AU724908B2 - Equine rhinovirus 1 proteins - Google Patents

Description

WO 97/22701 PCT/AU96/00815 1 EQUINE RHINOVIRUS 1 PROTEINS INTRODUCTION TO INVENTION This invention relates to the equine rhinovirus 1 (ERhV1) which has been sequenced and characterized. In particular, the invention relates to nucleotide and protein sequences of ERhVl and a range of clinical and diagnostic products derived from ERhVl.

BACKGROUND OF INVENTION Equine rhinovirus 1 (ERhVI) was first isolated from horses in the United Kingdom and subsequently from horses in mainland Europe, the USA and Australia. Most isolates were from the nasopharynx of horses with an acute, febrile respiratory disease. Virions had the characteristic size and morphology of picomaviruses and were acid-labile. Two other serologically distinct, acid-labile picornaviruses, ERhV2 and ERhV3, have also been isolated from horses.

Considerable uncertainty has surrounded the classification of ERhV1.

Physicochemical studies have shown that the nucleic acid density and base composition of ERhV1 differ from those of rhinoviruses. In contrast to rhinoviruses, ERhVl has a broad host-cell range in vitro and in vivo and there is no evidence of extensive antigenic variation. Infection of horses with ERhV1 causes a disease characterized by an acute febrile respiratory disease accompanied by anemia, fecal and urine shedding and viral persistence. The signs of systemic infection and persistence are not characteristic of rhinovirus infections in other species. The known host range of ERhV1 is broad and includes rabbits, guinea pigs, monkeys and humans, although in these species the virus does not appear to spread horizontally. There is both experimental and epidemiological evidence of ERhV1 infection of humans. A human volunteer inoculated intranasally with ERhV1 developed severe pharyngitis, lymphadenitis, fever and viremia, and high ERhV antibody titers were found in the sera of 3 of 12 stable workers whereas no ERhV1 antibody was found in the sera of 159 non-stable workers.

In order to clarify the taxonomic status of ERhV1, a detailed study was undertaken to determine the nucleotide and amino acid sequence of ERhV1. The resultant studies provided the complete nucleotide sequence of the gene encoding SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 2 the ERhV I polyprotein and the 3'-nontranslated region (NTR) as well as part of the nucleotide sequence of the 5'NTR. The amino acid sequence of the various ERhV I proteins was deduced from the nucleotide sequence.

The analysis of the nucleotide sequence of ERhV1 confirmed previous studies which indicated that many properties of ERhVl are not consistent with those of other members of the genus Rhinovirus. Indeed many of the physicochemical and biological properties of ERhV1 have suggested ERhVI is more closely related to foot-and-mouth disease virus (FMDV) the sole member of the Alpthovirus genus. In addition to the overall sequence similarity, several features of the ERhVl genome are similar to those of FMDV. The ERhV1 L protein is most similar to its counterpart in aphthoviruses in both length, 207 amino acids in ERhVl and 201 in FMDV, and in amino acid sequence identity. In aphthoviruses, the L protein catalyses its own cleavage from the polyprotein, and mediates cleavage of the p220 component of the cap-binding complex leading to inhibition of translation of capped mRNAs. Cardiovirus L proteins are only 67-76 amino acids long and are not auto catalytic. In contrast to the cardioviruses, aphthoviruses utilize two distinct initiation codons, which results in different forms of the L protein, Lab and Lb, differing from each other by 28 amino acids at their N-termini.

The second initiation codon occurs in a more favourable context, which is presumably the reason why Lb, the smaller of the two proteins, is the predominant species. Thus far, differences in the function of the two FMDV L proteins have not been detected. ERhVI also possesses a second ATG, 63 bases downstream from the first optimal ATG, which is also present in a context optimal for initiation of translation. Translation from this ATG would result in an L protein with 21 fewer amino acids at its N-terminus. Therefore, it is probable that ERhV1 possesses a second species of L protein, similar to the FMDV Lb protein. If so, the reason for the existence and conservation of two forms of the L protein in ERhV I and FMDV is an intriguing question. Curiously, ERhV1 has tandemly repeated ATG codons at each of the possible initiation sites, where the first ATG in each case does not SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 3 occur in a context optimal for translation. The role of these ATGs may be to ensure that translation is initiated from both possible initiation sites.

The 2A protease is only 16 amino acids in length in both FMDV and ERhV1, compared to 142-149 amino acids in other picornaviruses. In FMDV 2A protease cleaves at its C-terminus but, unlike the 2A protease of other picomaviruses, appears not to have a role in shut down of host cell macromolecular synthesis. The high degree of conservation of the FMDV and ERhV 2A proteins is intriguing and suggests an important role for this protein in the diseases produced by these viruses.

It may be expected that the tree derived from the complete polyprotein coding sequence would provide the most representative view of the taxonomic status of ERhV1 by reducing any bias imparted by using restricted parts of the genome with highly variable evolutionary rates. However, such analysis is restricted because there are only a few complete polyprotein sequences available.

The polymerase genes are the most conserved genes in positive strand RNA viruses and they have been used to construct a taxonomy, and to predict the ancient roots, of these viruses. In contrast to the polymerase gene, the VP1 gene encodes the major antigenic determinants of the virus and evolves more rapidly than other regions in the genome. The diversity of VP1 regions make them useful for the study of closely related picoraviruses. Thus, trees based on the polymerase and VP1 genes presumably reflect the extremes of evolutionary rates from which the taxonomic status and evolutionary origin of ERhV1 could be identified. The ERhV1 VP1 amino acid sequence was more similar to FMDV than to any other sequence in the data base; this was true even when representative segments across the entire sequence were separately analysed.

Therefore, we consider that the difference in the topology of the VP1, compared to the other two trees, is most unlikely to be a consequence of genetic recombination. The topographic differences between the three ERhV1 trees compared to those of aphthoviruses, particularly the VP1 derived trees, as well as the presence of only one VPg gene in ERhV1 genome, leads us to conclude that SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU9610081 4 ERhVI is probably a member of a distinct genus proposed to be called Equirhinovirus.

The reassessment of the taxonomic status of ERhVl focuses on a requirement to reassess the biology of the virus particularly with respect to the nature of clinical disease as well as means for control by vaccination and improved methods of diagnosis. For example, cardioviruses and aphthoviruses cause viremic infections accompanied by myocarditis. Clinical disease caused by ERhV1 is generally considered to be confined to the respiratory tract even though there is a viremia and the virus is shed in faeces and urine. Whether ERhV 1 infection produces systemic disease similar to that observed in aphthovirus or cardiovirus infections, including the production of myocarditis, needs to be investigated. There is serological. evidence that the incidence of ERhV 1 infection is as high as 50% in some horse populations however, the number of reported isolations of ERhV1 is very small. We have clear evidence that primary isolation of the virus from clinical specimens is known to be difficult, suggesting that the true incidence of ERhV1 disease is much greater than reported.

The determination of the complete nucleotide sequence of ERhV1 polyprotein has important practical applications in developing novel methods for the diagnosis and control of ERhV disease in horses and other species.

OBJECT AND STATEMENT OF MNVNTION In one aspect, the invention provides a substantially pure nucleotide sequence for ERhV1 being: a substantially pure nucleotide sequence for ERhVL being: CCGTCAAGCC CGTTGCCTGT ATAGCCAGGT AACCGGACAG CGGCTTGCTG GATTTTCCCG -375 GTGCCATTG;C TCTGG;ATGGT GTCACCAAGC TGACAAATGC GGAGTGAPLCC TCACAAAGCG -315 ACACGCCTGT GGTAGCGCTG CCCAAAAGGG AGCGGAACTC CCCGCCGAGG CGGTCCTCTC -255 TGGCCAAAAG CCCAGCGTTG ATAGCGCCTT TTGGGATGCA GG?.ACCCCAC CTGCCAGGTG 195 TGAAGTGGAG TGAGCGGATC TCCAATTTGG TCTGTTCTGA ACTACACCAT TTACTGCTGT -135 GAAGAATGCC CTGGAGGCAA GCTGGTTACA GCCCTGACCA GGCCCTGCCC GTGACTCTCG ACCG.GCGCAG GGTCAAAAAT TGTCTAAGCA GCAGCAGGAA CGCGGGAGCG TTTCTTTTCC TTTGTACTG ACATGATGGC GGCGTCTAAG GTGTATAGAG TTTGCGAGCA GACTCTGCTG GCAGGTGCCG TTCGCATGAT GGACAAATTC TTGCAAAAGA GAACTGTTTT TGTCCCCCAT 105 CTTGACAAAA CALATTCGTTT GACTGGACTC CACAATTATG ACAATACTTG CTGGTTGAAT 1.65 GCCTTGACAC AACTGACACA GATTCTTGGA ATTCGGCTTT TTGATGAACA CTTCGGCAAT 225 AGAGGTCTGT TCALCTcG.GAA AACAATTGAT TGGGTGAGTG ACCAGACTGG TATAAAAGAT 285 SUBSTITUTE SHEET (Rule 26) WO 97/22701PC/U6085 PCT/AU96/00815 CTAAAATCAG GAGCACCGCC GATGTCG-GTA CGATGGAGAA

CTTTCTGATT

ACAAGCGAGG

GAGGATGTAC

CTACACCAGA

ACTGGCAACC

CAGTACCAGA

GGCAGCAACA

TCTAGTTTGC

AACATTGAAG

TCTGTTGGAA

GACCCAGTTA

TGGCCCCATT

AAGAAGATGG

GATGTGGTTG

GTGCCGGAGT

GCTTACACAT

TCATCAGTGC

CACAACCCGT

GTGCCTGTGA

CCAGAGGCAC

CCTGATAATT

CGGTTTACAA

AAAccTTATT

TCGCTCASTG

CAGTACAGAG

AAGTTTCTGG

ATGGCGTGCA

CCTTATCCCT

GTCTCTGGAT

AACAGTAAAG

CCGGCGGACC

ACAGAGCCTC

TTGcTG.cc

GCCACACATG

AACACTTGCA

ACTCCGTCCT

ACCAAGACCA

TTGGCCGTTG

GCCTGTGCTT

GGCTGGGGTG

GGTTCCATCT

TTCCGCGCGC

GACAAAACCA

CTCAAATTGG

GCAGACCTGA

TCTGGGCCTG

GTTGGATCTG

TTGTTTTTGC

GATATGAAAA

AAAACATGTC

ATTCAATTGA

CTAGTAGTTC

TGAACCTTGG

ACAGAATTGA

CAACCTACTG

CCAGACTTGG

CTCAATCACA

GC-AGTTTTCA

TGCALGGTGAA

ACGAACACAC

ACCAACAACT

ATTGGTGAT

GGACCATTGT

CCATGTCGGT

CGATTAGAGT

CGACTCCACT

ATTTCATTGA

TTGAGGTTAC

CGGCAGAGCT

GCTCACTTAA

TTGCTTTTrGT

TCCATGCCGT

CCCCTGCTGA

GGCTTCAAGT

GCCGTGTGCT

TGCCCGACAA

GTCATGCTTT

GGTTCTTTGA

TTCTGGATCC

CCTACTTCTT

CTGTTGGAGA

CAGATGCTTG

CAGGGATGTG

CAGCGTTGCC

CACCTACCAA

TGCTGACTTC

CAGCGATGTA

GACATAAATT

CTGGAGATGT

ATGCCTTGTC

ACTCGTGGTG

ACCCCGGTCG

TGTTTCGGCA

TGTTGATGAC

GCCCTATGAC

AGCATTTGAG

CGGAAACAGT

CGCAGACCTG

AACCTCATCA

AACAAAACTA

AACAACAGTG

TTACTCCAAA

ACCCACGCTT

TGGTC-ACGCA

TGAGGTTGTC

TCCCTCATTT

ACATGAGAAA

TTCAGTTTTT

GCCCTACATT

TATTTTAATT

GGCTCCCATC

GGTGTCTGTG

ATACCCCAAG

TGTGGCAAAA

CAACACCTCT

ACATGGCACT

TTTCAACTTT

GCCTCCCCAC

GTGGGATGTT

CTTCATGGCC

TTATGCACTA

GGTTGCTGTT

GCAGGTTACC

GTCACCCGTG

TGTTGAGACA

GTTTGGCTCG

TTCTGATTTG

GGGCTTTGTG

GCAGTTAGAA

CCCCACTGTT

ATATACATCA

AGAAAAGGCA

TGATGCGCAT

TTGCCCTCGA

TCCCACTAAC

TGAGAGCAAC

AACGTCGCTA

GTGTACA.AAC

ATTACTCTAT

AAACCGGGAC

AAGAAAATAT

TTTGAGTCAC

CTCAGTGGCG

GGTTCAATTG

GGAGACAATG

AGCCAATCCT

CTGGCTGACA

GTTGGAGTCA

CCGGATGGTA

TCCAGGCACT

TGGATCTCTC

AAAGCCCACC

GCTCACTCCG

GCACTCAAGT

CCCCACCAGT

GGGCCAGGCC

TTGTCTGAAT

GAT G CAT GG

CCTGAATCAG

GTTGTGGTCC

CAGACATATT

GGGGACGAGC

TACGTAGCTA

ATTT'TCACTG

AGTGCAGCGC

GCCTTGAACT

GTTTATTCTG

ACAGCTCTAA

TCCGCCGGCC

AATGTGGGAG

GACATGCACG

CTTGAGCTTT

ACTGCCCAAC

GAATTGTCAA

TGTGGCAACA

GGTCTIGGCCC

ATGCAGTATT

AC CCAGAAAC

TGGGCAAGGA

GAGGTACATC

TTCAAAATTT

TGATTAGCCC

CTGGCTTGGG

AGAAGACAGA

CTATTATTAA

GACCACCATC

ACACATTTAA

CGTTGCCAGG

ACCTGGTCAA

GGCCGCTGTG

GGTCTG;AGCT

TGCTAA.ATTT

AACCAACAAA

TGACAGGACC

TTAATGGGCC

ATTCTTTAT

CACCGCGCCA

CATTTTGTTC

CALCTGTTTCA

GTTTGTCATC

GTGCAGCAGC

CCAAAACGCG

CAGCTTTTTC

CGGAACGGAC

CTTCAACTGA

CAGACTTCTC

AGGATGGTGA

TGCACACAGA

CA.AATTTGAC

TGGCTTGGGC

TCCAGTTTA.

TGGACACTTG

CAAAGTGTGT

CTACCTTCTC

ACCCAA.AACA

CCCACC.AAAG

CACTCCAACA

TTACATGCAA

TGAAGGCCAG

CGGGTGGTTC

AGAGACTACA

TTCACA7AGGA CACAGTGTCAk

GGTAGGTGAG

TGACAAACTC

GAACGGCTGG

TGTAGCAGCA

TGAGGAACCA

GAGGACAAAT

TCTGACTTTG

TGGCCAAACT

TCTTCCAAAT

GTCTTCAGTA

AGTTCCTGGC

CATTTCTGGA

GATGGATGTG

ATTTTTTGCA

CACTAAGGCA

CGATGAAGCA

TTTTAATGTA

GGTTGTGAAT

CATTGCCGTG

CCTTCGTCAC

ACCCGGTGAG

TGTCAGTTTC

AGGTTCTCCT

ACGTCTGCTA

ATTTACCACC

TGGAGCACCA

AATTCAAAAA

CCGTTCACAA

CTTTTACAAT

TGCACACTTT

GCGGTATGCT

GCGTCCAGCG

TTACTCTCTC

CAAAGCATCA

AAAAGATCTT

345 405 465 525 585 645 705 765 825 885 945 1005 1065 1125 1185 1245 1305 1365 1425 1485 1545 1605 1665 1725 1785 1845 1905 1965 2025 2085 2145 2205 2265 2325 2385 2445 2505 2565 2625 2685 2745 2805 2865 2925 2985 3045 3105 TGGGTGAPAGT GGCTCCCTGT GGAGGTGGAA ATTCAGTTAG GTGTTCAAGA TTGCAGGCTC ATGTGGCGTG TTGTGCCAGT ACCTACAATT GGCTTCCTGG GATAAAGGAG GGTGCTACTT CCCATTCCGC CGGCTTTTAC ATCAACAAAc AGTGTACTAA CCTGGCCCCA CTATTTTTTC GGTGAATTGA CTGGCATGCT SUJBSTrF"rB SHEET (Rule 26) PCT/AU96/0O81 WO 97/22701

AAAGCCAAGG

GCAACACTAG

GCTGATTTCG

TTGCAGCCTT

ACG;GCTGCCG

CCTGAAGGAG

CTTAAAAATT

TGGTTGCTC

TTGTACAAGT

GACATGCAGC

CCATTAGTTA

TCAGGGCAAG

GTTGGCAAGC

AACGGACAGG

AAATATT

GATAAAGGCA

TTACCCCTA

ACGGTGTCCG

CCACTTGCTC

ATTATAAATG

CTTATTGAGA

TTAAACAAT

TTGCAGTTT

TCCATTTTAC

TTCACCGCC

ATTTTTGTT

TATGATCCTT

ACTGAGACTG

ATTGAGCTTT

AAiTTCATATT

GGACGTCATT

GACGTGGTGT

ATTGTTCA6TC

ATGAATTCAC

TCCATCTTAA

TCGCGTGGGT

CATTCAGCTG

CAACTCTGGC

GTTCCGCGCC

GGCCCAGCGC

GTGGTTTTG

TTGCGTGCGG

GCTTTGACTG

ACCGCTCCCG

GAAGAGGGCC

TCTAATTTGG

GCTGGAAAGA

CAGAAACTTA TTCCCCGTTT TACAAAATGG CCAA.AATGCT TTTCAAACTT

CTGTGGCAGC

GATTGGAGGT

ATATGAAAAC

CCCAAATTCC

TGGTTGAGAA

CTGATTGGTT

AAGAACAACA

TAAAATTGAA

AGCGTGCTCT

ACTTGCCCCA

GCAAATCTTA

AGGACAGTGT

CTGTGGTGAT

GCCAGATGGT

TTCCATTTAC

TTIACTGTTTC

CTAAACCGGG

TTAAGCCAGC

GGCAGGCTGT

TGATACTGTC

CATGGTCTGA

TAATTGACAA

TACATGAAGA

TGGCTGCTGA

TAGTTTATTC

CAACTAAGCC

GTGTACCAGC

ACCTTGACAA

TGGTGCCCCT

ACAAGAAAGC

CATCAGATGC

TTTTTACAAA

CACACCAGGC

CATCGGATGG

ATTGTGGCGC

GCACTGGATC

CCCAGAAACA

GCTCCAAATT

CACTCTCTCA

CTAAACATAC

CGCATGTATA

AAAAAGAGGC

GGCTGCCCTA

GGCTGAAGGG

TCTATCAATC

CCCGCCTGAT

TATGAGGACA

CTTTGACACT

TATTCCTGGT

AAAGGGAGTG

GCAGGTGTCT

CATAAAGACT

GGCAGATGAT

GGAACCTGAT

CGCTGTGAAG

GAGCCGTCCA

TTTGGCAAAT

GTGGAGTTGT

TATG4GATGCA

CTCTACAACA

TTCTCCTGTT

TTGTCCTGAA

CTTTGTGCGC

TGGTCTTCCC

TAAALTTGGCT

AGAAGTTCA6A

TTTGTTCAGA

TAAAGATTCA

GTATCTTAAA

TTTTGCTATG

TATGTATCAA

AAAACCAAAG

AACTGACTTG

TGAATTGGTT

TCATTTGTTT

TGAGTGTGAG

GTGTCTACTT

TGAAATTGAA

ACGCACTGTG

GACTGTAATG

TGCAATTGTT

TGTTGCATTT

GGGCAACGTT

GG;TGAAATCA

ATTTGACAAG

TGGAGACAAG

CGCCCAGAAG

CATTTGTGGC

TGCTCAGCAA

CGCCGAACTC

ATTTTTGAAA

TGACGTGCCG

AAGGACCCAG

GGGTTTTTCT

AAGATTTCTG

TATTCTTIG

CTTCGGACAG

CTTG.TTGCCC

GCGCTCTATT

ACTCAAGAGG

GACAAAGGTC

GAGCCCOTTG

CTGATGGCTC

CCTCCTGACC

TTGGGCCAGG

GCTTTTGTAC

GTTATTTGTA

GCTCTTAAGA

ACTGTTGGTT

CCACACCCTA

CTTTCTGGTG

AACAGACAAG

AAGTGTACAA

GAAATTCTCA6

TGGGAGTCAT

TTTCTATCCA

C-T T11TA.AGA

ACCCAGGAAG

CAACAATCCA

ACTGACTGCT

GAATTTGATT

AAGGTAGAGT

CGAGTGTCAT

TTGAAGAAAA

TTTTTTGGCA

CCCAATGTT

GCTGGCTCAC

TGCTCCCTGG

AGTCGCCTTG

TTGGCTTACC

CGCCTGTCAG

GAGATTTCCG

GTTTTCTCCC

ATTCCTGGCC

AATAAGAGAA

CAAAAGGACA

GATGAGATCC

CCGATGCCCC

TAGTGGTGGT

TTTCCTACTT

ATTTGGTCAC

TGTCGTCATT

TGAATGACAT

TCAAGAAATG

CAGAATTGGA

AAGCGCGCCA

TCTTTTCCCT

TATGCGTCCT

AAGCAATTTC

CCACATATTT

ATCCGAATGG

CACCTATGGC

CTACAAATT

GGAGGTTTCG

CAAACCAGCT

TC=TGAAAA

GAGAAGTGAC

ACACACACAA

CTGATGAGGA

GGGCGTTGT

ATATGACCAG

TTCTTACTTC

CCCCTGACGA

TGAAAACACT

TCATGAAAAA

CTGCCTTGGG

TTGATACCAT

TTGAGCTTGA

CGGGGCCTAA

TGACCCAAGT

GTTTTTTGAC

TGTCCTATGC

CTGCCCGCAT

TGTCCAGAGA

ATGACGATGT

CCATTTTCAA

ACGGCGTGAA

CACAGGACCA

GGATTGGATT

TTGACAAGAT

GGAAAGACAT

GATTTATGGC

GCCCACTTGA

ATGTGGTGGT

TATGTTGATT

TC.AAGAGAAG

TGATGCGGCT

TTTCGAAACG

ATTTGCTTTG

GCTGACATCC

AAAATATCCC

GTGGTTTAAA

CCTGCAAATT

TCGGGGCGCA

GCTTCTCTTG

TGATGGCTAT

TGCTGACTTT

CCATTTGGAT

GCATTCATCT

GTTTGATGTG

TTTGAATCTC

TGACATGCCC

AGCTTTTGAG

AATGCCCATT

ACAGAAAATG

TTCAGAACGC

GAGAGCCAAG

ACTGATTGTT

GCAATCAGCT

GAAGATTAGG

TGTTCAGCCA

TGTTTATGAC

TGTGCTTGGT

AGTGAATGGA

AGTTAGAAAT

GACAGGAATC

AGTGAGGAAG

CGCTCAGACC

AATTGGTATC

CGCGCTGGAG

GAGGGTGTCT

ACCTGACTAT

GCTGGATGAA

GAAATGGCTC

TGACAACCAG

GGAGCAGGAC

CTGTGATTTT

TGGTGACTAC

GAAAGTTAGG

TGGTAGGCAG

3165 3225 3285 3345 3405 3465 3525 3585 364S 3705 3765 3825 3885 3945 4005 4065 4125 4185 4245 4305 4365 4425 4485 4545 4605 4665 4725 4785 4845 4905 4965 5025 5085 5145 5205 5265 5325 5385 5445 5505 5565 5625 5685 5745 5805 5865 S925 SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 7 CTCTTGGGCC GGTTTGTGGC AAAATTTCAT GAAGCAAATG GATTTGACAT TGGCTCAGCC 5985 ATTGGATGTG ACCCAGATGT GGACTGGACT CGGTTTGGCC TCGAGTTGGA GCGTTTCAGG 6045 TATGTATATG CCTGTGACTA CTCACGGTTC GATGCaAACC ATGCAGCTGA TGCAATGAGA 6105 GTTGTGCTTA ACTACTTTTT CTCTGAGGAC CACGGTTTCG ACCCTGGTGT GCCTGCTTTT 6165 ATTGAGTCAC TGGTTGATTC AGTGCATGCC TATGAAGAGA AAAGGTATAA CATCTACGGT 6225 GGCTTGCCAT CCGGGTGTTC CTGCACATCA ATTTTGAATA CCATCTTGAA CAATGTTTAC 6285 ATTCTTGCAG CTATGATGAA GGCTTATGAG AATTTTGAGC CAGATGACAT TCAGGTCATT 6345 TGCTATGGG ACGACTGCCT CATTGCTTCT GATTTTGAAA TTGATTTCCA ACAACTGGTG 6405 CCTGTCTTTT CTAGTTTTGG ACAGGTAATA ACTACAGCTG ACAAGACTGA TTTTTAAA 6465 CTGACAACGC TTTCGGAGGT GACCTTCCTT AAGCGCGCTT TTGTTCTGAC GGCCTTTTAC 6525 AAGCCAGTGA TGGATGTGAA GACCCTTGAA GCAATCTTAA GCTTTGTTCG CCCAGGCACA 6585 CAGGCTGAAA AGCTCCTGTC CGTGGCGCAG TTGGCAGGCC ACTGCGAACC GGAGCAGTAT 6645 GAGCGCCTGT TTGAGCCCTT TGCTGGGATG TATTTCGTCC CTACTTGGCG ACTTGCGCCT 6705 GCAGTGGTTG ATGAAGCTTG GATGCTAAAT TCTTTrTGAC TTTGTTTTTC TTTGTTTTCT 6765 TTTAGGCTTT TAAGGTGTTA AGTTTAAAGG TTAAGAGTTT TTAGAAGTTA AGATAGAGTT 6825 TAGTTTTTAG TTTTGAGC-poly(A) as disclosed in Fig. 2 and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants, degeneracy equivalents and deletion mutants thereof.

In another aspect, the invention provides a substantially pure amino acid sequence being: a substantially pure amino acid sequence being: M A A S K V Y R V C E Q T L L A G A V R M M D K F L Q K R T V F V P HL D K T I R L T G L H N Y D N T C W L N A L T Q L T Q I L G I R L F D E H F G N R G L F T R K TI D W V S D Q T G I K D L K S G A PP LVVVYKLWQ H GH LDVGTME K P RS IT LW S GP KVC L S DF WACVSA K PG HA V FYLLTS EGW IC VD DKKIYP E T P K T E D V L V F A P Y D F E S L G K D P P K L H Q R Y L 4 VP4 E K A F E L S G G G T S T P T T G N Q N M SG ON S GSI VQNF MQ Q Y QN SI DADLG DNV I S P EGO GS NT SSS TS S SQS SG LGG W F VP4 4 VP2 S S L L N LGT K L LA DK KT E E T TN I ED R I E T T VVG VT II N SQ GS VG TT Y C Y SK PD GR P Ps TVS D P VT RL PTL S RH YT SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/0081

F

K

V

A

N

H

S

P

R

K

E

I

T

p

Q

K V G E KM OGS N P S F L~ K W S L R T N N P W T V A P I E S D S Q V P G P Y F E L H G T F T G A R D E A Y PS sP V YA L W P H S F H EV A H SOG E L EE S S V H I V I L D AM V F MS S R FT N V TN T Y V A S A AT K M A CI A D FM T AL T F S LR T EP R F DV E T AQ L F K FT T KT T M C P T M W RV P GA H F RA P Q S V K P L P A L V I L

NOG

F I S G L S A K H A A V S T H P H A T L A W T T D A A M H A H L.

A A y Q y I L T P N S T A K P L F A V A D V

ASE

A V L P P V S N L L S V L E I A Y N L L P R W I C P L P G V K N G W D V V P E YE H T Q L S V F P H G P GOQ P T N O P GOQ T V P VP2 4 VP3 P EA PI R V PL YP KV V Q T Y S F C S F QM DV S L Q YR GS L N F V P P H S A GL N SA FS R T V VNVS N S KG RV L VP3 4 VPI S A G E L L D P E L P V L A L P T Y Y L G P D P G E D R F F GOS S IQ G AP VA G Y TS N WL R Y A D K D M L T N T G E

GOG

G S

GOW

T S P I V T N V T Y F F F VW V N S V R A PT K A HD K S L L K N A L S P F Y K VV V V E KL Q A Q I P vp 1 4 2A N I NK Q C TN Y

LAOG

D K T R H K F P T 2A 4 2B D V E S N POGP T G r T GM L K D

M

A

K

F K L F G L I P G A T L A E V FD K I S D I F L K V A T G L V A S A E R T F S A A A D L K D P Y FQ TA A S L A K L I Y M G v SUBSTITUTE SHEET (Rule 26) WO 97/22701 WO 9722701PCT/AU96/0081 2B 4 2C y

D

W

L

D

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V

FV vS F A L AQ E E P D G L F R G A KQ0D M D A v p p H S S S A K G L P G G E V I A F N L T V P A L S H K M P I L Q F L I E E YL K F L S I L 3A 43B D EQ SA 3B 4 3C T E T G V DONE L V E F D F D L E V NG I V H L F Q AR T V P N V L S R. I I G I S F F E T L K NMS D Q QAD0D T Q E E A S L LQ I S G Q G K S VW wsC L G QD P M A H L D F T P IT P G F V P.

P H P I F V T A F E 2C 4 3A F K QS W DMNK D S W E S yM T S L I V Y D P S T P AT D L T D C S A T I VL G DVV vvS T N E I E F F GS F YA AQ T H S AG T 3C 4 3D K Q GMN V L A YP I V K LOD E L R A A H P E W F A L R Q P L S Y Pp N G

DOK

V S T V E N L I S D ElI T R I F V V E K Q X T L V A V s L K y p Q R P. P Q A G Y C Q P V

PRR

N L G Q V Q

TOD

E P.

L A y Q L R K W L Y A L E P I S M G M V V I F P.

P L A V

NP.

E E S I A D L F T V L T K L A V V V L L Q A S T C T F D A L K L

QOD

Q K L L F A K T R. K A F K P K P K TOQE V KT L K I P.

Q Q L G

GP.

D A L K L T S R G S S P.

F K V V v Y NMS Y R V S s I L C S L I E P L K V P K

MMN

DOG

A G R O V P S Q ElI G F L Y X L E F

VP.

S P T V S P A L

R.P.

FOD

S A 0 N

Q

K

P.

D

L W P0Q L V K S L S D G Q K W L SUBSTITUTE SHEET (RULE 26) WO 97/22701 PCT/AU96/00815 A L T E K E A I C G I P G L 0 K M E Q A P Y A Q Q N K R R K D I CD F E E G R L K G Q K D R F M AG D Y S N L V Y Q S F L K D E L E K V R A G K T R L I D V P P M P H V V V L L G R F V A K F H E A N G F D I G S A I G D V D W T R F G L E L E RF R Y V Y A C D Y D A N H A .A D A M R V V L N Y F F S E D H G G V P A F I E S L V D S V H A Y E E K R Y N G L P S G C S C T S I L N T I L N N V Y I L M KA YE N FE P D D I Q V IC Y G D D C L D F E I D F Q Q L V P V F S S F G Q V I T T T D F F K L T T L S E V T F L K R A F V L T K P V M D V K T LE A I LS F V R P G T Q A L S V A Q L A G H CE P EQ YE R L F E P F 3D Y F VP T W R LA P A V V D E A W ML NS F

P

A

I

G

C

S

F

I

A

I

A

A

E

A

G L E L R P R Q D P R F D P Y G A M A S D K F Y K L G M as disclosed in Fig. 2.

In another aspect, the invention provides proteins derived from ERhV1 which exhibit virus like particle characteristics incorporating VP1 and having the following amino acid sequence: a protein or virus like particle incorporating VP1, derived from ERhV and having the following amino acid sequence: V TNV G E D GE P G E T E V H TD V S FL L D R F F D S P A T H V L D P F G S T A T Y F F S D L E L S I Q F K F VW V K W L P V GA P T K N S VR I Q K L A V A G M C S Q A C A S AL P Y T S M W A P T K E K A T Y N W L P G A H D K G G C Y L R Y A F R P A F T R P AD K T R H K F P R H A L V E TL E Q LA W A F T T T P T T D A W PT V V F RV V P V AH F G S AP AMY P T N I N V D M H N L T G L N T C V G E G E G G G A G S R N G W G L T SD R P I P C T SUBSTITTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 11 In another aspect, the invention provides proteins derived fiom ERhV I which exhibit virus like particle characteristics incorporating VP2 and having the following amino acid sequence: a protein or virus like particle incorporating VP2, derived from ERhV 1 and having the following amino acid sequence: D KK T E E T TN I E DR I E SQ G S V G T T Y C YS K PD VT R LG PT LS R H Y T F K G H A W IC P L P GD K L KK H H L V K N G W D V V V Q V N V A A V PE Y EH THE K A L T YQ Q L SV FP H Q LL N L P Y I G P G Q PT N L T L H N E LT G P G Q TV P V T M S V L P N P E T T G R V G M G P S K W R T P W A P VV G VT PP S TV EW PH S SF HE V FAH S G SE L E E N S S VH T I V IL ID A MV II N S D P Q S H QSH V KA P L C P A Y L V M I L S N G P

PLC

PAY

LVM

ILS

N G P In another aspect, the invention provides proteins derived from ERhVI which exhibit virus like particle characteristics incorporating VP3 and having the following amino acid sequence: a protein or virus like particle incorporating VP3, derived from ERhV1 and having the following amino acid sequence: A PI RVVS VP ES D S F M Y P K V V V PR Q VP G RF Y SF C S I S GK P Y FE V T MDV S L S A A E L H GT Y V R G S L N F N F I F T G A A A PP H S AA P KT RD E A MA N S A F S F N V P Y PS P A D V V N V S GW LQ V Y A L T A KG R V L V A VS AG PD F S

Q

S S V P D NS T TN F ID VA K NT S GD E P L A S L S S F F A T K A K F LV A CI H A V W D V F M A V Y S A E L T S T D I A V L R H P A D L P SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 12 In another aspect, the invention provides proteins derived from ERhV I which exhibit virus like particle characteristics incorporating V'P4 and having the following amino acid sequence: a protein or virus like particle incorporating VP4, derived from ERhV1 and having the following amino acid sequence: G G G T ST P TT G N Q NM S GN S G SI V Q N F Y M Q Q Y Q N S I D A D L G D N V I S P E G Q G S NT S S S TS S SQ S S GL G GW FS S L LN L G T KL LA 0 The invention also provides a virus like particle comprising any one or a combination of VPI, VP2, VP3 and VP4.

In another aspect, the invention provides a substantially pure nucleotide sequence for VPl being:

GTTACCAATG

CCCQTGGACA

GAGACACTTG

GGCTCGACTG

GATTTGGAAT

TTTGTGTGGG

TTAGAAGGAG

ACTGTTGTGT

ACATCAATGT

AAGGCAACCT

GCGCATGATA

ccTCGACCCA

ACTAACATCA

TGGGAGAGGA

TGCACGTGCA

AGCTTTCAAA

CCCAACTGGC

TGTCAATCCA

TGAAGTGGCT

GTGGAAATTC

TCAAGATTGC

GGCGTGTTGT

ACAATTGGCT

AAGGAGGGTG

TTCCGCCGGC

ACAAACAGTG

TGGTGAACCC

CACAGATGTC

TTTGACAGGT

TTGGGCACGT

GTTTAAATTT

CCCTGTTGGA

AGTTAGAATT

AGGCTCCCGT

GCCAGTCTTT

TCCTGGTGCA

CTACTTGCGG

TTTTACGCGT

TACT

GGTGAGACAG

AGTTTCTTGC

TCTCCTGCCA

CTGCTAAACA

ACCACCACTC

GCACCAACCA

CAAAAATTGG

TCACAAGCCT

TACAATGGCT

CACTTTGGTT

TATGCTTTCC

CCAGCGGACA

AGCCTCGTCA

TTGACCGGTT

CACATGTTCT

CTTGCACCTA

CGTCCTCTGT

AGACCACAGA

CCGTTGCAGG

GTGCTTCAGC

GGGGTGCACC

CCATCTTGCT

GCGCGCCAGC

AAACCAGACA

TGCTTTGTCA

CTTTGATGTT

GGATCCGTTT

CTTCTTTTCT

TGGAGAGGGC

TGCTTGGCAG

GATGTGCCCC

GTTGCCATAT

TACCAAAGAA

GACTTCTGAT

GATGTATTGC

TAAATTTCCC

and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

SUBSTITUTE SHEET (Rule 26) WO 97/22701 WO 9722701PCT/AU96/0081 13) In another aspect, the invention provides a substantially pure nucleotide sequence for VP2 being: GACAAGAAGA CAGAAGAGAC GTCACTATTA TTAATTCACA GGTAGACCAC CATCCACAGT CACTACACAT TTAAGGTAGG TGTCCGTTGC CAGGTGACAA CACCACCTGG TCAAGAACGG TCCGGGCCGC TGTGTGTAGC AAGTGGTCTG AGCTTGAGGA CAGTTGCTAA ATTTGAGGAC GGCCAACCAA CAAATCTGAC GAATTGACAG GACCTGGCCA ATGGTTAATG GGCCTCTTCC

TACAAACATT

AGGATCTGTT

GTCAGACCCA

TGAGTGGCCC

ACTCAAGAAG

CTGGGATGTG

AGCAGTGCCG

ACCAGCTTAC

AAATTCATCA

TTTGCACAAC

AACTGTGCCT

AAATCCAGAG

GAAGACAGAA

GGAACAACCT

GTTACCAGAC

CATTCTCAAT

ATGGGCAGTT

GTTGTGCAGG

GAGTACGA-AC

ACATACCAAC

GTGCATTTGG

CCGTGGACCA

GTGACCATGT

TTGAAACAAC

ACTGTTACTC

TTGGACCCAC

CACATGGTCA

TTCATGAGGT

TGAATCCCTC

ACACACATGA

AACTTTCAGT

TGATGCCCTA

TTGTTATTTT

CGGTGGCTCC

AGTGGTTGGA

CAAACCGGAT

GCTTTCCAGG

CGCATGGATC

TGTCAAAGCC

ATTTGCTCAC

GAAAGCACTC

TTTTCCCCAC

CATTGGGCCA

AATTTTGTCT

CATCG.ATGCA

and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

In another aspect, the invention provides a substantially pure nucleotide sequence for VP3 being: GCACCGATTA GAGTGGTGTC TGTGCCTGAA TCAGATTCTT AATTCGACTC CACTATACCC CAAGGTTGTG GTCCCACCGC ACAAATTTCA TTGATGTGGC AP.AACAGACA TATTCATTTT TATTTTGAGG TTACCAACAC CTCTGGGGAC GAGCCACTGT AGTGCGGCAG AGCTACATGG CACTTACGTA GCTAGTTTGT AGAGGCTCAC TTAATTTCAA CTTTATTTTC ACTGGTGCAG CTGGTTGCTT TTGTGCCTCC CCACAGTGCA GCGCCCAAAA TGCATCCATG CCGTGTGGGA TGTTGGCTTG AACTCAGCTT CCCTCCCCTG CTGACTTCAT GGCCGTTTAT TCTGCGGAAC GGATGGCTTC AAGTTTATGC ACTAACAGCT CTAACTTCAA AAAGGCCGTG TGCTGGTTGC TGTTTCCGCC GGCCCAGACT GACCTGCCCG ACAAGCAG

TTATGTCTTC

GCCAAGTTCC

GTTCCATTTC

TTCAGATGGA

CATCATTTTT

CAGCCACTAA

CGCGCGATGA

TTTCTTTTAA

GGACGGTTGT

CTGACATTGC

TCTCCCTTCG

AGTACCTGAT

TGGCCGGTTT

TGGAA.AACCT

TGTGTCGCTC

TGCACAGTAC

GGCAAAGTTT

AGCAATGGCG

TGTACCTTAT

GAATGTCTCT

CGTGAACAGT

TCACCCGGCG

and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

SIJBSTnmJTE ShEMET (Rule 26) WO 97/22701 PCT/AU96/00815 14 In another aspect, the invention provides a substantially pure nucleotide sequence for VP4 being: GGCGGAGGTA CATCCACTCC AACAACTGGC AACCAAAACA TGTCCGGAAA CAGTGGTTCA ATTGTTCAAA ATTTTTACAT GCAACAGTAC CAGAATTCAA TTGACGCAGA CCTGGGAGAC AATGTGATTA GCCCTGAAGG CCAGGGCAGC AACACTAGTA GTTCAACCTC ATCAAGCCAA TCCTCTGGCT TGGGCGGGTG GTTCTCTAGT TTGCTGAACC TTGGAACAAA ACTACTGGCT and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

In another aspect, the invention provides oligonucleotide primers derived from the nucleotide sequence of Fig. 2 being highly specific for ERhV1 or cross reactive with other ERhV types.

The oligonucleotide primers may have any one of the following nucleotide sequences: VP1F 5' GTTGTGTTCAAGATTGCAGGC 3' VP1R1 5' TTGCTCTCAACATCTCCAGC 3' VP1R2 5' TAGCACCCTCCTTTATCATGCG 3' In another aspect, the invention provides an oligonucleotide probe derived from the sequence of Fig. 2.

In another aspect, the invention provides diagnostic reagents, methods and kits characterised by the aforesaid oligonucleotide primers and probes.

In another aspect, the invention provides antigens comprising any one or a combination of the non-capsid proteins, being other than the individual VP to VP4 proteins, that are cleavage products of the polypeptide of Figure 2.

In another aspect, the invention provides vaccines and vectors incorporating any one or a combination of virion proteins VP1 to VP4.

In another aspect, the invention provides diagnostic tests for the detection of antibodies to ERhVl in blood of horses or other animals characterised by the use of the aforesaid antigens. Such diagnostic tests may be ELISA based.

In a particularly preferred embodiment, the invention provides a test to distinguish horses infected with ERhV1 in which said virus had replicated from horses which have been vaccinated with the vaccine incorporating any one or a SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 combination of virion proteins VP I to VP4; comprising the steps of applying an antigen being any one or a combination of non-capsid proteins, being other than VPl to VP4, that are cleavage products of the polypeptide of Figure 2 to a horse and testing for an immunoreaction thereto, wherein a positive immunoreaction would indicate that said horse had been infected with ERhV1 and a negative immunoreaction would indicate that said horse has not been infected with ERhV1.

In another aspect, the invention provides recombinant plasmids incorporating nucleotide sequences and subsequences derived from the nucleotide sequences of Fig. 2. The recombinant plasmid may comprise the P1-2A-3C region of the ERhV 1 genome.

In another aspect, the invention provides a host system characterised by incorporating the nucleotide sequence of Fig. 2 or part thereof. The host may be E.coli, vaccinia virus, baculovirus or yeast.

In another aspect, the invention provides a process for producing a protein product derived from ERhV1 comprising the steps of selecting out a gene of interest from the ERhV1 nucleotide sequence of Fig. 2 and expressing said protein product in a suitable host system.

DETAILED DESCRIPTION OF INVENTION The invention will now be described in detail with reference to Figs. 1 to 6: Fig. 1 Schematic representation of the ERhVI genome and (B) comparison of the genomic structures of picornaviruses showing the predicted proteolytic cleavage pattern of the polyprotein. The lengths of individual regions are drawn approximately to scale. The dashed line represents the unsequenced region of the ERhV1 Fig. 2a Nucleotide and predicted amino acid sequence of the ERhV1 polyprotein. The nucleotide sequences of the 3'-NTR and part of the 5'-NTR are also shown. Numbering is from the first ATG codon that occurs in a context optimal for translational initiation (Kozak, 1989). A polypyrimidine tract upstream of the putative initiating ATG and the two pairs of in-frame ATG codons are underlined. The predicted proteolytic cleavage sites are indicated by arrows.

SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 16 Fig. 2b Nucleotide sequence of the ERhVI 5'-nontranslated region.

The polyC tract (dotted underline), polypyrimidine tract (underline) and potential initiation codons (double underline) are indicated. Predicted coding sequence is shown in bold type. Numbering is from the ATG considered most likely to be used for translation initiation.

Fig. 3 Alignment of the predicted amino acid sequences of ERhV1.393/76 and FMDV.01K polyprotein. Proteolytic cleavage sites, which are predicted in the case of ERhVI, are indicated by the arrows. Identical residues highly conserved residues and less conserved residues are indicated.

Fig. 4 Unrooted phylogenetic trees inferred using the picornavirus nucleotide sequences of the complete polyprotein gene, the polymerase gene and VP1 gene of viruses representing the five recognised genera of the family Picornaviridae. The viruses used were: FMDV.O1K, FMDV.A12, FMDV.C3, FMDV.SAT3, EMCV, TMEV, Mengovirus, poliovirus 1.Mahoney (Polio poliovirus 2.Sabin (Polio 2), poliovirus 3.Leon (Polio coxsackievirus A9 (CV.A9), CV.B3, echovirus 22 (Echo 22), swine vesicular disease virus (SVDV), bovine enterovirus (BEV) hepatitis A virus (HAV) human rhinovirus 1B (HRV1B), HRV89 and HRV14.

Note: The branch lengths represent proportionate change only within each tree; they do not allow direct comparisons to be made between the three trees.

Fig. 5(A) Diagram outlining the strategy for nested, reverse transcriptionpolymerase chain reaction (RT-PCR) for the detection of ERhV genome. The genome structure of ERhV1 is shown schematically (top), and the first round PCR product (362bp), corresponding to VP 1 and 2A regions, and the second round PCR product (210bp), corresponding to part of VPl, are represented as black lines.

the sequence of specific oligonucleotide primers used for RT-PCR are shown. VP1R1 was used for the RT reaction.

Fig. 6 Construction of ERhV1 expression plasmid for E. coli and baculovirus transfer vector for insect cells. The ERhV I genome is shown (top) and oligonucleotide primers used to amplify P1.2A and 3C regions are depicted as SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 17 arrows. The P1.2A fragment and subsequently the P1.2A.3C fragment. obtained through the ligation of PI.2A and 3C, were cloned separately into the multiple cloning sites of the pET 15b and pBacblulII plasmid vectors to construct pET.P1.2A and pET.P1.2A.3C respectively for expression in E. coli and pBac.PI.2A and pBac.P1.2A.3C respectively for expression in insect cells.

The sequence of specific oligonucleotide primers used for the construction of expression plasmids are: VP4F 5' GCTGGATCCATGAGTGGCGGAGGTACATCCACT 3' R2A 5' GCTCTGCAGCAGGTCTGCTGATGCTTTGGA 3' 3CF 5' GCTCTGCAGATGATTAGGACTGAGACTGGTGT 3' 3CR 5' GCTGGATCCTTAGCCATAGTCAGGTTTGAA 3' Virus growth and purification ERhV1 strain 393/76 was isolated from a nasal swab taken from a thoroughbred horse in South Australia while it was being held in quarantine following importation from the United Kingdom. The mare had an acute, systemic febrile illness. The virus was passaged 14 times in equine fetal kidney (EFK) monolayer cell cultures and then once in Vero cells. ERhV1 virions were purified by a modification of the procedure described by Abraham and Colonno. Cells were harvested 48 hours after infection. The infected cells and supernatant fluid were frozen and thawed three times and clarified by centrifuging at 2,000 x g for 20 min at 4 C. Polyethylene glycol 6000 and NaCI were added to the supernatant to final concentrations of 7% and 380 mM, respectively, and the mixture was stirred overnight at 4 C. The precipitated virions were recovered by centrifuging at 10,000 x g for 15 min at 4 C and resuspended in 200-400 pl TNE buffer (10 mM Tris-HCI pH 8.0, 100 mM NaCI, 1 mM EDTA) containing 1% NP40. The suspension was clarified by centrifuging at 12,000 x g for 3 min before layering onto 15% to (wt/vol) linear sucrose gradients (35 ml) in TNE buffer and centrifuging at 100,000 x g for 4 h at 4 C. Gradients were fractionated and the fractions analyzed by SDS-PAGE. Viral fractions were pooled, centrifuged at 200,000 x g for 2 h at 4 C, and the viral pellet was resuspended in a small volume of TNE buffer, cDNA SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 18 synthesis and cloning. Viral RNA was reverse transcribed using an oligo-dT primer (Amersham) or ERhV1 specific primers P1 (5'-ATCCAGCAAGCCGCTGTCCGGTTAC-3') and (5'-CGAAGAGACACCTGCTTC-3'). Viral RNA was prepared as described in (1987) Anal-Biochem. 162, 156-159.

SViral RNA and 100 pmol of primer were mixed, boiled for 2 min and cooled at room temperature. First strand cDNA was synthesized using 200 U of Maloney murine leukemia virus reverse transcriptase (Promega) in the presence of 0.8 mM dNTPs and 30 U of human placental RNAse inhibitor (Pharmacia) in a reaction volume of 25 pl. Second strand cDNA was synthesized using a cDNA synthesis kit (Amersham). The cDNA fragments were ligated into pUC18, either as blunt ended fragments or after ligating BamH I adaptors (Pharmacia), and the lighted products used to transform E. coli strain DH5a (Stratagene). Colonies were selected by hybridization, initially with an [32P]-dCTP-labelled cDNA probe derived from reverse transcribed viral RNA, and subsequently with [32P]-dCTP-labelled cloned viral cDNA The sequence between two cDNA clones was obtained using the oligonucleotide primers P6 (5'-TTCTGGTGGAGAAGTGACAGC-3') and P7 (5'-GTGAGCCAGCAACAATTGC-3') in a polymerase chain reaction (PCR; 17) using the polymerase, Vent Exo+ (New England Biolabs).

DNA sequencing and analyses Double-stranded DNA was prepared using the alkaline lysis method and sequenced by dideoxy chain termination using modified T7 DNA polymerase (Pharmacia) and [35S]-dATP (Amersham). Sequence was read and analyzed using the GeneWorks software package (IntelliGenetics, Mountain View, CA). The GenBank database was searched using the FASTA searching and comparison program. The protein alignment shown in Fig. 3 was performed using the Genetics Computer Group, Inc. (Madison, Wisconsin, USA, 1994) GAP program with a gap creation penalty (GCP) of 3.0 and a gap extension penalty (GEP) of 0.1. The multiple alignments of nucleotide sequences were performed using ClustalW. For pairwise alignments the slow method was used with a GCP of 10 and a GEP of 0.1.

SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 19 For multiple alignments a GCP of 10 and a GEP of 0.05 was used. with alignment of sequences which were more than 60% divergent delayed and using weighted transitions. Phylogenetic relationships were examined using the maximum likelihood method with the DNAML program of the Phylogeny Inference Package (Phylip) version 3.5c (1993, J. Felsenstein, Department of Genetics, University of Washington, Seattle). The model used allowed for unequal expected frequencies of the four nucleotides, with the frequencies determined empirically from those present in the sequences analysed, and unequal rates of transitions and transversions.

A single rate of change was assumed for all sites. The program was allowed to perform global rearrangements to optimise the tree. Initial analyses were performed on polymerase sequences using a range of transition/transversion ratios to determine that which gave the maximal log likelihood. A ratio of 2.0 gave the maximal log likelihood and thus this ratio was used for all subsequent analyses of other sequences.

Cloning And Sequencing Of The ERHV1 Genome Sixty seven overlapping cDNA clones and one PCR product clone were obtained and sequenced from both ends. The nucleotide in each position was determined at least twice, and 95% of the sequence was obtained by sequencing in both directions. The predicted genomic structure of ERhVl was characteristic of picoraviruses, possessing one long open reading frame (ORF) flanked by and 3'-NTR's (Fig. 1).

The nucleotide and predicted amino acid sequences of the ERhV1 polyprotein are shown in Fig. 2a. Partial sequence of the 5'-NTR (433 bases) was also obtained Fig. 2b. There was a tract of 9 Cs at position -550 to -542. PolyC tracts of various lengths have been observed in similar locations in FMDV and EMCV. The actual length of the ERhVl polyC tract is uncertain as these sequences are known to be unstable when propagated in E. coli. A 14 nucleotide polypyrimidine tract, which possessed the TTTC motif common to all picornaviruses, was present near the potential translation initiation codons. A region of 450 nucleotides upstream of the most likely initiation codon is predicted to contain an internal ribosome entry site (IRES). This region showed most SUBSTITUTE SHEET (RULE 26) WO 97/22701 PCT/AU96/00815 sequence identity (48-50%) with corresponding sequences in FMDV and EMCV.

The 3'-NTR of ERhVI was 102 nucleotides excluding the polyA tail (data not shown).

In picornaviruses, there are two factors that influence which ATG codon initiates translation, a requirement for the ATG to be located at the 3'-end of the IRES, and that this ATG occurs in a sequence optimal for initiating translation, that is, a purine at position -3 and a G in position Two pairs of in-frame ATG codons were identified in the ERhV1 genome. The second ATG of the first pair is separated by 25 nucleotides from the beginning of the polypyrimidine tract (Fig.

2b), similar to the distance (25 to 27 nucleotides) found in the corresponding regions in FMDV and EMCV The second ATG of each pair occurs in an optimal context. Therefore, the second ATG of the first pair is most likely to be the translation initiation codon but it is possible that translation is also initiated from the second optimal ATG, by a process of leaky scanning, or even from the other two, non-optimal ATG codons. The predicted ERhVI coding sequence, beginning at the most likely initiation ATG, extended for 6,741 bases and would encode a polyprotein of 2,247 amino acids.

Alignment of the ERhV1 amino acid sequence with those of other picornaviruses showed that it was most similar to aphthoviruses and, to a lesser extent, to cardioviruses in all regions of the genome (data not shown). Fig. 3 shows a comparison of the predicted amino acid sequence of ERhV 1 with that of FMDV.O 1K. The two sequences were 40% identical. The more conserved regions include: the 3D/polymerase (50% identity), VP4 (49% identity) and some regions of the 2C protein. ERhVI encoded a 2A protein of 16 amino acids, 14 of which were identical with those of FMDV 2A. ERhV1 possessed only one copy of the VPg sequence. This is in contrast to FMDV which has 3 tandemly repeated, non-identical VPg sequences (27-29).

Table 1 shows the proteolytic cleavage sites of ERhVI predicted from the amino acid alignment (Fig. and compares these with those of FMDV, EMCV and Theiler's murine encephalomyelitis virus (TMEV). Most of the ERhVl cleavage sites could be assigned with reasonable confidence because of significant SUBSTITUTE SHEET (RULE 26) WO 97/22701 PCT/AU96/00815 21 amino acid similarity with FMDV in the regions flanking the predicted cleavage site; an exception was the 3A/3B cleavage site where there was less sequence similarity. As is the case with FMDV, the predicted ERhVI 3C protease cleavage sites were more variable than those of the cardioviruses, EMCV and TMEV.

Table 1. Comparison of the predicted proteolytic cleavage sites of the ERhV1 polyprotein with those of FMDV, EMCV and TMEV.

Cleavage sites* Proteins ERhVI FMDV EMCV

TMEV

Leader/IA(VP4) S/G K/G Q/G Q/G IA(VP4)/1B(VP2) A/D A/D A/D

L/D

IB(VP2)/1C(VP3) E/A E/G Q/S Q/S IC(VP3)/l1D(VPl) Q/V E/T Q/G

Q/G

ID(VP1)/2A T/N L/N E/S

E/N

2A/2B NPG/P NPG/P NPG/P NPG/P 2B/2C Q/V Q/L Q/S Q/G 2C/3A Q/S Q/I Q/G Q/S 3A/3B Q/S E/G Q/G Q/G 3B/3C E/T E/S Q/G Q/G 3C/3D Q/G E/G Q/G Q/G Cleavage data from: FMDV.OIK (Forss et al. 1984), TMEV (Pevear et al. 1987) and EMCV (Palmenberg et al. 1984). The single amino acid code is used.

Phylogenetic analyses A phylogenetic tree was derived from the nucleotide sequences of complete picornavirus polyproteins (Fig. 4a). Each branch of this tree was statistically, highly significant with the 95% confidence limits ranging from to ±15% of branch lengths. ERhVI wa§ found to be most closely related to the aphthoviruses, although it was clear that ERhV 1 was considerably more distant from individual members of this genus than the aphthoviruses were from each other. A phylogenetic tree was also derived from the nucleotide sequences of picornavirus polymerase genes (Fig. 4b). Each branch of this tree was statistically, SUBSTITUTE SIHEET (Rule 26) WO 97/22701 PCT/AU96/00815 highly significant (P<0.01) with 95% confidence limits ranging from ±14% to ±38% of the branch lengths. Again, ERhV grouped with the aphthoviruses and the topology of the tree was the same as that obtained using data of the entire polyprotein (Fig. 4a). The VPI nucleotide sequences were also similarly analyzed (Fig. 4c). Most branches were statistically, highly significant although, that between the ERhVI branch point and the branch point for the echovirus 22-hepatovirus cluster was less so The 95% confidence limits of the branch lengths of this tree were considerably greater than for the other two trees, ranging from ±18% to This tree did not group ERhV1 with the aphthoviruses. With the exception of bovine enterovirus (BEV), the tree had the same topology as those derived from the complete polyprotein and the polymerase sequences. It was also apparent that picomaviruses formed three clusters: enteroviruses-rhinoviruses, echovirus 22-hepatovirus and cardioviruses-aphthoviruses-ERhV 1.

Diagnostic reagents Oligonucleotide primers: We have designed short oligonucleotide primers and used them in polymerase chain reactions (PCR) for the diagnosis of ERhV infected horses. Any of the ERhV nucleotide sequence may be used for the 'design primer sets for use as diagnostic reagents. They may be highly specific for ERhV I or they may be designed to be more cross reactive so as to amplify single strand RNA template from other ERhV types ERhV 2, 3 and 4. As a specific example we have used the primer set shown in Fig. 5 to diagnose ERhV disease in several groups of seriously ill horses in circumstances in which, despite exhaustive efforts, we could not isolate the virus using conventional cell culture procedures.

We now consider ERhV a very under reported disease simply because, most of the time, nasal samples collected from horses experiencing severe, systemic clinical disease because of ERhV infection do not yield the virus in cell culture. In one particular group of horses, we detected the presence of ERhV by PCR and confirmed that the horses were both actively infected and seriously ill with ERhV by use of paired serum samples which showed that there was a concomitant rise in SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 23 ERhVI serum neutralising antibody. Vigorous attempts to isolate the virus in cell cultures yielded negative results.

Oligonucleotide probes: Virus specific oligonucleotides are used as probes to detect the presence of the virus in infected samples from diseased horses and other animals. This may be especially important given the systemic nature of the illness it is a foot-and-mouth-like, generalized disease with virus distributed throughout the body in many organs and tissues; it is not just a simple "common cold-like" illness as the name rhinovirus implies The significance of the sequence in moving the virus out of the Rhinovirus genus and into a new genus proposed to be called "Equirhinovirus" in the Picornaviridae family does not represent merely a taxonomic change but represents a paradigm shift in how ERhV and related viruses must now be regarded as pathogens for the horse and other animal species.

Diagnostic antigens: Individual virion proteins, in particular VP 1, VP2 and VP3, can be expressed in any one of a number of heterologous expression systems to provide antigens to detect specific antibody to ERhV I present in blood.

Such expression systems, which are well established for E. coli, yeast, vaccinia virus and baculovirus, allow for the production of large quantities of protein to a high degree of purity. The expressed virion proteins may be used in simple immunoassays, such as ELISA, to detect ERhVI specific antibody. Virion proteins expressed in this way also serve as effective vaccines against ERhV1 disease.

Vaccines Production of virus like particles (VLPs): We have used the sequence information to construct recombinant plasmids containing the P1-2A-3C region of the genome (see Fig. la and Fig. These plasmid constructions are of course critically dependent on the ERhV1 sequence that has been determined although the strategy that we are adopting, in general, is similar to that described in J. Virol 66, 4557-4564. Some early plasmid constructions have been inserted into E. coli and baculovirus expression systems based on prior art with similar viruses such as poliomyelitis of humans and foot-and-mouth disease virus of cattle and other cloven hoofed animals. The RT PCR double stranded DNA of the P1-2A-3C region of the ERhV1 genome is transcribed, within the transformed E. coli or insect cell for SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 24 baculovirus, into messenger RNA as a single transcript which is then translated into a mini polyprotein. The 3C protease activity results in the cleavage of the mini polyprotein into its constituent parts namely 1A (VP4), 1B (VP2), IC (VP3) and ID(VP 2A and 3C (see Fig. la and Fig. 6) and that the VP component parts then self assemble into VLPs virus particles that lack nucleic acid and are therefore non infectious are unable to cause disease. Two important applications of ERhV VLPs are as follows: The VLPs are very useful as highly effective, safe, high antigen-mass vaccines for the control ERhVI disease. If ERhV1 disease is confirmed, as we believe to be the case, as significant and responsible for much hither to undiagnosed illness that results in many lost training days, many expensive treatments, much serious illness because of secondary infections following on the primary ERhV1 infection, and much poor performance, then the utility of the vaccine based on the VLPs that are the subject of this invention will be very great and likely to have world-wide application.

With improved methods for the diagnosis of ERhV1 infection such as by PCR and ELISA as described herein, it is likely that other members of the proposed new Equirhinovirus genus within the family Picornaviridae including for example ERhV2, ERhV3, may be similarly diagnosed. Indeed suitably selected PCR primer sets based on the ERhV1 sequence could be used to detect these other equine rhinoviruses. The sequencing of these genomes could provide a basis for their specific diagnosis. It is also evident that the construction of VLP's based on expression plasmids similar to those described herein for ERhV1, could be readily adapted to these other equine rhinoviruses leading for example to production of combined ERhV vaccines to cover all antigenic types as may be extant or as may emerge by antigenic variation, as is very much a part of the biology of FMDV, in the future. Polyvalent VLP vaccines incorporating a range of ERhV antigenic types are obvious extensions based on the work described herein.

ERhV VLPs can be used as a delivery vector that will provide not only protection against ERhV disease but will be used to deliver other therapeutic and useful substances to the horses following administration by parenteral or other SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 routes. Such delivery vectors can be produced by inserting into. for example the PI region at some appropriate site, double stranded DNA coding for antigenic epitopes of other virus and infectious agents of horse as well as epitopes derived from other non infectious sources for example reproductive hormones.

ERhV1 DIAGNOSTIC TESTS For the detection of ERhVl antibodies in infected or vaccinated horses various standard tests can be used. VLP's may be used in such tests for example in an ELISA test for antibody.

Other diagnostic tests based on recombinant antigens derived from the ERhV1 sequence can be devised along similar lines to those reported for FMDV in which the absence of protein 2C from clarified inactivated whole virus FMD, FMDV or FMDV VLP vaccines maybe used as the basis for distinguishing infected from vaccinated animals where the vaccine is a non-replicating form of ERhV1 or a deletion mutant of ERhV1 in which a particular non-structural protein gene has been deleted. Precedent for this comes from studies of FMDV as reported in for example Lubroth, Grubman, Burrage, Newman Brown, 1996, Absence of protein 2C from clarified foot-and-mouth disease virus vaccines provides the basis for distinguishing convalescent from vaccinated animals, Vaccine 14(5), 419-427.

PREPARATION AND USE OF VIRUS-LIKE PARTICLES AND OTHER PROTEINS BASED ON ERhV1 SEQUENCE From the sequence of ERhVI it is possible to clone certain segments of the viral genome into a variety of vectors for expression in a variety of different expression systems. There is a straight forward and strong literature for FMDV that provides a very clear precedent for what can be done for ERhV1. Examples include the expression of FMDV P1-2A in a baculovirus (Abrams CC Belsham GJ, 1994, The antigenicity of foot-and-mouth disease virus P1-2A polyprotein and empty capsids produced in vaccinia virus and baculovirus expression systems. In VIIth Meeting of the European Study Group on the Molecular Biology of Picomaviruses, 6-11 August 1994, Korpilampi, Finland) or vaccina virus systems (Abrams CC, King AMQ Belsham GJ, 1995, Assembly of foot-and-mouth SUBSTITUTE SHEET (RULE 26) WO 97/22701 PCT/AU96/00815 26 disease virus empty capsides synthesized by a vaccinia virus expression system, Journal of General Virology 76:3089-3098) to obtain VLPs or viral proteins. We have prepared similar plasmids in which P1-2A. P1-2A-3C and these two sequences in a myristolated form have been inserted into p fastbac 1 baculovirus vector (Gibco/BRL) and into a PET vector (Novogene) for expression in insect cells and E.coli respectively.

These expressed products either as protein antigens or as VLPs, have utility as the basis for diagnostic tests or vaccines.

Accordingly, such references are herein incorporated in support of the full description and enablement of the invention where the disclosed methods of preparing diagnostics, vaccines, vectors, host systems and kits are fully described and applicable to the like aspects of the current invention.

Applications in human medicine: ERhV is also a human pathogen. We have unpublished data to confirm that humans have serum neutralising antibody to ERhV1 that is indicative of infection.

One of the laboratory workers concerned with the conduct of the sequencing and who handled infectious virus has specific antibody in high amounts (serum neutralising antibody titre 1 640 to ERhVl). We are currently extending these studies and anticipate finding a significant incidence of infection in humans world wide particularly among those humans who work with horses. The improved diagnostic methods outlined above, perhaps also the vaccine, are expected to have application in human medicine.

SUBSTITUTE SHEET (Rule 26)

Claims (22)

1. A substantially pure nucleotide sequence for ERhVI being: CCGTCAAGCC CGTTGCCTGT GTGCCATTGC TCTGGATGGT ACACGCCTGT GGTAGCGCTG TGGCCAAAAG CCCAGCGTTG TGAAGTGGAG TGAGCGGATC GAAGAATGCC CTGGAGGCAA ACCGGCGCAG GGTCAAAAAT TTTTGTACTG ACATGATGGC GCAGGTGCCG TTCGC&LTGAT CTTGACAAAA CAATTCGTTT GCCTTGACAC AACTGACACA AGAGGTCTGT TCACTCGGAA CTAAA;ATCAG GAGCACCGCC GATGTCGGTA CGATGGAGAA CTTTCTGATT TCTGGGCCTG ACAAGCGAGG GTTGGATCTG GAGGATGTAC TTGTTTTTGC cTAcAccAGA GATATGAAAA ACTGGCAACC AAAACATGTC CAGTACCAGA ATTCAATTGA GGCAGCAACA CTAGTAGTTC TCTAGTTTGC TGAACC=TGG AACATTGAAG ACAGAATTGA TCTGTTGGAA CAACCTACTG GACCCAGTTA CCAGACTTGG TGGCCCCATT CTCAATCACA AAGAAGATGG GCAGTTTTCA GATGTGGTTG TGCAGGTGAA GTGCCGGAGT ACGAACACAC GCTTACACAT ACCAACAA.CT TCATCAGTGC ATTTGGTGAT CACAACCCGT GGACCATTGT GTGCCTGTGA CCATGTCGGT CCAGAGGCAC CGATTAGAGT CCTGATAATT CGACTCCACT CGGTTTACAA ATTTCATTGA AAACCTTATT TTGAGGTTAC TCGCTCAGTG CGGCAGAGCT CAGTACAGAG GCTCACTTAA AAGTTTCTGG TTGCTTTTGT ATGGCGTGCA TCCATGCCGT CCT'rATCCCT CCCCTGCTGA GTCTCTGGAT GGCTTCAAGT AACAGTAAAG GCCGTGTGCT ATAGCCAGGT GTCACCAAGC CCCAAAAGGG ATAGCGCCTT TCCAATTTGG GCTGGTTTACA TGTCTAAGCA GGCGTCTAAG GGACAAATTC GACTGGACTC GATTCTTGGA AACAATTGAT ACTCGTGGTG ACCCCGGTCG TGTTTCGGr-A TGTTGATGAC GCCCTATGAC AGCATTGAG CGGAAACAGT CGCAGACCTG AACCTCATCA AACAAAACTA AACAACAGTG TTACTCCAA.A ACCCACGCTT TGGTCACGCA TGAGGTTGTC TCCCTCATTT ACATGAGAAA TTCAGTTTTT GCCCTACATT TATTTTAATT GGCTCCCATC GGTGTCTGTG ATACCCCAAG TGTGGCAAAA CAACACCTCT ACATGGCACT TTTCAACTTT GCCTCCCCAC GTGGGATGTT CTTCATGGCC TTATGCACTA GGTTGCTGTT AACCGGACAG TGACAAATGC AGCGGAACTC TTGGGATGCA TCTGTTCTGA GCCCTGACCA GCAGCAGGAA GTGTATAGAG TTGCAAAAGA CACAATTATG ATTCGGCTTT TGGGTGAGTG GTGTACAAAC ATTACTCTAT AAACCGGGAC AAGAAAATAT TTTGAGTCAC CTCAGTGGCG GGTTCAATTG GGAGACAATG AGCCAATCCT CTGGCTGACA GTTGGAGTCA CCGGATGGTA TCCAGGCACT TGGATCTGTC AAAGCCCACC GCTC-ACTCCG GCACTCAAGT CCCCACCAGT GGGCCAGGCC TTGTCTGALT GATGCAATGG CCTGAATCAG GTTGTGGTCC CAGACATATT GGGGACGAGC TACGTAGCTA ATTTTCACTG AGTGCAGCGC GGCTTGAACT GTTTATTCTG ACAGCTCTAA TCCGCCGGCC CGGCTTGCTG GGAGTGAACC CCCGCCGAGG GGAACCCCAC ACTACACCAT GGCCCTGCCC CGCGGGAGCG TTTGCGAGCA GAACTGTTTT ACA6ATACTTG TTGATGAACA ACCAGACTGG TGTGGCAACA GGTCTGGCCC ATGCAGTATT ACCCAGAAAC TGGGCAAGGA GAGGTACATC TTCAAAATTT TGATTAGCCC CTGGCTTGGG AGAAGACAGA CTATTATTAA GACCACCATC ACACATTTAA CGTTGCCAGG ACCTGGTCAA GGCCGCTGTG GGTCTGAGCT TGCTAAATTT AACCAACAAA TGACAGGACC TTAATGGGCC ATTCTTTTAT CACCGCGCCA CATTTTGTTC CACTGTTTCA GTTTGTCATC GTGCAGCAGC CCAAAACGCG CAGCTTTTTC CGGAACGGAC CTTCAACTGA CAGACTTCTC GATTTTCCCG TCACAAAGCG CGGTCCTCTC CTGCCAGGTG TTACTGCTGT GTGACTCTCG TTTCTTTTCC GACTCTGCTG TGTCCCCCAT CTGGTTGAAT CTTCGGCAAT TATAAAAGAT TGGACACTTG CAAAGTGTGT CTACCTTCTC ACCCAAAACA CCCACCAAAG CACTCCAACA TTACATGCAA TGAAGGCCAG CGGGTGGTTC AGAGACTACA TTCACAPAGGA CACAGTGTCA GGTAGGTGAG TGACAAACTC GAACGGCTG TGTAGCAGCA TGAGGAACCA GAGGACAAAT TCTGACTTTG TGGCCAAACT TCTTCCAAAT GTCTTCAGTA AGTTCCTGGC CATTTCTGGA GATGGATGTG ATTTTTTGCA CACTAAGGCA cGATGAAGCA TTTTAATGTA GGTTGTGAAT CATTGCCGTG CCTTCGTCAC -375 -315 -255 -195 -135 4S 105 165 225 285 345 405 46S 525 585 645 705 765 825 885 945 1005 1065 1125 1185 1245 1305 1365 1425 1485 1545 1605 1665 1725 1785 1845 1905 1965 2025 2085 2145 2205 SUBSTITUTE, SIHEET (Rule 26) PCT/AU96/0081 WO 97/22701 CCGGCGGACC ACAGAGCCTC TTGCTTGACC GCCACACATG AACACTTGCA ACTCCGTCCT ACCAAGACCA TTGGCCGTTG GCCTGTGCTT GGCTGGGGTG GGTTCCATCT TTCCGCGCGC GACAAAACCA CTCAAATTGG GCAGACCTGA AAAGCCAAGG GCAACACTAG GCTGATTTCG TTGCAGCCTT ACGGCTGCCG CCTGAAGGAG CTTAAAAATT TGGTTTGCTC TTGTACAAGT GACATGCAGC CCATTAGTTA TCAGGGCAAG GTTGGCAAGC AACGGACAGG AAATATTT= GATAAAGGCA TTACCCCTA ACGGTGTCCG CCACTTGCTC ATTATAAATG CTTATTGAGA TTTAAACAAT TTGCAGTTT TCCATTTTAC TTTCACCGCC ATTTTTGTT TATGATCCTT ACTGAGACTG ATTGAGCTTT AATTcATATT GGACGTCATT GACGTGGTGT TGCCCGACAA GTCATGCTTT GGTTCTTTGA TTCTGGATCC CCTACTTCTT CTGTTGGAGA CAGATGCTTG CAGGGATGTG CAGCGTTGCC CACCTACCAA TGCTGACTTC CAGCGATGTA GACATAAATT CTGGAGATGT ATGCCTTGTC CAGAAACTTA CTGTGGCAGC GATTGGAGGT ATATGAAAAC CCCAAATTCC TGGTTGAGAA CTGATTGGTT AAGAACAACA TAAAATTGAA AGCGTGCTCT ACTTGCCCCA GCAAATCTTA AGGACAGTGT CTGTGGTGAT GCCAGATGGT TTCCATTTAC TTACTGTTTC CTAAACCGGG TTAAGCCAGC GGCAGGCTGT TGATACTGTC CATGGTCTGA TAATTGACAA TACATGAAGA TGGCTGCTGA TAGTTTATTC CAACTAAGCC GTGTACCAGC ACCTTGACAPA TGGTGCCCCT ACAAGAAAGC CATCAGATGC GCAGGTTACC GTCACCCGTG TGTTGAGACA G=TGGCTCG TTCTGATTTG GGGCTTGTG GCAGTTAGAA CCCCACTGTT ATATACATCA AGAAAAGGCA TGATGCGCAT TTGCCCTCGA TCCCACTAAC TGAGAGCAAC AACGTCGCTA TTCCCCGTTT TATGAGGACA CTTTGACACT TATTCCTGGT AAAGGGAGTG GCAGGTGTCT CATAAAGACT GGCAGATGAT GGAACCTGAT CGCTGTGAAG GAGCCGTCCA TTTGGCAAAT GTGGAGTTGT TATGGATGCA CTCTACAACA TTCTCCTGTT TTGTCCTGAA CT'rGTGCGC TGGTCTTCCC TAAATTGGCT AGAAGTTCAA TTTGTTCAGA TAAAGATTCA GTATCTTAAA TTTTGCTATG TATGTATCAA AAAACCAAAG AACTGACTTG TGAATTGGTI TCATTTGTTT TGAGTGTGAG AATGTGGGAG GACATGC.ZCG CTTGAGCTTT ACTGCCCAAC GAATTGTCAA TGGGTGAAGT GGAGGTGGAA GTGTTCAAGA ATGTGGCGTG ACCTACAATT GATAAAGGAG CCCATTCCGC ATCAACAAAC CCTGGCCCCA GGTGAATTGA TACAAAATGG AAGGACCCAG GGGT'rTTTCT AAGATTTCTG TATTCTTTTG CTTCGGACAG CTTGTTGCCC GCGCTCTATT ACTCAAGAGG GACAZAAGGTC GAGCCCGTTG CTGATGGCTC CCTCCTGACC TTGGGCCAGG GCTTTTGTAC GTTATTTGTA GCTCTTAAGA ACTGTTGGTT CCACACCCTA CTTTCTGGTG AACAGACAAG AAGTGTACAA rA.AATTCTCA TGGGAGTCAT TTTCTATCCA CTTTTTAAGA ACCCAGGAAG CAACAATCCA ACTGACTGCT GAATTTGATT AAGGTAGAGT AGGATGGTGA TGCACACAGA CAAATTTGAC TGGCTTGGGC TCCAGTTTAA GGCTCCCTGT ATTCAGTTAG TTGCAGGCTC TTGTGCCAGT GGCTTCCTGG GGTGCTACTT CGGCTTTTAC AGTGTACTAA CTATTTTTTC CTGGCATGCT CCAAAATGCT TAGTGGTGGT TTTCCTACTT ATTTGGTCAC TGTCGTCATT TGAATGACAT TCAAGAAATG CAGAATTGGA AAGCGCGCCA TCTTTTCCCT TATGCGTCCT AAGCAATTTC CCACATATTT ATCCGAATGG CACCTATGGC CTACAAATTT GGAGGTTTCG CAAACCAGCT TCTTTGAAA GAGAAGTGAC ACACACACAA CTGATGAGGA GGGCGTTTGT ATATGACCAG TTCTTACTTC CCCCTGACGA TGAAAACACT TCATGAAAAA CTGCCTTGGG TTGATACCAT TTGAGCTTGA CGGGGCCTAA ACCCGGTGAG TCTCAGTTTC AGGTTCTCCT ACGTCTGCTA ATTTACCACC TGGAGCACCA AATTCAAAAA CCGTTCACAA CT-ITTACAAT TGCACACTTT GCGGTATGCT GCGTCCAGCG TTACTCTCTC CAAAGCATCA AAAAGATCTT TTTCAAACTT TATGTTGATT TCAAGAGAAG TGATGCGGCT TTTCGAAACG ATTTGCTTG GCTGACATCC AAAATA TCCC GTGGTTAAL CCTGCAAATT TCGGGGCGCA GCTTCTCTTG TGATGGCTAT TGCTGACTTT CCATTTGGAT GCATTCATCT GTTTGATGTG TTTGAATCTC TGACATGCCC AGCTTTGAG AATGCCCATT ACAGAAAATG TTCAGAACGC GAGAGCCAAG ACTGATTGTT GCAATCAGCT GAAGATTAGG TGTTCAGCCA TGTTTATGAC TGTGCTTGGT AGTGAATGGA AGTTAGAAAT 2265 2325 2385 2445 2505 2565 2625 2685 2745 2805 2865 2925 2985 3045 3105 3165 3225 3285 3345 3405 3465 3525 3585 3645 3705 3765 3825 3885 3945 4005 4065 4125 4185 4245 4305 4365 4425 4485 4545 4605 4665 4725 4785 4845 4905 4965 5025 GTGTCTACTT CGAGTGTCAT SUBSTIUTE SHEET (Rule 26) WO 97/22701PC/U/085 PCT/AU96/00815 ATTGTTCATC TTTTTACAAA ATGAATTCAC CACACCAGGC TCCATCTTAA CATCGGATGG TCGCGTGGGT ATTGTGGCGC CATTCAGCTG GCACTGGATC CAACTCTGGC CCCAGAAACA GTTCCGCGCC GCTCCAAATT GGCCCAGCGC CACTCTCTCA GTGGTTTTTG CTAAACATAC TTGCGTGCGG CGCATGTATA GCTTTGACTG AAAAAGAGGC ACCGCTCCCG GGCTGCCCTA GAAGAGGGCC GGCTGAAGGG TCTAATTTGG TCTATCAATC GCTGGAAAGA CCCGCCTGAT CTCTTGGGCC GGTTTGTGGC ATTGGATGTG ACCCAGATGT TATGTATATG CCTGTGACTA GTTGTGCTTA ACTACTTTTT ATTGAGTC-AC TGGTTGATTC GGCTTGCCAT CCGGGTGTTC ATTCTTGCAG CTATGATGAA TGCTATGGGG ACGACTGCCT CCTGTCTTTT CTAGTTTTGG CTGACAACGC TTTCGGAGGT AAGCCAGTGA TGGATGTGAA CAGGCTGAAA AGCTCCTGTC GAGCGCCTGT TTGAGCCCTT GCAGTGGTTG ATGAAGCTTG TTTAGr.CTTT TAAGGTGTTA TGAAATTGAA ACGCACTGTG GACTGTAATG TGCAATTGTT TGTTGCATTT GGGCAACGTT GGTGAAATCA ATTTGACAAG TGGAGACAAG CGCCCAGAAG CATTTGTGGC TGCTCAGCAA CGCCGAACTC ATTTTTGAAA TGACGTGCCG AAAATTTCAT GGACTGGACT CTCACGGTTC CTCTGAGGAC AGTGCATGCC CTGCACATCA GGCTTATGAG CATTGCTTCT ACAGGTAATA GACCTTCCTT GACCCTTGAA CGTGGCGCAG TGCTGGGATG GATGCTAAPLT AGTTTAAAGG TTGAAGAAAA TTTTTTGGCA CCCAATGTTT GCTGGCTCAC TGCTCCCTGG AGTCGC-CTTG TTGGCTTACC CGCCTGTCAG GAGATTTCCG GTTTTCTCCC ATTCCTGGCC AATAAGAGAA CAAAAGGACA GATGAGATCC CCGATGCCCC GAAGCA.AATG CGGTTTGGCC GATGCCAACC CACGGTTTCG TATGAAGAGA ATTTGAATA AATTTTGAGC GATTTTGAAP. ACTACAGCTG AAGCGCGCTT GCAATCTTAA TTGGCAGGCC TATTTCGTCC TCTTTTTGAC TTAAGAGTTT TGACCCAAGT GTTTTTTGAC TGTCCTATGC CTGCCCGCAT TGTCCAGAGA ATGACGATGT CCATTTTCAA ACGGCGTGAA CACAGGACCA GGATTGGATT TTGACAAGAT GGAAAGACAT GATTTATGGC GCCCACTTGA ATGTGGTGGT GATTTGACAT TCGAGTTGGA ATGCAGCTGA ACCCTGGTGT AAAGGTATAA CCATCTTGAA CAGATGACAT TTGATTTCCA ACAAGACTGA TTGTTCTGAC GCTTTGTTCG ACTGCGAACC CTACTTGGCG TTTGTTTTTC TTAGAAGTTA GACAGGAATC AGTGAGGAAG CGCTCAGACC AATTGGTLATC CGCGCTGGAG GAGGGTGTCT ACCTGACTAT GCTGGATGAA GAAATGGCTC TGACAACCAG GGAGCAGGAC CTGTGATTTT TGGTGACTAC GAAAGTTAGG TGGTAGGCAG TGGCTCAGCC GCGTTTCAGG TGCAATGAGA GCCTGCTTT CATCTACGGT CAATGTTTAC TCAGGTCATT ACAACTGGTG TTTTTTAAA GGCCTTTTAC CCCAGGCACA GGAGCAGTAT ACTTGCGCCT TTTGTITTCT AGATAGAGTT 5085 5145S 5205 52465 5325 5385 S445 5505 5565 5625 5685 5745 5805 5865 5925 5985 6045 6105 6165 6225 6285 6345 6405 6465 6525 6585 6645 6705 6765 6825 TAGTTTTTAG TTTTGAGC-poly (A) as disclosed in Fig. 2 and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants, degeneracy equivalents and deletion mutants thereof. SUBSTIUTE SHEE (Rule 26) PCT/AU96/0081 WO 97/22701

2. A substantially pure amino acid sequence being: M L T R P I V E E G S S F K v A N H s P R K E 1 T P Q V Y R V v F V P A LT Q R KT I V Y K L G P K V T S E G F A P Y L 4 VP4 L S GG N F Y M GOS N T E Q T L L D K T T Q I L w V S D Q H G H L S D F I c V D F E S L A G A V R R L T G L I R L F D TOG I K D D V G T M A C V S A K K I Y P K D P P K TOGN Q N I D AD L S 0 S SOG O T S T P QQ YQ N sSSS T S VP4 4 VP2 LOGT V GV P S T w PH S S V Iv I D AM Y V A A A T A D F T AL K L LA D K K T E E T T T I I NS QG SV GT T V S D P V T R L 0 P T L S Q S H G HMAW IC P L V V K AMK H L V KNG W O P L C V A AV P E YE E PA Y T YQQ L S V F H L V M P Y I GPO GQ P L I L S E L TG P GQ T VP2 4 VP3 V NG P L P NP E AP I S V P D N S T PL Y P K N F I D V A KOQTY S F T S G D E P L FO0 M DV S L S S F F AQ YROG S K A K F L V A F V PPMH I HMA V W D V 0 L N S A M A V Y S A E R T VV N T S T D I A V N S K O R VP3 4 VP1 R H PA D L P D K Q V T Vs V V P P I S G S A A F N F A P K F N V O WL V A V S A G P D F S L N vG E D SUBSTITE SHEET (Rule 26) PCT/AU96/0081 WO 97/22701 G E P G E T E P R H L L D R F F D V ET D P F G S T A Q L A E L SEIQ F K F T T P V G AP T K T T D L A V AG M C P T V L P Y T S M W R V V T Y N W L P G A H F Y L R YA FR A P A D K TR H K F P T N 2A 4 2B D V E S N PG P T I G EL T G M L K D L M L F K L AT L A V A D F GLE V F D T K T I P G K I S D L A L S P V D M H V H T 0 V S F Y D w L D V v V A N T p L H L E L E L S N L T G S W AR L L N T C T T P S S V G E G F A W Q L E GG G N V F K I A GS R S P V F Y NG WG A G S IL L TS D A M Y CP R P IP P VP1 4 2A I N X Q CT NY S F S K AS A DL N K A KAE T Y S P A A MR TK DPV G F FF5S Y FQ E V T D AA T AA A 23 4 2C P E GV VE K QV W F I K T L VA L A LY S E L EK Y RQ W F KD MQ0Q P L VN L P QS R S YL A NL MA Q P PD PT YFD G N GA D F K YF C D KG I P F TS P V S C P E AL K R T VG S N Q L L N E N D M P I1 N G L I E M I L S E V S D L F R K CT T ElI L R A F V S E T R R A K F H R L F V SS F F E T FPAL L K N S D A Q E 0Q A DD E P D T Q E E A G L F S L L Q I R GA S G Q G K K Q D SV W S C M DAL G Q D P V P P MARH L D H S S F T PI T S A K P G F V R G L P P H PI F G G E V T A F E 2C 4 3A M P I F K Q S W F L I D N K D S Y L K W E S Y M A T F F W V V R A C T K D K F T L L K LA G A L S F Y K vV V K LQ Q IP S L R KR Kw PL Y R A L PE P A I S Y NG Q MV V V I R F R L P L Q AV Q NR D EE R S I A A D StJBSTITMYrE SMEBT (Rule 26) PCT/AU96/00815 WO 97/22701 F D T D E L I Q P R Q K R D A P Q L L 0 D G G M D T K L L S I 3A 1 31 E QS 33 4 3C E T G N E L F D F E V N V HL L A D G F V S I Q S G L K Q F R F T A F G E D K D Q T S L I V I F Y D P S T K P CL V Y S M Y Q L F K T P K P K T Q E V K T L K I R P A TD T D C S T I V L D V VS T NE I F F G S Y A AQ H SA G 3C 4 3D K QG N L AY P V KL D L RA A E A I C N K RR M AG D A G K T V A K F R FG L A DA M I E S L C S C T N F E P F QQ L L T T L V K T L L A G H w R L A S I V Y H Y C L K M VyR G Y V A L D P D F A A Q G L C D L V D V N G R F L N V a N T Q V F S T F L S E Q M K D N K K L R T Q K S C G F C D D Y G KHK K V D K F E Y Q P P F 0 R Y Y F A Y I L S Y L V T G I L T A A I S L V L Y H L E F V R S P T V S P A L R V A P G D S R E Q G R F L PHK G S Y A S E E K N V G D Q V A F P G L F P A V V 0 E A W M L NS F Y F V P T SUBSTITUT SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 33

3. A protein or virus like particle incorporating VP 1, derived from ERhV I and having the following amino acid sequence: V T N V G E D G E P G E T E V H T D V S F L L D R F F D S PA T H V L D P F G S T A T Y F F S D L E L S I Q F K F V W V K W L P V G AP T K N S V R I Q K L AV AG M C S Q AC A S A LP Y T S M W A P T K E K A T Y N W L P G A H D K G G C Y L R Y A F R P A F T R P AD K T R H K F R H A L S P V D M H E T L E L S N L T G L AW A R L L N T C T T T P55 V G E G T D A W Q LE G G G T V V F K I A G S R V V P V F Y N G W G H F G S IL L T S D PA M Y C P R P IP T N I N K Q C T

4. A protein or virus like particle incorporating VP2, derived from ERhVlI and having the following amino acid sequence: D K K T E E T T N I E D R I E T T V V G V T I I N S Q G S V G T TY C Y S K P D GR P P S TV S D P V T R L G P T L S RHR Y T F K VG E W P H S Q S H G H AW I C P L P G D K L K K MG S F H E VV K A H H L V K NG WD V VV Q V N P S F ARHS G P L C V A A V P E Y E H T H E K A L K W S E L E E P A Y T Y QQ L S V F P H Q L L N L RT N S S V H LV M P Y I G P G Q P T N L T L H N P W T I V I L I L S E L T G P G Q T V P V T M S VA P I D A MV NG P L P N P E SUB STITUTE SHEE T (Rule 26) PCT/AU96/0081 WO 97/22701 A protein or virus like particle incorporating VP3, derived from ERhVlI and having the following amino acid sequence: A PI R V V S V P E S D S F M S S V P D N S T P L Y P K V VV P P R Q VP G RF T NF I D V A K QOT Y S F C S IS G K P YF E V T NT S G D E P L FQ0 M DV S L S A ASE L H G T Y VA S L S S FF A Q Y R G S L N F N F I F TG A AA T K A K F L V A F V P P H S A AP K T R DE A MA C I H AV W D VG L N SA F S F N V P Y PS P AD F M AV Y S A E R T V V N V S G W L Q V Y A L T A L T S T D I A V N S K G R V L V A V S AG P D F S L R H P AD L P D K

6. A protein or virus like particle incorporating VP4, derived from ERhVl and having the following amino acid sequence: G G G T S T P T T G N Q N M S G N S G S I V Q N F Y MQ0 Q Y QN S I D AD L G D NV I SP E G Q G S N T S S S T S S S S S G L GG W F S S L L N L G T K L L A

7. A substantially pure nucleotide sequence for VPI being: GTTACCAATG CCCGTGGACA GAGACACTTG GGCTCGACTG GATTTGGAIAT TTTGTGTGGG TTAGAAGGAG ACTGTTGTGT ACATCAATGT AAGGCAACCT GCGCATGATA CCTCGACCCA ACTAACATCA TGGGAGAGGA TGCACGTGCA AGCTTTCAAA CCCAACTGGC TGTCAATCCA TGAAGTGGCT GTGGAAATTC TCAAGATTGC GGCGTGTTGT ACAATTGGCT AAGGAGGGTG TTCCGCCGGC AcAAACAGTG TGGTGAACCC CACAGATGTC TTTGACAGGT TTGGGCACGT GTTTAAATTT CCCTGTTGGA AGTTAGAATT AGGCTCCCGT GCCAGTCTTT TCCTGGTGCA CTACTTGCGG TTTTACGCGT TACT GGTGAGACAG AGTTTCTTGC TCTCCTGCCA CTGCTAAACA ACCACCACTC GCACCAACCA CAAAA6ATTGG TCACAAGCCT TACAATGGCT CACTTTGGTT TATGCTTTCC CCAGCGGACA AGCCTCGTCA TTGACCGGTT CACATGTTCT CTTGCACCTA CGTCCTCTGT AGACCACAGA CCGTTGCAGG GTGCTTCAGC GGGGTGCACC CCATCTTGCT GCGCGCCAGC AA6ACCAGACA TGCTTTGTCA CTTTGATGTT GGATCCGTTT CTTCTTTTCT TGGAGAGGGC TGCTTGGCAG GATGTGCCCC GTTGCCATAT TACCAAAGAA GACTTCTGAT GATGTATTGC TAAATTTCCC SUBSTITUT SHEET (Rule 26) WO 97/22701 PCT/AU96/0081 and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

8. A substantially pure nucleotide sequence for VP2 being: GACAAGAAGA GTCACTATTA GGTAGACCAC CACTACACAT TGTCCGTTGC CACCACCTGG TCCGGGCCGC AAGTGGTCTG CAGTTGCTAA GGCCAACCAA GAATTGACAG ATGGTTAATG CAGAAGAGAC TTAATTCACA CATCCACAGT TTAAGGTAGG CAGGTGACAA TCAAGAACGG TGTGTGTAGC AGCTTGAGGA ATTTGAGGAC CAAATCTGAC GACCTGGCCA GGCCTCTTCC TACAAACATT AGGATCTGTT GTCAGACCCA TGAGTGGCCC ACTCAAGAAG CTGGGATGTG AGCAGTGCCG ACCAGCTTAC AAATTCATCA TTGCACAAC AACTGTGCCT AAATCCAGAG GAAGACAGAA GGAACAACCT GTTACCAGAC CATTCTCAAT ATGGGCAGTT GTTGTGCAGG GAGTACGAAC ACATACCAAC GTGCATTTGG CCGTGGACCA GTGACCATGT TTGAAACAAC ACTGTTACTC TTGGACCCAC CACATGGTCA TTCATGAGGT TGAATCCCTC ACACACATGA AACTTTCAGT TGATGCCCTA TTGTTATTT CGGTGGCTCC AGTGGTTGGA CAAACCGGAT GCT'rTCCAGG CGCATGGATC TGTCAAAGCC ATTTGCTCAC GAAAGCACTC TTTCCCCAC CATTGGGCCA AATTTTGTCT CATCGATGCA and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

9. A substantially pure nucleotide sequence for VP3 being: GCACCGATTA GAGTGGTGTC TGTGCCTGAA TC-AGATTCTT TTATGTCTTC AATTCGACTC CACTATACCC CAAGGTTGTG GTCCCACCGC GCCAAGTTCC ACAAATTTCA TTGATGTGGC AAAACAGACA TATTCATTTT GTTCCATTTC TATTTTGAGG TTACCAACAC CTCTGGGGAC GAGCCACTGT TTCAGATGGA AGTGCGGCAG AGCTACATGG CACTTACGTA GCTAGTTTGT CATCATTTTT AGAGGCTCAC TTAATTTCAA CTTTATTTTC ACTGGTGCAG CAGCCACTAA CTGGTTGCTT TTGTGCCTCC CCACAGTGCA GCGCCCAAAA CGCGCGATGA TGCATCCATG CCGTGTGGGA TGTTGGCTI'G AACTCAGCTT TTTCTTTTAA CCCTCCCCTG CTGACTTCAT GGCCGTTTAT TCTGCGGAAC GGACGGTTGT GGATGGCTTC AAGTTTATGC ACTAACAGCT CTAACTTCAA CTGACATTGC AAAGGCCGTG TGCTGGTTGC TGTTTCCGCC GGCCCAGACT TCTCCCTTCG GACCTGCCCG ACAAGCAG AGTACCTGAT TGGCCGGTTT TGGAAAACCT TGTGTCGCTC TGCACAGTAC GGCAAAGTTT AGCAATGGCG TGTACCTTAT GAATGTCTCT CGTGAACAGT TCACCCGGCG SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 36 and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents. A substantially pure nucleotide sequence for VP4 being: GGCGGAGGTA CATCCACTCC AACAACTGGC AACCAAAACA TGTCCGGAAA ATTGTTCAAA ATTTTTACAT GCAACAGTAC CAGAATTCAA TTGACGCAGA AATGTGATTA GCCCTGAAGG CCAGGGCAGC AACACTAGTA GTTCAACCTC TCCTCTGGCT TGGGCGGGTG GTTCTCTAGT TTGCTGAACC TTGGAACAAA CAGTGGTTCA CCTGGGAGAC ATCAAGCCAA ACTACTGGCT and functional equivalents of said nucleotide sequence including naturally occurring derivatives, variants and degeneracy equivalents.

11. Oligonucleotide primers derived from the nucleotide sequence of claim 1 being highly specific for ERhVl or cross-reactive with other ERhV types.

12. An nucleotide

13. An nucleotide

14. An nucleotide oligonucleotide sequence: VP1F 5' oligonucleotide sequence: VP1R1 oligonucleotide sequence: VP1R2 primer according to claim 11 having the following GTTGTGTTCAAGATTGCAGGC 3' primer according to claim 11 having the following 5' TTGCTCTCAACATCTCCAGC 3' primer according to claim 11 having the following 5' TAGCACCCTCCTTTATCATGCG 3' Oligonucleotide probes derived from the nucleotide sequence of claim 1.

16. Diagnostic reagents, methods and kits characterised by the oligonucleotide primers and probes of claims 11 to

17. Antigens comprising any one or a combination of the non-capsid proteins, being other than the individual VP1 to VP4 proteins, that are cleavage products of the polypeptide of claim 2. SUBSTITUTE SHEET (Rule 26) WO 97/22701 PCT/AU96/00815 37

18. Vaccines characterised by the incorporation of any one of a combination of virion proteins VPI to VP4.

19. Vectors characterised by the incorporation of any one or a combination of virion proteins VP1 to VP4.

20. A diagnostic test for the detection of antibodies to ERhV I in blood of horses and any other animal species characterised by the use of the antigens of claim 17.

21. A diagnostic test according to claim 20 being an enzyme linked immunosorbent assay.

22. A test to distinguish horses infected with ERhVI in which said virus had replicated from horses which have been vaccinated with the vaccine of claim 18 comprising the steps of applying an antigen of claim 17 to a horse and testing for an immunoreaction thereto, wherein a positive immunoreaction would indicate that said horse had been infected with ERhVI and a negative immunoreaction would indicate that said horse has not been infected with ERhV1.

23. Recombinant plasmids comprising nucleotide sequences and subsequences derived from the nucleotide sequence of claim 1.

24. A recombinant plasmid according to claim 22 comprising the P1-2A-3C region of the ERhVI genome. A host system characterised comprising the nucleotide sequence of claim 1 or part thereof.

26. A process for producing a protein product derived from ERhVl comprising the steps of selecting out a gene of interest from the ERhVI nucleotide sequence of claim I and expressing said protein product in a suitable host system. SUBSTITUTE SHEET (Rule 26)