such as an adverse immune response, and those associated with administration of purified blood components, such as infection with as yet undiscovered viruses.

The ET-NANB derived proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis. Here an ET-NANB protein', or mixture of proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected proteins is monitored, during WO 89/12462 PCT/US8902648 42 a several- week period following immunization, by periodic serum sampling to detect the presence an anti- ET-NANB serum antibodies, as described in Section IIA above.

The antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection.

The anitserum is also useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.

E. Monoclonal Antibodies For both in vivo use of antibodies to ET-NANB virus particles and proteins and anti-idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a ET-NANB virus (where infection has been shown. for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy.

Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.

Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal O 89/12462 PCT/US89/02648 43 anti-virus particle antibodies, the antibodies must bind to ET-NANB virus particles. For monoclonal antiidiotype antibodies, the antibodies must bind to antivirus particle antibodies. Cells producing antibodies of the desired specificity are selected.

The following examples illustrate various aspects of the invention, but are in no way intended to limit the scope thereof.

Material The materials used in the following Examples were as follows: Enzymes: DNAse I and alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN); EcoRI, EcoRI methylase, DNA ligase, and DNA Polymerase I, from New England Biolabs (NEB, Beverly MA); and RNase A was obtained from Sigma (St.

Louis, MO).

Other reagents: EcoRI linkers were obtained from NEB; and nitro blue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) 5-bromo-4-chloro-3-indolyl-B-D-galaetopyranoside (Xgal) and isopropyl B-D-thiogalactopyranoside (IPTG) were obtained from Sigma.

cDNA synthesis kit and random priming labeling kits are available from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN).

Eamele 1 Preparing cDNA Library A. Source of ET-NANB virus Two cynomolgus monkeys (cynos) were intravenously injected with a 10% suspension of a stool pool obtained from a second-passage cyno (cyno #37) infected with a strain of ET-NANB virus isolated from Burma cases whose stools were positive for ET-NANB, as WO 89/12462 PCT/US89/02648 44 evidenced by binding of 27-34 nm virus-like particles (VLPs) in the stool to immune serum from a known ET- NANB patient. The animals developed elevated levels of alanine aminotransferase (ALT) between 24-36 days after innoculation, and one excreted 27-34 nm VLPs in its bile in the pre-acute phase of infection.

The bile duct of each infected animal was cannulated and about 1-3 cc of bile was collected daily. RNA was extracted from one bile specimen (cyno #121) by hot phenol extraction, using a standard RNA isolation procedure. Double-strand cDNA was formed from the isolated RNA by a random primer for firststrand generation, using a cDNA synthesis kit obtained from Boehringer-Mannheim (Indianapolis, IN).

B. Cloning the Duplex Fragments The duplex cDNA fragments were blunt-ended with T4 DNA polymerase under standard conditions (Maniatis, p. 118), then extracted with phenol/chloroform and precipitated with ethanol. The blunt-ended material was ligated with EcoRI linkers under standard conditions (Maniatis, pp. 396-397) and digested with EcoRI to remove redundant linker ends, Non-ligated linkers were removed by sequential isopropanol precipitation.

Lambda gtlO phage vector (Huynh) was obtained from Promega Biotec (Madison, WI). This cloning vector has a unique EcoRI cloning site in the phage cI repressor gene. The cDNA fragments from above were introduced into the EcoRI site by mixing 0.5 1.0 ug EcoRI-cleaved gtl0, 0.5-3 ul of the above duplex fragments, 0.5 pl 10X ligation buffer, 0.5 pl ligase (200 units), and distilled water to 5 pl. The mixture was incubated overnight at 14 0 C, followed by in vitro packaging, according to standard methods (Maniatis, pp.

256-268).

WO 89/12462 PCT/ US89/02648 The packaged phage were used to infect an E.

coli hfl strain, such as strain HG415. Alternatively, E. coli, strain C600 hfl, avialable from Promega Biotec, Madison, WI, could be used. The percentage of recombinant plaques obtained with insertion of the EcoRI-ended fragments was less than 5% by analysis of random plaques.

The resultant cDNA library was plated and phage were eluted from the selection plates by addition of elution buffer. After DNA extraction from the phage, the DNA was digested with EcoRI to release the heterogeneous insert population, and the DNA fragments were fractionated on agarose to remove phage fragments. The 500-4,000 basepair inserts were isolated and recloned into lambda gtl0 as above, and the packaged phage was used to infect E. coli strain HG415. The percentage of successful recombinants was greater than 95%. The phage library was plated on E.

coli strain HG415, at about 5,000 plaques/plate, on a total of 8 plates.

WO 89/12462 PCT/US89/02648 46 Example 2 Selecting ET-NANB Cloned Fragments A. cDNA Probes Duplex cDNA fragments from noninfected and ET- NANB-infected cynomolgus monkeys were prepared as in Example 1. The cDNA fragments were radiolabeled by random priming, using a random-priming labeling kit obtained from Boehringer-Mannheim (Indianapolis, IN).

B. Clone Selection The plated cDNA library from Example 1 was transferred to each of two nitrocellulose filters, and the phage DNA was fixed on the filters by baking, according to standard methods (Maniatis, pp. 320- 323). The duplicate filters were hybridized with either.infected-source or control cDNA probes from above. Autoradiographs of the filters were examined to identify library clones which hybridized with radiolabeled cDNA probes from infected source only, did not hybridize with cDNA probes from the noninfected source. Sixteen such clones, out of a total of about 40,000 clones examined, were identified by this subtraction selection method.

Each of the sixteen clones was picked and replated at low concentration on an agar plate. The clones on each plate were transferred to two nitrocellulose as duplicate lifts, and examined for hybridization to radiolabeled cDNA probes from infected and noninfected sources, as above. Clones were selected which showed selective binding for infected-source probes binding with infected-source probes and substantially no binding with non-infected-source probes). One of the clones which bound selectively to probe from infected source was isolated for further study. The selected vector was identified as lambda gtlO-1.1, indicated in Figure 1.

vQ 89/12462 PCTUS9/02648( 47 ExamDle 3 ET-NANB Seauence Clone lambda gtlO-1.1 from Example 2 was digested with EcoRI to release the heterologous insert, which was separated from the vector fragments by gel electrophoresis. The electrophoretic mobility of the fragment was consistent with a 1.33 kb fragment. This fragment, which contained EcoRI ends, was inserted into the EcoRI site of a pTZ-KF1 vector, whose construction and properties are described in co-owned U.S. patent application for "Cloning Vector System and Method for Rare Clone Identification", Serial No. 125, 650, filed November 25, 1987. Briefly, and as illustrated in Figure 1, this plasmid contains a unique EcoRI site adjacent a T7 polymerase promoter site, and plasmid and phage origins of replication. The sequence immediately adjacent each side of the EcoRI site is known. E. coli BB4 bacteria, obtained from Stratagene (La Jolla, CA, were transformed with the plasmid.

Radiolabeled ET-NANB probe was prepared by excising the 1.33 kb insert from the lambda gtlO-1.1 phage in Example 2, separating the fragment by gel electrophoresis, and randomly labeling as above.

Bacteria transfected with the above pTZ-KFl and containing the desired ET-NANB insert were selected by replica lift and hybridization with the radiolabeled ET-NANB probe, according to methods outlined in Example 2.

One bacterial colony containing a successsful recombinant was used for sequencing a portion of the 1.33 kb insert. This isolate, designated pTZ- KFl(ET1.1), has been deposited with the American Type Culture Collection, and is identified by ATCC deposit no. 67717. Using a standard dideoxy sequencing procedure, and primers for the sequences flanking the EcoRI site, about 200-250 basepairs of sequence from WO 89/12462 PCT/US89/02648 48 the 5'-end region and 3'-end region of the insert were obtained. The sequences are given above in Section II. Later sequencing by the same techniques gave the full sequence in both directions, also given above.

Example 4 Detecting ET-NANB Sequences cDNA fragment mixtures from the bile of noninfected and ET-NANB-infected cynomolgus monkeys were prepared as above. The cDNA fragments obtained from human stool samples were prepared as follows.

Thirty ml of a 10% stool suspension obtained from an individual from Mexico diagnosed as infected with ET- NANB as a result of an ET-NANB outbreak, and a similar volume of stool from a healthy non-infected individual, were layered over a 30% sucrose density gradient cushion, and centrifuged at 25,000 xg for 6 hr in an SW27 rotor, at 15 0 C. The pelleted material from the infected-source stool contained 27-34 nm VLP particles characteristic of ET-NANB infection in the infected-stool sample. RNA was isolated from the sucrose-gradient pellets in both the infected and noninfected samples, and the isolated RNA was used to produce cDNA fragments as described in Example 1.

The cDNA fragment mixtures from infected and non-infected bile source, and from infected and noninfected human-stool source were each amplified by a novel linker/primer replication method described in coowned patent application serial number 07/208,512 for "DNA Amplification and Subtraction Technique," filed June 17, 1988. Briefly, the fragments in each sample were blunt-ended with DNA Pol I then extracted with phenol/chloroform and precipitated with ethanol. The blunt-ended material was ligated with linkers having the following sequence: V/O 89/12462 PCT/UtS8/02648 49 5'-GGAATTCGCGGCCGCTCG-3' The duplex fragements were digested with NruI to remove linker dimers, mixed with a primer having the sequence 5'-GGAATTCGCGGCCGCTCG-3', and then heat denatured and cooled to room temperature to form single-strand DNA/primer complexes. The complexes were replicated to formeduplex fragments by addition of Thermus aquaticus (Taq) polymerase and all four deoxynucleotides. The replication procedures, involving successive strand denaturation, formation of strand/primer complexes, and replication, was repeated times.

The amplified cDNA sequences were fractionated by agarose gel electrophoresis, using a 2% agarose matrix. After transfer of the DNA fragments from the agarose gels to nitrocellulose paper, the filters were hybridized to a random-labeled 32 P probe prepared by treating the pTZ-KF1(ET1.1) plasmid from above with EcoRI, (ii) isolating the released 1.33 kb ET-NANB fragment, and (iii) randomly labeling the isolated fragment. The probe hybridization was performed by conventional Southern blotting methods (Maniatis, pp.

382-389). Figure 2 shows the hybridization pattern obtained with cDNAs from infected and non-infected bile sources (2A) and from infected and noninfected human stool sources As seen, the ET-NANB probe hybridized with fragments obtained from both of the infected sources, but was non-homologous to sequences obtained from either of the non-infected sources, thus confirming the specificity of derived sequence.

Southern blots of the radiolabeled 1.33 kb fragment with genomic DNA fragments from both human and cynomologus-monkey DNA were also prepared. No probe hybridization to either of the genomic fragment WO 89/12462 PUT/US89/02648 mixtures was observed, confirming that the ET-NANB sequence is exogenous to either human or cynomolgus genome.

Example Expressing ET-NANB Proteins A. Preparing ET-NANB Coding Sequences The pTZ-KFl(ET1.1) plasmid from Example 2 was digested with EcoRI to release the 1.33 kb ET-NANB insert which was purified from the linearized plasmid by gel electrophoresis. The purified fragment was suspended in a standard digest buffer (0.5M Tris HC1, pH 7.5; 1 mg/ml BSA; 10mM MnC1 2 to a concentration of about 1 mg/ml and digested with DNAse I at room temperature for about 5 minutes. These reaction conditions were determined from a prior calibration study, in which the incubation time required to produce predominantly 100-300 basepair fragments was determined. The material was extracted with phenol/chloroform before ethanol precipitation.

The fragments in the digest mixture were bluntended and ligated with EcoRI linkers as in Example 1.

The resultant fragments were analyzed by electrophoresis (5-10V/cm) on 1.2% agarose gel, using PhiX174/HaeIII and lambda/HindIII size markers. The 100-300 bp fraction was eluted onto NA45 strips (Schleicher and Schuell), which were then placed into ml microtubes with eluting solution (1 M NaC1, mM arginine, pH and incubated at 67 0 C for 30-60 minutes. The eluted DNA was phenol/chloroform extracted and then precipitated with two volumes of ethanol. The pellet was resuspended in 20 ul TE (0.01 M Tris HC1, pH 7.5, 0.001 M EDTA).

W'O 89/12462 PCIYUS89/02648 51 B. Cloning in an Expression Vector Lambda gtll phage vector (Huynh) was obtained from Promega Biotec (Madison, WI). This cloning vector has a unique EcoRI cloning site 53 base pairs upstream from the beta-galactosidase translation termination codon. The genomic fragments from above were introduced into the EcoRI site by mixing 0.5-1.0 pg EcoRI-cleaved gtll, 0.3-3 ul of the above sized fragments, 0.5 pl 10X ligation buffer (above), 0.5 pl ligase (200 units), and distilled water to 5 ul. The mixture was incubated overnight-at 14 0 C, followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).

The packaged phage were used to infect E. coli strain KM392, obtained from Dr. Kevin Moore, DNAX (Palo Alto, CA). Alternatively, E. Coli strain Y1090, available from the American Type Culture Collection (ATCC #37197), could be used. The infected bacteria were plated and the resultant colonies were checked for loss of beta-galactosidase activity-(clear plaques) in the presence of X-gal using a standard X-gal substrate plaque assay method (Maniatis). About 50% of the phage plaques showed loss of beta-galactosidase enzyme activity (recombinants).

C. Screening for ET-NANB Recombinant Proteins ET-NANB convalescent antiserum was obtained from patients infected during documented ET-NANB outbreaks in Mexico, Borneo, Pakistan, Somalia, and Burma. The sera were immunoreactive with VLPs in stool specimens from each of several other patients with ET- NANB hepatitis.

A lawn of E. coli KM392 cells infected with about 104 pfu of the phage stock from above was prepared on a 150 mm plate and incubated, inverted, for 5-8 hours at 370C. The lawn was overlaid with a nitrocellulose sheet, causing transfer of expressed ET- WO 89/12462 PCT/US89/02648 52 NANB recombinant protein from the plaques to the paper. The plate and filter were indexed for matching corresponding-plate and filter positions.

The filter was washed twice in TBST buffer mM Tris, pH 8.0, 105 mM NaCI, 0.05% Tween 20), blocked with AIB (TBST buffer with 1% gelatin), washed again in TEST, and incubated overnight after addition of antiserum (diluted to 1:50 in AIB, 12-15 ml/plate).

The sheet was washed twice in TBST and then contacted with enzyme-labeled anti-human antibody to attach the labeled antibody at filter sites containing antigen recognized by the antiserum. After a final washing, the filter was developed in a substrate medium containing 33 ul NBT (50 mg/ml stock solution maintained at 50C) mixed with 16 ul BCIP (50 mg/ml stock solution maintained at 50C) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaC1, 5 mM MgC12). Purple color appeared at points of antigen production, as recognized by the antiserum.

D. Screening Plating The areas of antigen production determiied in the previous step were replated at about 100-200 pfu on an 82 mm plate. The above steps, beginning with a 5-8 hour incubation, through NBT-BCIP development, were repeated in order to plaque purify phage secreting an antigen capable of reacting with the ET-NANB antibody. The identified plaques were picked and eluted in phage buffer (Maniatis, p. 443).

E. Epitope Identification A series of subclones derived from the original pTZ-KF1 (ET1.1) plasmid from Example 2 wbre isolated using the same techniques described above. Each of these five subclones were immunoreactive with a pool of anti-ET antisera noted in C. The subclones contained short sequences from the "reverse" sequence set forth V4)lO89/124622 PCT/US89/02648 53 previously. The beginning and ending points of the sequences in the subclones (relative to the full "reverse" sequence), are identified in the table below.

TABLE 1 Subclone Position in "Reverse" Sequence 0 5'-end 3'-end Y1 522 643 Y2 594 667 Y3 508 665 Y4 558 752 Y5 545 665 Since all of the gene sequences identified in the table must contain the coding sequence for the epitope, it is apparent that the coding sequence for the epitope falls in the region between nucleotide 594 and 643 Genetic sequences equivalent to and complementary to this relatively short sequence are therefore particularly preferred aspects of the present invention, as are peptides produced using this coding region.

A second series of clones identifying an altogether different epitope was isolated with only Mexican serum.

WO 8/12462 PCIV US89/02060 TABLE 2 Subclone Position in "Forward" Sequence ET 2-2 ET 8-3 ET 9-1 ET 13-1 2 2 2 2 3' end 193 135 109 101 The coding system for this epitope falls between nucleotide 2 (5'-end) and 101 (3'-end).

Genetic sequences related to this short sequence are therefore s1:e preferred, as are peptides produced using thi coding region.

While the invention has been described with reference to particular embodiments, methods, construction and use, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims (9)

1. Ajprotein derived from an enterically transmitted non-A/non-B viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETl.1) carried in E. coi strain BB4 and having ATCC deposit no. 67717.

2. The protein of claim 1, which is erQded by a coding region within said 1.33 kb t~coRI insert.

3. AArecombinant protein derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to a duplex DNA having a first sequence: 1 11 21 31 41 51l CGAATTCCGCCAACTGATGGAAGGCACTAATCTGGCAAGACCTGTCCC'r'GTTGCAGCTGT 61 71 81 91 101 ill TCTACCACCCTGCCCCGAGCTCGAACAGGGCCTTCTCTACCTGCCCCAGGAGCTCACCAC 121 131 141 151 161 171 CTGTGATAGTGTCGTAACATTTGAATTAACAGACATTGTGCACTGCCGCATGGCCGC CCC WO 89/12462 WO 8912462PCT/US89/0264SW 56 181 191 201 211 221 231 GAGCCAGCGCAAGGCCGTGCTGTCCACACTCGTGGGCCGCTACGGCGTCGCACAAAGCTC 241 251 261 271 281 291 TACAATGCTTCCCACTCTGATGTTCGCGACTCTCTCGCCCGTTTTATCCCGGCCATTGGC 301 311 321 331 341 351 CCCGTACAGGTTACAACTTGTGAATTGTACGAGCTAGTGGAGGCCATGGTCGAGAAGGGC 361 371 381 391 401 411 CAGGATGGCTCCGCCGTCCTTGAGCTTGATCTTTGCAACCGTGACGTGTCCAGGATCACC 421 431 441 451 461 471 TTCTTCCAGA.AAGATTGTAACAAGTTCACCACAGGTGAGACCATTGCCCATGGTAAAGTG 481 491 501 511 521 531 GGCCAGGGCATCTCGGCCTGGAGCAAGACCTTCTGCGCCCTCTTTGGCCCTTGGTTCCGC 541 551 561 571 581 591 .GCTATTGAGAAGGCTATTCTGGCCCTGCTCCCTCAGGGTGTGTTTTACGGTGATGCCTTT 601 611 621 631 641 651 GATGACACCGTCTTCTCGGCGGCTGTGGCCGCAGCAAAGGCATCCATGGTGTTTGAGAAT 661 671 681 691 701 711 GACTTTTCTGAGTTTGACTCCACCCAGAATAACTTTTCTCTGGGTCTAGAGTGTGCTATT 89/12462 ~r S9'24 PCr,/US8(,'/L)2648 57 721 731 741 751 761 771 ATGGAGGAGTGTGGGATGCCGCAGTGGCTCATCCGCCTGTATCACCTTATAAGGTCTGCG 781 791 801 811 821 831. TGGATCTTGCAGGCCCCGAAGGAGTCTCTGCGAGGGTTTTGGAAGAAACACTCCGGTGAG 841 851 861 871 881 891 CCCGGCACTCTTCTATGGAATACTGTCTGGAATATGGCCGTTATTACCCACTGTTATGAC 901 911 921 931 941 951 TTCCGCGATTTTCAGGTGGCTGCCTTTAAAGGTGATGATTCGATAGTGCTTTGCAGTGAG

962. 971 981 991 1001 1011 TATCGTCAGAGTCCAGGAGCTGCTGTCCTGATCGCCGGCTGTGGCTTGAAGTTGAAGGTA 4 0 1021 1031 1041 1051 1061 1071 GATTTCCGCCCGATCGGTTTGTATGCAGGTGTTGTGGTGGCCCCCGGCCTTGGCGCGCTC 1081 1091 1101 1111 1121 1131 CCTGATGTTGTGCGCTTCGCCGQCCGGCTTACCGAGAAGAATTGGGGCCCTGGCCCTGAG 1141 1151 1161 1171 1181 1191 CGGGCGGAGCAGCTCCGCCTCGCTGTTAGTGATTTCCTCCGCAAGCTCACGAATGTArGCT 1201 1211 1221 123i 1241 1251 CAGATGTGTGTGGATGTTGTTTCCCGTGTTTATZGGGGTTTCCCCTGGACTCGTTCATAAC WO 89/12462 [ICT/ US89/O264K 58

1261. 1271 1281 1291 1301 1313. CTGATTGGCATGCTACAGGCTGTTGCTGATGGCAAGGCACATTTCACTGAGTCAGTAAAA 1321 1331 1341 CCAGTGCTCGACCGGAATTCGAGC, a second sequence 1 11 21 31 41 51 GCTCGAATTCCGGTCGAGCACTGGTTTTACTGACTCAGTGAAATGTGCCTTGCCATCAGC 61 71 81 91 101 ill AACAGCCTGTAGCATGCCAATCAGGTTATGAACGAGTCCAGGGGAAACCCCATAAACACG 121 131 141 151 161 171 GGAAACAACATCCACACACATCTGAGCTACATTCGTGAGCTTGCGGAGGAAATCACTAAC 183. 191 201 211 221 231 AGCGAGGCGGAGCTGCTCCGCCCGCTCAGGGCCAGGGCCCCAATTCTTCTCGGTAAGCCG 241 251 261 271 281 291 GCCGGCGAAGCGCACAACATCAGGGAGCGCGCCAAGGCCGGGGGCCACCACAACACCTGC 301 311 321 331 341 351 ATACAAACCGATCGGGCGGAAATCTACCTTCAACTTCAAGCCACAGC CGGCGATCAGGAC 361 371 381 391 401 413. AGCAGCTCCTGGACTCTGACGATACTC-ACTGCAAAGCACTA TCGAATCATCACCTTTAAA WO 89/12462 PTU8/24 PCY/US89/02648 p 59 4221 432. 442. 452. 461 4721 GGCAGCCACCTGAAAATCGCGGAAGTCATAACAGTGGGTAATAACGGCCATATTCCAGAC 481 4921 5021 51.1 521 531 AGTATTCCATAGA.AGAGTGCCGGGCTCACCGGAGTGTTTCTTCCAAAACCCTCGCAGAGA 541 552. 561. 572. 581 591 CTCCTTCGGGGCCTGCAAGATCCACGCAGACCTTATAAGGTGATACAGGCGGATGAGCCA 602. 611 621 631. 641 651 CTGCGGCATCCCACACTCCTCCATAATAGCACACTCTAGACC CAGAGAAAAGTTATTCTG 661 671 681 6.91 701 711 GGTGGAGTCAAACTCAGAAAAGTCATTCTCAAACACCATGGATGCCTTTGCTGCGGCCAC 721 731 741 751 761 771 AG CCG CCGAGAAGACGGTGTCATCAAAGGCATCACCGTAAAACACACCCTGAGGGAGCAG 781 791 801 811 821 831 GGCCAGAATAGCCTTCTCAATAGCGCGGAACCAAGGGCCAAAGAGGGCGCAGAAGGTCTT 841 851 861 871 881 891 GCTCCAGGCCGAGATGCCCTGGCCCACTTTACCATGGGCAATGGTCTCACCTGTGGTGAA 901 912. 922. 931 941 951 CTTGTTACAATCTTTCTGGAAGAAGGTGATCCTGCACGTCACGGTTGCAAAGATCAAG WO 89/12462 WO 8912462PCT/ US89/02648W 971 1001 1011 CTCAAGGACGGCGGAGCCATCCTGGCCCTTCTCGACCATGGCCTCCACTAGCTCGTACAA 1021 1031 1041 1051 1061 1071 TTCACAAGTTGTAACCTGTACGGGGCCAAITGGCCG'GGATAAAACGGGCGAGAGAGTCGCG 1081 1091 1101 1111 1121 1131 AACATCAGAGTGGGAAGCATTGTAGAGCTTTGTGCGACGC"CGTAGCGGCCCACGAGTGTG 1141 1151 1161 1171 1181 1191 GACAGCACGGCCTTGCGCTGGCTCGGGGCGGCCATGCGGCAGTGCACAATGTCTGTTAAT 1201 1211 1221 1231 1241 1251 TCAAATGTTACGACACTATCACAGGTGGTGAGCTCCTGGGGCAGGTAGAGAAGGCCCTGT 1261 1271 1281 1291 1301 1311 TCGAGCTCGGGGCAGGGTGGTAGAACAGCTGCAACAGGGACAGGTCTTGCCAGATTAGTG 1321 1331 1341 CCTTCCATCAGTTGGCGGAATTCG, a third sequence 1 CGGTTGTTCA GTACCAGTTT 51 CGCTCTATCA CCCAAGCCGA 101 GTTGCGTAAT GCCTGGCGCC 151 CTGCCGCCAG AGTCACCCAG 201 TCCCTCCCCC CTCACCTGCT 251 CCACCTTCTT GGCGCCCCGA 301 CTGGGCTCGT CCCCGCCATC ACTGCAGGTG TGTGGACGTT GTCGCGGCTT GGGCGCCGGG GCTGCTCCAC ACCAGATCCC AGGCCCGACT TGCCTGGATC GTCGTGGTCC TGCTGCTTTT TTGTCATTGA ATGCAGCGGG AGCCATCGAC TAGC C CCAC C CGGCAAGTCC CGACGCGTGA ACCCCGCATA TGAGGCTCCA CCGCCACCGT TTTGAGCACG TCCTGGTGGC WO 89/12462 ~yO 9/1462PC]V/US89/02648 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1253 1L30) 1351 140: 145: 155. 160 165 170 175 180 185 190 19E 20! 21( 21! ATGTTACCCA TACCCCATGA TGAGCCTGCC CCAACCCCGG GAGACCACTA TCGGGCTCAT TCATTGACGC GTTAATAACT AGTTATTCCC TCCCGCCGTC GGCCACAGAC CGAACAGGGC TCGTAACATT AGCCAGCGCA ACAAAGCTCT TTTTATCCCG AGCTAGTGGA *GAGCTTGATC *AGATTGTAAC *GCCAGGGCAI LTGGTTCCGCC L GTTTTACGG) L CAGCAAAGG( 1 ACCCAGAATJ 1. TGGGATGCC( 1 GGATCTTGC, 1 TCCGGTGAG, 1 TATTACCCA 1 GTGATGATT 1 GCTGTCCTG 1 GATCGGTTT '1 CTGATGTTG i1 GGCCCTGAG )l CAAGCTCAC il ATGGGGTTI )l GTTGCTGAM 51 CTTGACAAJ TCGCTGCCCT TCCAGACCAC GTCGGGCAGA CTCAGTIPACG TTATTGCCAC GCCATTGTTG ACCAGGCCTG TTTTCCTCGC CGTGGCAACC TTGCCAGATT CTGTCCCTGT CTTCTCTACC TGAATTAACA AGGCCGTGCT ACAATGCTTC GCCATTGGCC GGCCATGGTC TTTGCA!.ACCG AAGTTCACCP CTCGGCCTGC CTATTGAGAJ GATGCCTTTC ATCCATGGT( SACTTTTCTC! 3CAGTGGCTCQ N. GGCCCCGAA( C CCGGCACTC C TGTTATGAC C GATAGTGCT A TCGCCGGCT G TATGCAGGT T GCGCTTCGC C GGGCGGAGC !G AATGTAGCUI TC CCCTGGAC'I .G GCAAGGCAC kT TCAATCTTC GCGGATGTAT TAGCCGGGTT AACTAGTGTT GTCCACGAGG AGCAGATGCC CTCTGACGCG CTTCGCGAGG TGGTGGCGAA CTGACGCCAA AGTGCCTTCC TGCAGCTGTT TGCCCCAGGA GACATTGTGC GTCCACACTC CCACTCTGAT CCGTACAGGT GAGAAGGGCC TGACGTGTCC CAGGTGAGAC AGCAAGACCI GGCTATTCTC ATGACACCGI TTTGAGAATC C GGGTCTAGAC k TCCGCCTGTJ 3 GAGTCTCTG( r TCTATGGAA' T TCCGCGATT' T TGCAGTGAG G TGGCTTGAA G TTGTGGTGG C GGCCGGCTT A GCTCCGCCT 'C AGATGTGTG !C GTTCATAAC :A TTTCACTG; ;T GTCGGGTGC GCGAGCTCAT Cc CTCCGTTCGT T( CACCCAGGCG Gi CGCAGGGCGC T~ CGGGGCCTTA T' CCACACTGAG A TGGGCATCTC C ATTGGTCACC A TGTTGACACC C ATCAGTTGGC T CTACCACCCT G GCTCACCACC TI ACTGCCGCAT G GTGGGCCGCT P GTTCGCGACT C TACAACTTGT C AGGATGGCTC AGGATCACCT CATTGCCCAT TCTGCGCCCT GCCCTGCTCC CTTCTCGGCG ACTTTTCTGA 3TGTGCTATTA k. TCACCTTATA 2GAGGGTTTTG r ACTGTCTGGA T TCAGGTGGCT T ATCGTCAGAG G TTGAAGGTAG C CCCCGGCCTT A CCGAGAAGAA 'C GCTGTTAGTG !T GGATGTTGTT !C TGATTGGCAT kG TCAGTAAAAC ;A ATGAATAACA CGTGGTGCA GTTCTGGGG CCAAGG CCG PCCTACACG 1'CAGTCGTC AGTGCGTCA GATGCAATC GCGCCCATC TGGCTGCCT GAGGAGCTT CCCCGAGCT 'GTGATAGTG ~GCCGCCCCG ~CGGCGTCGC TCTCGCCCG AATTGTACG GCCGTCCTT 1CTTCCAGAA 37GTAAAGTGG ZTTTGGCCCT CTCAGGGTGT GCTGTGGCCG GTTTGACTCC TGGAGGAGTG AGGTCTGCGT GAAGAAACAC ATATGGCCGT GCCTTTAAAG TCCAGGAGCT ATTTCCGCCC GGCGCGCTCC TTGGGGCCCT ATTTCCTCCG TCCCGTGTTT GCTACAGGCT CAGTGCTCGA TGTCTTTTGC WO 89/12462 ~VO 89/2462 rCriUS$9/0264Vo

2202. 2251

2302. 23521 24021 2451

2501. 2551 TGCGCCCATG CCTCATGTTT GCCGCCGTCG GACCGGGTTG CCCCTTCGCC G CCAAC CCGC CCCGCCGTTG AACCGCGGTC GGTTCGCGAC TTG CCTATGC TGGGCGGCGC ATTCTCAGCC CCCGATGTCA CCGACCACTC CCTCACGTCG GCTCCGGCCC CATGCGCCCT TGCCCGCGCC AGCGGCGGTT CTTCGCAATC CCGCTGCGGC GCGTCCGCTT TAGACCTACC CG, CGGCCTATTT ACCGCCCGGT CCGGCGGTGG CCCTATATTC CGGGGCTGGA GGCGTGACCA ACAGCTGGGG TGTTGCTGCT CAGCCGTCTG TTTCTGGGGT ATCCAACCA.A CCTCGTGTTC GGCCCAGCGC CCGCGCCGCT or a fourth sequence 1 51 101 152. 201 250 301. 350 401 451 501 552. 601 651 701 751 802 852 90J 100: 105: 1120 3 5 115 120 125 GCGGCCGCTC TAAGCAAGGA CAGATGCCAT CAGAGACCAT CTTGCGGCGT TGAGGAGCTG GAGCTTGAGC CAGTGTTGTG CCCCTAGCCA AGACGCACAA TGCGCGCTTT TCTTTGAGCT GTCCTCGAGT CCAGAAGGAT *AAGTCGGTCA *GGCCCCTGGT *AAGCTGTGTT *GTGGCTGGCG L TGACTCGAC'I L AAGAGTGTGC I. TCGGCGTGGI 1 GAAGCATTCI) I. TGGCAATCW' 1 TTCAAGGGCf, 1 AGGCGCCGG', 1 TCCGGCCGA' GGTGGGGTTC ATrTAATTCGC TG;TTAATAAT CGGTCATTCC TTCCACCTTC GGCCACCGGC AGGGCCTTCT ACATTTGAGC AAGCJAAAGCT GGCTTTATGA ATTCCCACTC TGTAGAGGCG TGGATTTGTG TGTAACAAGT GGGTATCTCC TCCGTTGCGA CTACGGGGA'I CCAGCCATGC CAGAATAAC3 TATGCCCCAC STCCTGCAGGC SGGTGAGCCG( C' TGCCCATTG( ACGACTCGG! r TCGCTTATAI r TGGGCTGTA' CACCr'TCTCTT C GGCCGCTCGT G TTCTTCCTTT C GCGAGCAACC C ATGCCAAATA GGCGCCGGTG C CTATCTGCCAC TAACTGACAT GTTTTGTCCA TGCGGGTCAC TCGGGCGGGT ATGGTGGAGA CAGCCGAGAT TCACGACCGG GCCTGGAGTA TTGAGAAGGC GCTTATGACG CATGGTGTTT TTTCCCTAGG TGGCTTGTCA SCCCAAAAGAG GCACGTTGCT 2 TATGAGTTCC r' CGTCCTCTGT 37 CAGGCTGTGG r' GCCGGGGTTG :CCATTCCGA P TTGCGTGAG C :GGGTGGCGA C TGACCGCAA I) ~GCGCCTTCC TGTGCTACC :AGGAGCTAG VGTGCACTGC :GCTGGTAGG ACCGATGTCC TACTGCCACC A.GGGCCAAGA GTCTCCCGCA CGAGACAATT AGACCTTTTG TATTCTATCC ACTCAGTATT GAAA.ATGATT TCTTGAGTGC GGTTGTACCA TCTTTGAGAG CTGGAATACG GGGACCTCCA AGTGAATACC TTTGAAGCCG TCGTCGCCCC LLAGAGAAGT TGGGTATCT GTTGGTCAC ~GTTGACGTG kTCAGCTTGC ICCCTGCCCT CCTCCTGTGA CGCATGGCGG CCGGTATGGC GCGCCTCCCT ACCTGTGAAC C GGTT CAG CC TAACCTTTTT GCGCATGGCA TGCCCTGTTT CTTTTACCAC CTCTGCTGCC TTTCTGAGTT GCCATTIATGG TGCCGTCCGG GGTTCTGGAA GTGTGGAACA GGTTG CCGCC GCCAGAGCCC AAGGCTGACT GGGGCTCGGG 1301 GCCCTACCCG ATGTCGTTCG ATTCGCCGGA CGGCTTTCGG AGAAGAACTG 1351 GGGGCCTGAT CCGGAGCGGG CAGAGCAGCT CCGCCTCGCC GTGCAGGATT 1401 TCCTCCGTAG GTTAACGAAT GTGGCCCAGA TTTGTGTTGA GGTGGTGTCT 1451 AGAGTTTACG GGGTTTCCCC GGGTCTGGTT CATAACCTGA TAGGCATGCT 1501 CCAGACTATT GGTGATGGTA AGGCGCATTT TACAGAGTCT GTTAAGCCTA 1551 TACTTGACCT TACACACTCA ATTATGCACC GGTCTGAATG AATAACATGT 1601 GGTTTCCTGC GCCCATGGGT TCGCCACCAT GCGCCCTAGG CCTCTTTTGC 1651 CGAGCGGCCG C, or a sequence complementary thereto. 4. A recombinant protein derived substantially as hereinbefore described with reference to Figure I. A protein which is Immunoreactive with antibodies present in individuals infected with enterically transmitted nonA/nonB and (b) derived from a viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-KFI(ETI.1) carried in E. coli strain BB4, and having ATCC deposit no.

67717. 6. The protein of claim 5, which is encoded by a coding region within said 1.33 kb EcoRI insert. 7. A method of detecting infection by enterically transmitted nonA/nonB hepatitis viral agent in a test individual, comprising: providing a peptide antigen which is immunoreactive with antibodies present in individuals infected with enterically transmitted nonA/nonB hepatitis and derived from a viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-KF1(ETI.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717, reacting serum from the test individual with such antigen, and Sexamining the antigen for the presence of bound antibody. S 30 8. The method of claim 7, wherein the serum antibody is an IgM or IgG antibody, or a mixture of both, the antigen provided is attached to a support, said reacting includes contacting such serum with the support S. .and said examining includes reacting the support and bound serum antibody with a reporter-labeled anti-human antibody. 9. A method for detecting infection by enterlcally transmitted nonA/nonB hepatitis viral agents in a mammal substantially as hereinbefore described with reference to any of the Examples. A kit for ascertaining the presence of serum antibodies which Sare diagnostic of enterically transmitted nonA/nonB hepatitis infection, comprising e a support with surface-boundArecombinant peptide antigen which is immunoreactive with antibodies present in Individuals Infected with enterically transmitted nonA/nonB viral hepatitis agent and derived from a viral hepatitis agent whose genome contains a region which Is homologous to the 1.33 kb DNA EcoRI Insert present In plasmid pTZ-KFl(ETI.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717, a reporter-labeled anti-human antibody. p%A i-e-A 11. AADNA fragment derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI Insert present In plasmid pTZ-KF1(ET1.1) carried in E. coil strain BB4 and having ATCC deposit no. 67717. 12. The fragment of claim 11, which is derived from said 1.33 kb EcoRI insert. 13. A DNA fragment derived from an enterically transmitted nonA/nonB viral hepatitis agent whose S k 'CS 4695aljj el WO 89/12462 \A~)~39/2462ICTI/ US89/02648 geniome contains a region which is homologous to a duplex DNA, fragment within a first sequence: 1 11 21 31 41 51 CGAAT'TCCGCCAACTGATGGAGGCACTAATCTGGC-t.CCGCCT4TcAcG 61 71 81 91 101 ill TCTACCACCCTGCCCCGAGCTCGAACAGGGCCTTCTCTACCTGCCCCAGGAGCTCACCAC 121 131 141 151 161 171 CTGTGATAGTGTCGTAACATTTGAATTAACA'GACATTGTGCACT, CCGCATGGCCGCCCC 181 191 201 211 221 231 G 'AGCCAGCGCAAGGCCGTGCTGTCCACACTCGTGGGCCGC9>4.! GCGTCGCACAAAGCTC 241 251 261 271 281. 291 TACAATGCTTCCCACTCTGATGTTCGCGACTCTCTCGCCCGTTTTATCCCGGCCATTGGC 301. 311 321 331 341 351 CCCGTACAGGTTACAACTTGTG 1 ATTGTACGAGCTAGTGGAGGCCATGGTCGAGAAGGGC 361. 371 381 391 401 411 CAGGATGGCTCCGCCGTCCTTGAGCTTGATCTTTGCAACCGTGACGTGTCCAGGATCACC 421 431 441 451 461 471 TTCTTCCAGAAAGATTGTAACAAGTTCACCACAGGTGAGACCATTGCCCATGGTAAAGTG 481 491 501 511 521 531 WO 89/12462 C/U8/24 PCT/US89/0264ff 66 GGCCAGGGCATCTCGGCCTGGAGCAAGACCTTCTGCGCCCTCTTTGGCCCTTGGTTCCGC 541 552. 561 571 581 591 GCTATTG- ,AAGGCTATTCTGGCCC.TGCTCCCTCAGGGTGTGTTTTACGGTGATGCCTTT 601 611 621 631 641 651 GATGACACCGTCTTCTCGGCGGCTGTGGCCGCAGCAAAGGCATCCATGGTGTTTGAGAAT 661. 671 681 691 701 711 GACTTTTCTGAGTTTGACTCCACCCAGAATAACTTTTCTCTGGGTCTAGAGTGTGCTATT 721 731 741 751 761 771 ATGGAGGAGTGTGGGATGCCGCAGTGGCTCATCCGCCTGTATCACCTTATAAGGTCTGCG 781 791 801 811 821 831 TGGATCTTGCAGGCCCCGAAGGAGTCTCTGCGAGGGTTTTGGAAGAAACACTCCGGTGAG 841 851 861 871 881 891 CCCGGCACTCTTCTATGGAATACTGTCTGGAATATGGCCGTTATTACCCACTGTTATGAC 901 911 921 931 941 951 TTCCGCGATTTTCAGGTCGCTGCCTTTAAAGGTGATGATTCGATAGTGCTTTGCAGTGAG 961 971 981 991 1001 1011 TATCGTCAGAGTCCAGGAGCTGCTGTCCTGATCGCCGGCTGTGGCTTGAAGTTGAAGGTA 1021 1031 1041 1051 1061 1071 GATTTCCGCCCGATCGGTTTGTATGCAGGTGTTGTGGTGGCCCCCGGCCTTGGCGCGCTC WO 89/12462 PCT/ US89/02648 67 1081 1091 1101 1111 1121 1131 CCTGATGTTGTGCGCTTCGCCGGCCGGCTTACCGAGAAGAATTGGGGCCCTGGCCCTrGAG 1141 1151 1161 1171 1181 1191 CGGGCGGAGCAGCTCCGCCTCGCTGTTAGTGATTTCCTCCGCAAGCTCACGAATGTAGCT 1201 1211 1221 1231, 1241 1251 CAGATGTC4TGTGGATGTTGTTTCCCGTGTTTATGGGGTTTCCCCTGGACTCGTTCATAAC 1261 1271 1281 1291, 1301 1311 CTGATTGGCATGCTAkCAGGCTGTTGCTGATGGCAAGGCACATTTCACTGAGTCAGTAAAA 1321 1331 1341 CCAGTGCTCGACCGGAATTCGAGC, a second sequence 1 11 21 31 41 51 GCTCGAATTCCGGTCGAGCACTGGTTTTACTGACTCAGTGAAATGTGCCTTGCCATCAGC 61 71 81 91 101 ill AACAGCCTGTAGCATGCCAATCAGGTTATGAACGAr2TCCAGGGGAAACCCCATAAACACG 121 131 141 I 161 171 GGAAACAACATCCACACACATCTGAGCTACATTCGTGAGCTTGCGGAGGAAATCACTAAC 181 191 201 211 221 231 WO 89/12402 WO 89t2462PCI'! US$9/0264W' 68 AGCGAGGCGGAGCTGCTCCGCCCGCTCAGGGCCAGGGCCCCAATTCTTCTCGGTAAGCCG 241 251. 261 271 281 291 GCCGGCGA,'AGCGCACAACATCAGGGAGCGCGCCAAGGCCGGGGGCCACCACAACACCTGC 301 311 321 331 341 351 ATACA.AACCGATCGGGCGGAAATCTACCTTCAACTTCAAGCCACAGCCGGCGATCAGGAC 361 371 381 391. 401 411 AGCAGCTCCTGGACTCTGACGATACTCACTGCAAAGCACTATCGAATCATCACCTTTAAA 421 431 441 451 461 7 GGCAGCC)A.CCTGAAAATCGCGG,-AAJTCATAACAGTGGGTAATAACGGCCATATTCCAGAC 481 491 501. 511 521 531 AGTATTCCATAGAAGAG;TGCCGGGCTCACCGGAGTGTTTCTTCCAAAACCCTCGCAGAGA WO 89/12462 'C/U8/24 1-'Ct*/(JS89/02(w4g 69 541 551 561 571 581. 591 CTCCTTCGGGGCCTGCAAGATCCACGCAGACCTTATAAGGTGATACAGGCGGATGAGCCA 601 611 621 631 642. 651 CTGCGGCATCCCACACTCCTCCATAATAGCACACTCTAGACCCAGAGAAAAGTTATTCTG 66)1 671 681 691 701 711 GGTGGAGTCAAACTCAGAAAA GTCATTCTCAAACACCATGGATGCCTTTGCTGCGGCCAC 721. 731 741 751 761 771 AGCCGCCGAGAAGACGGTGTCATCAAAGGCATCACCGTAAAACACACCCTGAGGGAGCAG 781 791 801 811 821 831 GGCCAGAATAGCCTTCTCAATAGCGCGGAACCAAGGGCCAAAGAGGGCGCAGAAGGTCTT 841 8531 861 871 881 891 GCTCCAGGCCGAGATGCCCTGGCCCACTTTACCATGGGCAATGGTCTCACCTGTGGTGAA 901 911 921 931 941 951 CTTGTTACAATCTTTCTGGKAGAAGGTGATCCTGGACACGTCACGGTTGCAAAGATCAAG 9621 971 981 991 1001 1011 CTCAAGGACGGCGGAGCCATCCTGGCCCTTCTCGACCATGGCCTCCACTAGCTCGTACAA 1021 1031 1042 1051 1061 1071 TTCACAAGTTGTAACCTGTACGGGGCCAATGGCCGGGATAAAACGGGCGAGAGAGTCGCG WO $9/12462 WO 8912462ICT/ US89/026410 1081. 1091 1101 1111 1121 1131 AACATCAGAGTGGGAAGCATTGTAGAGCTTTGTGCGACGCCGTAGCGGCCCACGAGTGTG 1141 1151 1161 .1171 1181 1191 GACAGCACGGCCTTGCGCTGGCTCGGGGCGGCCATGCGGCAGTGCACAATGTCTGTTA.AT 1201 1211 1221 1231 1241 1251 TCAALATGTTACGACACTATCACAGGTGGTGAGCTCCTGGGGCAGGTAGAGAAGGCCCTGT 1261 1271 1281 1291 1301 1311 TCGAGCTCGGGGCAGGGTGGTAGAACAGCTGCAACAGGGACAGGTCTTGCCAGATTAGTG 1321 1331 1341 CCTTCCATCAGTTGGCGGAATTCG, a third sequence 1 CGGTTGTTCA GTACCAGTTT ACTGCAGGTG TGCCTGGATC CGGCAAGTCC 51 CGCTCTATCA 101 GTTGCGTAAT 151 CTGCCGCCAG 201 TCCCTCCCCC 251 CCACCTTCTT 301 CTGGGCTCGT 351 ATGTTACCCA 401 TACCCCATGA 451 TGAGCCTGCC 501 CCAACCCCGG 551 GAGACCACTA 601 TCGGGCTCAT 651 TCA'TTGACGC CCCAAGCCGA GCCTGGCGCC AGTCACCCAG CTCACCTGCT GGCGCCCCGA CCCCGCCATC TCGCTGCCCT TCCAGACCAC GTCGGGCAGA CTCAGTGACG TTATTGCCAC GCCATTGTTG ACCAGGCCTG TGTGGACGTT GTCGCGGCTT GGGCGCCGGG GCTGCTCCAC ACCAGATCCC AGGCCCGACT GCGGATGTAT TAGCCGGGTT AACTAGTGTT GTCCACGAGG AGCAGATGCC CTCTGACGCG CTTCGCGAGG GTCGTGGTCC TGCTGCTTTT TTGTCATTGA ATGCAGCGGG AGCCATCGAC TAGCCCCACC GCGAGCTCAT CTCCGTTCGT CACCCAGGCG CGCAGGGCGC CGGGGCCTTA CCACACTGAGi TGGGCATCTC CGACGCGTGA ACCCCGCATA TGAGGCTCCA CCGCCACCGT TTTGAGCACG TCCTGGTGGC CCGTGGTGCA TGTTCTGGGG GCCAAGGCCG TACCTACACG TTCAGTCGTC AAGTGCGTCA CGATGCAATC 701 GTTAATAACT TTTTCCTCGC TGGTGGCGAA ATTGGTCACC AGCGCC.CATC wo 89/12462 WO 89/2462 C/ US$9/02648 751 802. 851 AGTTATTCCC TCCCGCCGTC CGTGGCAACC CTGACGCCAA TGTTGACACC CTGGCTGCCT TTGCCAGATT AGTGCCTTCC ATCAGTTGGC GGCCACAGAC CTGTCCCTGT TGCAGCTGTT CTACCACCCT 901 CGAACAGGGC CTTCTCTACC TGCCCCAGGA 951 1001 1051 1101 TCG'IAACATT TGAATTAACA GACATTGTGC AGCCAGCGCA AGGCCGTGCT A CAAAG CTCT TTTTATCCCG ACAATGCTTC GCCATTGGCC GTCCACACTC CCACTCTGAT CCGTACAGGT GCTCACCACC ACTGCCGCAT GTGGGCCGCT GTTCGCGACT TACAACTTGT 1151 AGCTAGTGGA GGCCATGGTC GAGAAGGGCC AGGATGGCTC 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2152 2202 2252 230: 235: 24 0: 245:. 250 255 GAGCTTGATC T AGATTGTAACA GCCAGGGCAT C TGGTTCCGCG C GTTTTACGGT G CAGCAAAGGC P ACCCAGAATA P TGGGATGCCG C GGATCTTGCA C TCCGGTGAGC TATTACCCAC GTGATGATTC GCTGTCCTGAI GATCGGTTTG CTGATGTTGT4 GGCCCTGAGC CAAGCTCACG ATGGGGTTTC GTTGCTGATG *CTTGACAAAT *TGCGCCCATG L CCTCATGTTT L GCCGCCGTCG L GACCGGGTTG 1. CCCCTTCGCC I. GCCAACCCGC I. CCCGCCGTTG 1 AACCGCGGTC TTGCAACCG 'T AGTTCACCA C *TCGGCCTGG P TATTGAGAA C ATGCCTTTG I LTCCATGGTG IJ ~CTTTTCTCTC AGTGGCTCA ;GCCCCGAAG :CGGCACTCT VGTTATGACT ;ATAGTGCTT TCGCCGGCTG TATGCAGGTG GCGCTTCGCC GGGCGGAGCA AATGTAGCTC CCCTGGACTC GCAAGGCACA TCAATCTTGT GGTTCGCGAC TTGCCTATGC TGGGCGGCGC ATTCTCAGCC CCCGATGTCA CCGACCACT,.C CCTCACGTCG GCTCCGGCCC GACGTGTCC A :AGGTGAGAc C ~GCAAGACCT 'I ;GcTATTcTG C ~TGACACCGT C LTTGAGAATG I ;GGTCTAGAG rCCGCCTGTA ;AGTCTCTGC TCTATGGAAT TCCGCGATTT TGCAGTGAGT TGGCTTGAAG TTGTGGTGGC GGCCGGCTTA GCTCCGCCTC AGATGTGTGT GTTCATAACC TTTCACTGAG GTCGGGTGGA CATGCGCCCT TGCCCGCGCC AGCGGCGGTT CTTCGCAATC CCGCTGCGGC ~GGATCACCT :ATTGCCCAT 'CTGCGCCCT ;CCCTGCTC TTCTCGGCG ~CTTTTCTGA ['GTGCTATTA VCACCTTATA 3AGGGTTTTG PCTGTCTGGA TCAGGTGGCT FTCGTCAGAG TTGAAGGTAG CCCCGGCCTT CCGAGAAGAA GCTGTTAGTG GGATGTTGTT TGATTGGCAT TCAGTAAAAC ATGAATAACA rGAGGAGCTT 'CCCCGAGCT EGTGATAGTG 6GCCGCCCCG hCGGCGTCGC CTCTCGCCCG GAATTGTACG CGCCGTCCTT TCTTCCAGAA GGTAAAGTGG CTTTGGCCCT CTCAGGGTGT GCTGTGGCCG GTTTGACTCC TGGAGGAGTG AGGTCTGCGT GAAGAAACAC ATATGGCCGT GCCTTTAAAG TCCAGGAGCT ATTTCCGCCC GGCGCGCTCC TTGGGGCCCT ATTTCCTCCG TCCCGTGTTT GCTACAGGCT CAGTGCTCGA TGTCTTTTGC CGGCCTATTT TGTTGCTGCT ACCGCCCGGT CCGGCGGTGG CCCTATATTC CGGGGCTGGA CAGCCGTCTG TTTCTGGGGT ATCCAACCAA CCTCGTGTTC GCGTCCGCTT GGCGTGACCA GGCCCAGCGC TAGACCTACC CG, ACAGCTGGGG CCGCGCCGCT WO 89/12.462 PC1'/US89/0)2640 or a fourth sequence 1 GCGGCCGCTC 51 TAAGCAAGGA 101 CAGATGCCAT 151 CAGAGACCAT 201 CTTGCGGCGT 250 TGAGGAGCTG .0 301 350 401 451 501 551 601 651 701 751 801 851 902 100] 105: 110: 115: 120 125 130 135 140 145 150 15E GAG CTT GAG C CAGTGTTGTG CCCCTAGCCA AGACGCACAA TGCGCGCTTT TCTTTGAGCT GTCCTCGAGT CCAGAAGGAT AAGTCGGTCA GGCCCCTGG'I AAGCTGTGT'I *GTGGCTGGCC *TGACTCGACI *AAGAGTGTGC GGTGGGGTTC CACCTCTCTT ATTAATTCGC GtGC"CGCTCGT TGTTAATAAT TTCTTCCTTT CGGTCATTCC GCGAGCAACC TTCCACCTTC ATGCCAAATA GGCCACCGGC GGCGCCGGTG AGGGCCTTCT CTATCTGCCA ACATTTGAGC TAACTGACAT AAGGAAAGCT GTTTTGTCCA GGCTTTATGA TGCGGGTCAC ATTCCCACTC TCGGGCGGGT TGTAGAGGCG ATGGTGGAGA TGGATTTGTG CAGCCGAGAT TGTAACAAGT TCACGACCGG GGGTATCTCC GCCTGGAGTA TCCGTTGCGA TTGAGAAGGC CTACGGGGAT GCTTATGACG CCAGCCATGC CATGGTGTT'I CAGAATAACT TTTCCCTAGC TATGCCCCAG TGGCTTGTCP CTGACCGCAA TGTTGACGTG A C C T C GCGCCTTCC TGTGCTACC AGGAGCTAG 'GTGCACTGC :GCTGGTAGG .CCGATGTCC 'ACTGCCACC ~GGGCCAAGA TCTCCCGCA ZGAGACAATT kGACCTTTTG rATTCTATCC ~ACTCAGTATT GAAATGATT T CTTGAG TG C GGTTGTACCP TCTTTGAGAC CTGGAATACC GGGACCTCCI AGTGAATACC TTTGAAGCCC TCGTCGCCC( CGGCTTTCGI CCGCCTCGCI TTTGTGTTG CATAACCTG TACAGAGTC GGTCTGAAT ATCAGCTTGC TCCCTGCCCT CCTCCTGTGA CGCATGGCGG CCGGTATGGC GCGCCTCCCT ACCTGTGAAC CGGTTCAGCC TAACCTTTTT GCGCATGGCA TGCCCTGTTT CTTTTACCAC CTCTGCTGCC TTTCTGAGTT GCCATTATGG TGCCGTCCGG GGTTCTGGAA GTGTGGAACA GGTTGCCGCC SGCCAGAGCCC AAGGCTGACT SGGGGCTCGGG 3 AGAAGAACTG CGTGCAGGATT A~ GGTGGTGTCT A TAGGCATGCT T GTTAAGCCTA G AATAACATGT CCCATTCCGA GTTGCGTGAG CGGGTGGCGA ACAGAGAAGT GTGGGTATCT GGTTGGTCAC TCGGCGTGGA TCCTGCAGGC CCCAAAAGAG GAAGCATTCT GGTGAGCCGG GCACGTTGCT TGGCAATCAT TGCCCATTGC TATGAGTTCC TTCAAGGGCG ACGACTCGGT CGTCCTCTGT AGGCGCCGGT TCGCTTATAG CAGGCTGTGG TCCGGCCGAT TGGGCTGTAT GCCGGGGTTG GCCCTACCCG ATGTCGTTCG ATTCGCCGGA GGGGCCTGAT CCGGAGCGGG CAGAGCAGCT TCCTCCGTAG GTTAACGAAT GTGGCCCAGA AGAGTTTACG GGGTTTCCCC GGGTCTGGTT CCAGACTATT GGTGATGGTA AGGCGCATTT TACTTGACCT TACACACTCA ATTATGCACC 1601 1651 GGTTTCCTGC GCCCATGGGT TCGCCACCAT GCGCCCTAGG CGAGCGGCCG C, CCTCTTTTGC 73. or a sequence complementary thereto. 14. The DNA fragment of claim 13, wherein said fragment contains a coding sequence homologous to nucleotides 2 through 101 of said first sequence, nucleotides 594 through 643 of said second sequence, or a sequence complementary to said coding sequences. A kit comprising, in a container or separate containers, a pair of single-strand primers derived from non-homologous regions of opposite strands of a\DNA duplex fragment derived from an enterically transmitted viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-KF1(ET1.1) carried in E. coli strain BB4 and having ATCC deposit no. 67717. 16. The kit of claim 15, which are derived from opposite strands of the EcoRI duplex insert in said plasmid. 17. A method for detecting the presence of an enterically transmitted nonA/nonB hepatitis viral agent in a biological sample, comprising preparing a mixture ofAduplex DNA fragments derived from the sample, denaturing the duplex fragments, adding to the denatured DNA fragments, a pair of single-strand primers derived from non-homologous regions of opposite strands of a DNA duplex fragment derived from an enterically transmitted viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-KF1(ETI.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717, hybridizing said primers to homologous-sequence region of opposite S* strands of such duplex DNA fragments derived from enterically transmitted nonA/nonB hepatitis agent, reacting the primed fragment strands with DNA polymerase in the 30 presence of DNA nucleotides, to form new DNA duplexes containing the primer sequences, and repeating said denaturing, adding, hybridizing and reacting steps, until a desired degree of amplification of sequences is achieved. 18. The method of claim 17, wherein the primers are derived from opposite strands the EcoRI duplex insert in said plasmid. 19. A, method for detecting the presence of an enterically transmitted nonAinonB hepatitis viral agent in a biological sample substantially as hereinbefore described with reference to any of the Examples. S- 695a/jj 74. The method of claim 17, for detecting the presence of viral agent in a sample of cultured cells infected with the agent. 21. A method for detecting an enterically transmitted nonA/nonB hepatitis viral agent In a sample of cultured cells infected with the agent substantially as hereinbefore described with re"'-ence to any of the Examples. 22. A vaccine for immunizing an individual against enterically transmitted nonA/nonB hepatitis viral agent.comprising, in a pharmacologically acceptable adjuvant, aArecombinant protein derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcRI insert present in plasmid pTZ-KFl(ET1.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717. 23. The vaccine of claim 22, wherein the protein is derived from the EcoRI insert in said plasmid. 24. A method of immunizing a mammal against an enterically transmitted nonA/nonB hepatitis viral agent which method comprises administering to said mammal an effective amount of a protein according to any one of claims 1 to 6 or of a vaccine according to claim 22 or 23. 25. In a method of isolating an enterically transmitted nonA/nonB viral agent or a nucleic acid fragment produced by the agent, an improvement which comprises: utilizing, as a source of said agent, bile obtained from a human or cynomolgus monkey having an active infection of enterically transmitted non-A/non-B hepatitis. 26. The method of claim 25, wherein the bile is obtained from an S infected cynomolgus monkey. 27. .Human olyclonal anti-serum obtained from a human immunized with a-protein derived from an enterically transmitted non-A/non-B viral 30 hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-KF'I(ET1.1) carried in E. coli strain BB4 and having ATCC deposit no. 67717. 28. A method for treating enterically transmitted nonA/nonB hepatitis viral agent in a mammal requiring such treatment or prophylaxis, which method comprises administering to said mammal an effective amount of a composition comprising human polyclonal anti-serum according to claim 27 or obtained from the plasma or serum of a mammal immunized according to claim 24. o ,.4695a/jj A> DATED this FIRST day of JULY 1992 Genelabs Incorporated The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services and his Successors Patent Attorneys for the Applicant SPRUSON FERGUSON I I- .h i ,I 'G r 4, r :1U 4695a/jj