Hepatitis-C virus testing

New styles of hepatitis C virus (HCV), referred to as HCV-3 and HCV-4, have been identified and sequenced. Antigenic regions of HCV-2, HCV-3 and HCV-4 polypeptides have been identified. Immunoassays for HCV and antibodies thereto are described, which allow more complete screening of blood samples for HCV, and allow HCV genotyping.

TECHNICAL FIELD 
This application is a 371 of PCT/GB92/02143 filed Nov. 20, 1992, published 
as WO93/10239 May 27, 1993. 
The present invention relates to the discovery of new types of hepatitis C 
virus, that we have termed type 3 (HCV-3) and type 4 (HCV-4). In 
particular, it relates to the etiologic agent of hepatitis C virus type 3 
and 4, and to polynucleotides and immunoreactive polypeptides which are 
useful in immunoassays for the detection of HCV-3 and HCV-4 in biological 
samples; and also to the use of antigenic HCV-3 and HCV-4 specific 
polypeptides in vaccines. 
BACKGROUND OF THE INVENTION 
Acute viral hepatitis is a disease which may result in chronic liver 
damage. It is clinically diagnosed by a well-defined set of patient 
symptoms, including jaundice, hepatic tenderness, and an increase in the 
serum levels of alanine aminotransferase and aspartate aminotransferase. 
Serologic immunoassays are generally performed to diagnose the specific 
type of viral causative agent. Historically, patients presenting with 
symptoms of hepatitis and not otherwise infected by hepatitis A, hepatitis 
B, Epstein-Barr or cytomegalovirus were clinically diagnosed as having 
non-A, non-B hepatitis (NANBH) by default. 
For many years, the agent of non-A, non-B hepatitis remained elusive. It 
has now been established that many cases of NANBH are caused by a distinct 
virus termed hepatitis C virus (HCV). European Patent Application 
EP-A-0318216 discloses CDNA sequences derived from HCV, polynucleotide 
probes and polypeptides for use in immunoassays. Further information is 
provided in European Application EP-A-0388232. 
The HCV genome encodes a large polyprotein precursor, which contains 
structural and non-structural regions. The single protein is apparently 
cleaved into a variety of proteins after production. Most of the 
structural and non-structural proteins have now been identified from in 
vitro RNA translation and expression as recombinant proteins. The C and E 
regions encode for nucleocapsid structural proteins and for envelope 
structural proteins, respectively. At least five additional regions 
follow, which encode for non-structural (NS) protein of undefined 
function. The organisation is believed to be as follows (A. Alberti, 
Journal of Hepatology, 1991; 12; 279 to 282) 
##STR1## 
Certain immunoreactive proteins have been described as recombinant 
proteins, for example C22 (in the core region), C33 (in NS3 region), 5-1-1 
and C100 (both in the NS4 region), and NS5 (NS5 region). Diagnosis of 
hepatitis C is still largely based on methods which detect antibodies 
against the product of the C-100 clone. This clone was ligated with 
overlapping clones to produce a larger viral antigen (C100) corresponding 
to part of the NS3-NS4 genomic region. C100 was then fused with the human 
superoxide dismutase (SOD) gene, expressed in use as a large recombinant 
fusion protein (C100-3) and used on solid phase to develop radio-labelled 
(RIA) and enzyme-linked immunosorbent assays (ELISA). 
Polynucleotides useful for screening for HCV are disclosed in European 
Patent Specification EP-A-0398748. European Patent Specification 
EP-A-0414475 purports to disclose the propagation of HCV in culture cells 
and the production of antigens for use in diagnostics. European Patent 
Specification EP-A-0445423 discloses an improved immunoassay for detecting 
HCV antibodies. 
Blood banks in the United Kingdom have recently begun routine testing of 
blood donors for antibodies to components of HCV. One assay involves the 
detection of HCV antibodies to C100-3 polypeptides. The C100-3 antibody 
recognises a composite polyprotein antigen within non-structural regions 
of the virus and is a consistent marker of HCV infection. However, in 
acute infections this antibody is unreliable because of the delay 
(typically 22 weeks) in seroconversion after exposure. Furthermore, the 
C100-3 antibody test lacks specificity for the hepatitis C virus. 
Second generation antibody tests employ recombinant antigens or synthetic 
linear peptides representing structural antigens from the highly conserved 
core region of the virus as well as non-structural antigens. However, it 
is found that some second-generation ELISA tests can yield false-positive 
reactions. The recombinant immunoblot assay (RIBA-2) incorporating four 
antigens from the HCV genome, provides a method for identifying 
genuine-anti-HCV reactivity. However, the result can be "indeterminate." 
The present workers have reported (The Lancet, 338; Oct. 19, 1991) varying 
reactivity of HCV-positive blood donors to 5-1-1, C100, C33C and C22 
antigens, and compared these with the results of the direct detection of 
HCV RNA present in the blood samples using polymerase chain reaction (PCR) 
to amplify HCV polynucleotides. However, the work demonstrates that the 
unambiguous diagnosis of HCV infections is not yet possible. 
Recently there has been discovered a second type of HCV (References 1, 2) 
called K2 that differs considerably in sequence from the published 
prototype (Reference 3) or the first type K1 sequences (References 4 and 
5). 
SUMMARY OF THE INVENTION 
The present invention is based on the discovery of previously unknown type 
3 and 4 variants of HCV, by a comparison to sequences amplified by PCR in 
certain regions of the HCV genome and confirmed by phylogenetic analysis. 
The invention has thus identified polynucleotide sequences and peptides 
which are HCV-3 and HCV-4 specific. These may be used to diagnose HCV-3 
and HCV-4 infection and should thus be included in any definitive test for 
HCV infection. 
One aspect of the invention provides polynucleotide sequences unique to 
hepatitis C virus types 3 and 4 (HCV-3 and HCV-4). The sequences may be 
RNA or DNA sequences. In principal any HCV-3 or HCV-4 specific 
polynucleotide sequence from non-coding, core, E1, E2 or NS1-5 genome 
regions can be used as a hybridisation probe. The sequences may be 
recombinant (i.e. expressed in transformed cells) or synthetic and may be 
comprised within longer sequences if necessary. Equally, deletions, 
insertions or substitutions may also be tolerated if the polynucleotide 
may still function as a specific probe. Polynucleotide sequences such as 
core, NS3, NS4 and NS5 which code for antigenic peptides are particularly 
useful. 
Another aspect provides an antigenic HCV-3 or HCV-4 specific peptide, 
particularly from the core, NS3, NS4 or NS5 regions (e.g. the HCV-3 or 
HCV-4 counterparts of C100 peptide, 5-1-1 peptide, C33 peptide or C22 
peptide or epitopes thereof) or peptides including these antigens. 
The peptide may be a fusion peptide which comprises at least two of the 
antigenic HCV-3 or HCV-4 specific peptides. A fusion peptide may also 
comprise at least one of the antigenic peptides fused to 
.beta.-galactosidase, GST, trpE, or polyhedrin coding sequence. 
A further aspect of the invention provides labelled antigenic HCV-3 or 
HCV-4 specific peptide (or mixtures thereof, particularly from the core 
and NS4 regions) for use in an immunoassay. 
A further aspect of the invention provides antibodies to HCV-3 or HCV-4 
specific antigens, particularly monoclonal antibodies for use in therapy 
and diagnosis. Thus labelled antibodies may be used for in vivo diagnosis. 
Antibodies carrying cytotoxic agents may be used to attack HCV-3 or HCV-4 
infected cells. 
A further aspect of the invention provides a vaccine comprising immunogenic 
HCV-3 or HCV-4 specific peptide. 
The HCV-3 or HCV-4 specific polynucleotide sequences may be used for 
identification of the HCV virus itself (usually amplified by PCR) by 
hybridisation techniques. 
Oligonucleotides corresponding to variable regions in the NS-4 region could 
be used for type-specific PCR. Outer sense and inner sense primers may be 
used in combination with the two conserved anti-sense primers for a 
specific detection method for HCV types 1, 2, 3 and 4. 
Immunoreactive HCV-3 or HCV-4 specific peptides (particularly from the core 
and NS4 regions) may be used to detect HCV-3 and HCV-4 antibodies in 
biological samples, and may also provide the basis for immunogens for 
inclusion in vaccines (especially the E1 polypeptide). The term "peptide" 
is used herein to include epitopic peptides having the minimum number of 
amino acid residues for antigenicity, oligopeptides, polypeptides and 
proteins. The peptide may be a recombinant peptide expressed from a 
transformed cell, or could be a synthetic peptide produced by chemical 
synthesis. 
In particular, the invention allows blood donor screening by conventional 
assays (using HCV type 1 encoded antigens) to be supplemented with a 
second test that contains two oligopeptides corresponding to first and 
second antigenic regions found in the NS-4 sequence of HCV type 3 
(positions 1691 to 1708; sequence KVPDKEVLYQQYDEM (SEQ ID NO:1) and 
positions 1710 to 1728; sequence ECSQAAPYIEQAQVIAHQF (SEQ ID NO:2)) and 
two derived from the equivalent regions of HCV type 2, 
R(A/V)V(V/I)(A/T)PDKE(I/V)LYEAFDEM (SEQ ID NO:3 or 4) and 
ECAS(K/R)AALIEEGQR(M/I)AEML (SEQ ID NO:5 or 6). 
The corresponding HCV-4 antigens from substantially positions 1691 to 1708 
and 1710 to 1728 may be used for HCV-4 detection. 
Thus, the present invention has also identified corresponding 
polynucleotide and peptide sequences which may be used to identify 
hepatitis C type 2 viral infection. 
Production and detection of the antigen-antibody immune complex may be 
carried out by any methods currently known in the art. For example, a 
labelling system such as enzyme, radioisotope, fluorescent, luminescent or 
chemiluminescent labels may be employed, usually attached to the antigen. 
Labelled anti-antibody systems may also be used. The recombinant antigen 
may be either used in liquid phase or absorbed onto a solid substrate. 
Oligopeptides corresponding to the antigenic regions of all three major 
types may also be used separately to serologically distinguish individuals 
infected with different HCV types. Such an assay could be in the format of 
an indirect enzyme immunoassay (EIA) that used sets of wells or beads 
coated with peptides of the two major antigenic regions for HCV types 4, 3 
(SEQ ID NO:1 or 2) and 2 (SEQ ID NO:3, 4, 5 or 6), and with type 1 
(KPA(V/I)IPDREVLYREFDEM (SEQ ID NO:7 or 8) and RPAV(I/V)PDREVLYQEFDEM (SEQ 
ID NO:9) and ECSQHLPYIEG(M/A)AEQF) (SEQ ID NO: 10 or 11). Minor degrees of 
cross-reactivity, should they exist, can be absorbed out by dilution of 
the test serum in a diluent that contained blocking amounts of soluble 
heterologous-type oligopeptides, to ensure that only antibody with 
type-specific antibody reactivity bound to the solid phase. 
Immunogens for use in vaccine formulations may be formulated according to 
techniques currently known in the art, including the use of suitable 
adjuvant and immune-stimulation systems. 
Furthermore, the present invention also encompasses assay devices or kits 
including peptides which contain at least one epitope of HCV-3 or HCV-4 
antigen (or antibodies thereto), as well as necessary preparative 
reagents, washing reagents, detection reagents and signal producing 
reagents. The antigen may be from the core or NS4 regions. The assay 
device may be in the form of a plate having a series of locations 
respectively containing HCV-1, HCV-2, HCV-3, and optionally HCV-4, 
specific antigens. 
The invention also provides a method of in vitro testing for HCV which 
comprises reverse transcribing any HCV polynucleotide present and 
amplifying by polymerase chain reaction (PCR), and detecting the amplified 
HCV polynucleotide employing an HCV-2, HCV-3 or HCV-4 specific 
polynucleotide probe. 
The invention further provides a method of in vitro HCV typing which 
comprises 
carrying out endonuclease digestion of an HCV-containing sample employing 
ScrFI or HaeIII/RsaI endonuclease; and 
comparing the restriction patterns with characteristic type-specific 
patterns. 
The endonuclease digestion may also employ HinfI in a separate or the same 
digestion. 
The invention furthermore provides a method of in vitro HCV typing which 
comprises 
carrying out endonuclease digestion of an HCV-containing sample employing 
ScrFI endonuclease, the restriction pattern being characteristic of HCV-1, 
HCV-2 and HCV-3; 
carrying out endonuclease digestion employing HinfI endonuclease, the 
restriction pattern being characteristic of HCV-4.

I) ANALYSIS OF HEPATITIS C VIRUS AND PHYLOGENETIC RELATIONSHIP OF TYPES 1, 
2 AND 3 
Introduction 
Sequence analysis of the 5' non coding region of hepatitis C virus (HCV) 
amplified from the plasma of individuals infected in Britain revealed the 
existence of three distinct groups of HCV, differing by 9-14% in 
nucleotide sequence. Two of the groups identified were similar to those of 
HCV variants previously termed type 1 and type 2, while the third group 
appeared to represent a novel virus type. Sequence comparisons were then 
made between the three virus types in other regions of the viral genome. 
In the NS-5 region, a high degree of nucleotide and amino acid sequence 
diversity was observed, with samples classified here as type "3" (SEQ ID 
NO: 13) again forming a distinct group that was phylogenetically distinct 
from type 1 and type 2 variants. Type 3 sequences were similarly 
differentiated in the NS-3 (SEQ ID NO: 14) and core (SEQ ID NO: 15 and 19) 
regions from HCV type 1 sequences. The designation of virus types, 
including an observed sub-division of type 1 sequences into geographically 
distinct variants is discussed in relation to the new sequence data 
obtained in this study. 
Discussion 
Replication of nucleotide sequences by polymerase chain reaction (PCR) is a 
recently established technique. Synthetic complementary primer sequences 
are hybridised to single-stranded DNA on either side of a genome region to 
be copied. The second strand is built up under the action of a heat-stable 
polymerase in the region between the primers. Heating then dissociates the 
two-strands and the replication process starts again. The PCR technique 
allows tiny amounts of polynucleotide to be amplified provided that there 
is sufficient sequence information to synthesise the primer sequences. 
The major problem associated with the use of the PCR to assess sequence 
variation using the PCR is the possibility that mismatches between the 
primers and the variant sequence will prevent amplification. We have used 
several strategies to overcome this problem. For initial virus detection, 
we used primers in the 5'NCR, which are reported to be highly conserved 
amongst type 1 variants (4, 11, 13, 16, 23, 24, 26, 33), and between K1 
and K2 (23). Sequence analysis of the blood donors allowed the 
identification of type 1 and type 2 variants by comparison with published 
sequence data. This analysis also revealed the existence of a third "type" 
of HCV (SEQ ID NO: 12) that appeared to be as distinct from type 1 as type 
2 was (FIGS. 1, 2; Table 3). Based on our initial tentative 
classification, we sought corroboration of our findings in other (coding) 
and more variable regions of the viral genome. 
Analysis of the NS-5 region, which was based on several sequences of each 
of the three types (FIGS. 3, 4; Table 3), conformed the existence of 3 
major groups, with type 3 sequences (SEQ ID NO: 13) forming a relatively 
homogeneous group that was quite distinct from types 1 and 2. The proposed 
separation of type 1 sequences into PT and K1 "sub-types" and type 2 
sequences into K2a and K2b is supported by this analysis, in which the 
single type 2 blood donor sequence obtained in this study appears most 
similar to K2b. Differential n of HCV type 1 sequences into two groups is 
also clearly shown in the core (FIG. 7) and NS-3 regions (FIG. 5), in both 
cases with the type 3 sequences (SEQ ID NO: 15 and 14 , respectively) 
appearing considerably more distant. 
The clustering of phylogenetically distinct groups, their mixed 
distributions in a single geographic area (1, 7, 23, 27, 35) and our own 
finding of dual or triple infections in individual hemophiliacs all 
strongly suggest that the three types described here are distinct viruses 
rather than simply representing geographical or epidemiologically 
clustered variants of a single, highly variable but monophyletic group. 
Our own phylogenetic analysis of the 5'NCR reveals the existence of three 
distinct groups. This contrasts with analyses of coding region, where 
there appears to be a very prominent differentiation of type 1 sequences 
into two "subtypes". However, unlike type 2 and 3 variants, the two 
subtypes are geographically distinct, one sub-type comprising sequences 
obtained exclusively from Japanese patients, and the other comprising 
predominantly USA/European sequences (Table 2). Indeed the only exception 
to this geographical classification is the HC-J1 sequence (26); one 
apparent exception (Pt-1) was obtained from a Japanese hemophiliac treated 
with imported factor VIII of USA origin (7,23), which is likely to have 
contained HCV variants corresponding to the other sub-type. There is 
insufficient sequence data to indicate whether the two proposed type 2 
subtypes, K2a and K2b (7,23) represent geographically distinct variants. 
The genomic organisation of HCV corresponds to that of flaviviruses and 
pestiviruses, with a single open reading frame encoding a polyprotein that 
is subsequently cleaved into structural and non-structural proteins. Weak 
sequence homologies have been detected with several other virus groups 
that have positive-sense RNA genomes (19,21). Although the overall degree 
of sequence dissimilarity between types 1, 2 and 3 cannot be measured by 
comparison of the small regions of sequence analysed in this study, a 
rough estimate of the extent of divergence in protein coding regions is 
given by an examination of the divergence of the partial core sequence. 
This shows that the difference between HCV type 1 and type 3 (SEQ ID NO: 
15) core region (approximately 10% amino acid sequence divergence) is 
comparable to that which exists between different serotypes of the 
flavivirus, tick-borne encephalitis virus (14%; ref.20), but lower than 
that which is found between serotypes of a mosquito borne flavivirus, 
dengue fever virus (33%) , and the West Nile (WN) subgroup (28-43% 
divergence). The 5'NCR sequences of the different members of WN subgroup 
are also considerably more diverse than those of the three types of HCV 
(=50% similarity; ref.5), although within each of the members e.g. Murray 
Valley encephalitis virus, the 5'NCR is extremely well conserved (&gt;95% 
similarity; ref.5). On the basis of these analogies, we speculate that the 
major types of HCV represent distinct "serotypes," each capable of human 
infection irrespective of the immune response mounted against other HCV 
types. 
METHODS 
Samples. Plasma from 18 different blood donors (E-b1 through E-b18), that 
were repeatedly reactive on screening by Abbott 2nd generation 
enzymeimmunoassay (EIA), and confirmed or indeterminate by a recombinant 
immunoblot assay (RIBA; Ortho; ref 1) were the principal samples used in 
this study. Sequences in the NS-3 region from 5 anti-HCV positive IVDUs 
(abbreviated as i1-i5 in ref. 31), 5 hemophiliacs who had received 
non-heat treated clotting concentrate, and who were also anti-HCV positive 
(h1-h5), 3 pools of 1000 donations collected in 1983 (p1-p3), and 5 
separate batches of commercially available non-heat treated factor VIII 
(f1-f5) correspond to those described previously (31). 
Primers. The primers used for cDNA synthesis and polymerase chain reaction 
(PCR) are listed in Table 1 (SEQ ID NO: 22 through 43). They were 
synthesised by Oswel DNA Service, Department of Chemistry, University of 
Edinburgh. 
RNA Extraction and PCR. HCV virions in 0.2-1.0 ml volumes of plasma were 
pelleted from plasma by ultracentrifugation at 100,000 g for 2 hours at 
4.degree. C. RNA was extracted from the pellet as previously described 
(2,31). First strand cDNA was synthesized from 3 ul of RNA sample at 
42.degree. C. for 30 min. with 7 units of avian myeloblastosis virus 
reverse transcriptase (Promega) in 20 ul buffer containing 50 mM Tris-HCl 
(pH 8.0), 5 mM MgCl.sub.2, 5 mM dithiothreitol, 50 mM KCl, 0.05 ug/ul BSA, 
15% DMSO, 600 uM each of DATP, dCTP, dGTP and TTP, 1.5 uM primer and 10 U 
RNAsin (Promega). 
PCR was performed from 1 ul of the CDNA over 25 cycles with each consisting 
of 25 sec. at 94.degree. C., 35 sec. at 50.degree. C. and 2.5 min. at 
68.degree. C. The extension time for the last cycle was increased to 9.5 
min. The reactions were carried out with 0.4 unit Taq polymerase 
(Northumbria Biologicals Ltd.) in 20 ul buffer containing 10 mM Tris-HCl, 
pH 8.8, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.1% Triton X-100, 33 uM each of 
dATP, dCTP, dGTP and dTTP and 0.5 uM of each of the outer nested primers. 
One ul of the reaction mixture was then transferred to a second tube 
containing the same medium but with the inner pair of nested primers, and 
a further 25 heat cycles were carried out with the same programme. The PCR 
products were electrophoresed in 3% low melting point agarose gel (IBI) 
and the fragments were detected by ethidium bromide staining and UV 
illumination. For sequence analysis, single molecules of cDNA were 
obtained at a suitable limiting dilution at which a Poisson distribution 
of positive and negative results was obtained (30). 
Direct Sequencing of PCR Products. The PCR products were purified by 
glass-milk extraction ("GeneCleanl"; Bio101, Inc.). one quarter of the 
purified products was used in sequencing reactions with T7 DNA polymerase 
(Sequenase; United States Biologicals) performed according to the 
manufacturer's instructions except that the reactions were carried out in 
10% DMOS and the template DNA was heat denatured before primer annealing. 
Phylogenetic Methods. The sequences were compiled by version 2.0 of the 
programs of Staden (32) and analysed by programs available in the 
University of Wisconsin Genetics Computer Group sequence analysis package, 
version 7.0 (6). Phylogenetic trees were inferred using two different 
programs available in the PHYLIP package of Felsenstein (version 3,4 June 
1991; ref.9). The program DNAML finds the tree of the highest likelihood 
(the maximum likelihood tree) given a particular stochastic model of 
molecular evolution and has been shown to perform well in simulation 
studies (28). In the analyses performed here the global (G) option was 
used as this searches a greater proportion of all possible trees. The 
second program used was NEIGHBOR which clusters (following the algorithm 
of Saitou & Nei: ref.29) a matrix of nucleotide distances previously 
estimated using the program DNADIST (which itself was set, using the D 
option, to use the same stochastic model as underlies DNAML in order to 
estimate distances corrected for the probabilities of multiple 
substitution). In all cases the maximum likelihood and neighbour joining 
procedures produced congruent trees and thus only the former have been 
presented here. 
To establish the interrelationships of the major types of HCV, we have 
separately analysed several regions of the viral genome that differ in 
sequence variability and evolutionary constraint. Thus the conclusions 
drawn from the sequence comparisons are not subject to spurious 
evolutionary phenomena that may affect a particular region. However, one 
problem with the analysis presented here was the absence of a viral 
sequence that was sufficiently distantly related to HCV to serve as an 
out-group. Thus, although we describe the interrelationships of different 
sequence variants of HCV, it should be stressed that we have no means of 
deciding which sequence is ancestral to the others. The trees are thus 
drawn in the less familiar un-rooted form to indicate this. 
RESULTS 
1) Analysis of the 5' non-coding region. Samples were obtained from 18 
blood donors that were repeatedly reactive in the Abbott 2nd Generation 
enzyme immunoassay and which were confirmed or indeterminate in the Chiron 
4-RIBA (E-b1 through E-b18, ref.10). HCV sequences present in stored 
plasma samples from each donor were amplified with primers corresponding 
to sites in the 5'NCR (SEQ ID NO: 22 through 25) (12,25) that are well 
conserved between all known HCV type 1 and type 2 variants 
(4,11,13,16,23,24,26,33). Sequencing of the PCR product, after limiting 
dilution to isolate single molecules of CDNA before amplification, allowed 
approximately 190 bps in the centre of the region to be compared with 
equivalent published sequences (FIG. 1). 
Within the sequences, constant as well as variable regions can be found. 
Six sequences from donors E-b13 through E-b18 closely resembled those 
previously described as type 1 (4,11,13,16,23,24,26,33) and others 
resembled type 2(23) sequences (E-b9 through E -b12). However, eight 
sequences (E-b1 through E-b8) were distinct from both types, and have been 
provisionally termed type 3(SEQ ID NO: 12). Division of the sequences into 
three types is supported by formal phylogenetic analysis using the maximum 
likelihood (FIG. 2) and neighbour joining algorithms (data not shown) of 
the blood donor sequences along with previously published sequences 
(identified in table 2). Sequence variability within the three groups is 
in each case considerably less than that which separates the types. No 
sequence intermediate between the three types were found. This tree shows 
that the provisionally identified type 3 group (SEQ ID NO: 12) is equally 
distinct from type 1 as is type 2. Using the DNAML model, the corrected 
distances between sequences within each type were in each case less than 
3%. Between groups, they ranged from 9% (between type 1 and type 3 (SEQ ID 
NO: 12), and between type 1 and 2), to 14% between type 2 and type 3 (SEQ 
ID NO: 13) (table 3). 
2) Analysis of the NS-5 Region. The nucleotide sequence of the NS-5 region 
has been found to vary significantly between the previously described K1 
and K2 variants of HCV (7). To investigate whether type 3 (SEQ ID NO: 13) 
sequences were equally distant from the other two types in this region as 
well as in the 5'NCR, we compared sequences from four type 3 blood donors 
(E-b1, E-b2, E-b3 and E-b7) and one type 2 donor (E-b12) with previously 
published sequences (FIG. 3; FIG. 4; table 3). 
A remarkable variation was observed between sequences of the three types in 
this region. Again, type 3 sequences (SEQ ID NO: 13) form a separate group 
from type 1 and type 2 in this region. However, unlike the 5'NCR, there 
appear to be subdivisions within the type 1 and type 2 groups. Type 1 
sequences are split between those found in Japanese infected individuals 
(e.g. HCV-J; HCV-BK; sequence numbers 12, 13, 16-20 in table 2) and those 
of U.S.A. origin (HCV-1, Pt-1, H77, H90; sequence numbers 1-4; FIG. 4). 
There is also some evidence for a split between type 2 sequences, those 
corresponding to their previous designation as K2a (7) appearing distinct 
prom type K2b sequences and the Scottish blood donor, E-b12. 
Table 3 shows that the average nucleotide distances between the two groups 
of HCV type 1 sequences is 25% (indicated here as type 1a USA! and type 
1b Japanese!), with variation of only 4-7% within each group. The 
nucleotide sequence divergence within the two type 1 groups is similar to 
that which exists between K2a and K2b (table 3). However, both of these 
distances are considerably less than those which exist between type 1 and 
type 2 sequences (52-62%), and type 3 (SEQ ID NO: 13) (48-49%), and the 
distance between type 2 and type 3 (SEQ ID NO: 13) sequences (53-60%). 
3) Analysis of the NS-3 region. Amplification reactions were carried out 
using previously published primer sequences in the NS-3 region (37), and a 
pair of empirically derived inner primers (SEQ ID NO: 28 and 29) (31). 
Although these primers amplified HCV sequences from a high proportion of 
anti-C-100 positive sera from haemophiliacs (31), they were less effective 
with sera from IVDUs (31), and with blood donor samples (3 positive out of 
15 tested; data not shown). Two conserved sites in the amplified fragment 
were identified by sequence analysis of the NS-3 region from the 
haemophiliac and IVDU patients, and two new primers corresponding to these 
were specified (207 (SEQ ID NO: 31), 208 (SEQ ID NO: 30); Table 1). The 
combination of 288 (SEQ ID NO: 28)-208 (SEQ ID NO: 30) (first round) and 
290 (SEQ ID NO: 29)-207 (SEQ ID NO: 31) (second round) primers 
successfully amplified samples from four donors infected with HCV type 3 
(E-b1, E-b2, E-b6 and E-b7) but none of those infected with HCV type 2 
(data not shown). This enabled a comparison of the new type (SEQ ID NO: 
14) with our own (31) and previously published type 1 sequences (FIGS. 5, 
6; table 3). For clarity, only seven of the type 1 sequences obtained in 
this study (E-b16, E-b17, i3, i3, h5, h3 and h1) are shown in the tree. 
These sequences are representative of the range of variation found in this 
region in individuals infected in Britain; comparison of the tree 
previously published (31) with FIG. 6 shows that the former forms a very 
small component of the overall tree obtained once Japanese type 1 and type 
3 sequences are added. 
The maximum likelihood tree shows that type 1 and type 3 (SEQ ID NO: 14) 
have diverged considerably from each other. As was found in the NS-5 
region, subtypes of type 1 sequences are found in NS-3. Again, sequences 
of Japanese origin (HCV-J, HCV-BK and JH) are distinct from the prototype 
(PT) sequence, and those found in Scottish blood donors (E-b16, E-b17, 
p1-3), IVDUs (i1-5) and haemophiliacs (h1-5), all of which correspond to 
the prototype sequence (FIG. 5). However, the average subtype difference 
(23%) is lower than those that exist between HCV-1 and HCV-J with the four 
type 3 sequences (SEQ ID NO: 14) (37-43%). As reported previously (31), 
the majority of nucleotide substitutions that exist between type 1 
sequences are silent (i.e. do not affect the encoded amino acid sequence), 
while numerous amino aced substitutions exist between type 1 and type 3 
(SEQ ID NO: 14) sequences (FIG. 5). The analysis of the NS-3 region 
includes the sequence of clone A (35) which was obtained from Japanese 
patients with NANB hepatitis, and which was reported to be distinct from 
existing HCV type 1 sequences. In FIG. 6, this sequence appears to be 
distinct from both HCV type 1 and type 3 (SEQ ID NO: 14), with corrected 
sequence distances of 33-43% and 36% respectively. Although it is not 
possible to assign this sequence to any known group at this stage, these 
distances are not inconsistent with the hypothesis that it represents a 
type 2 sequence, or an equally distinct novel HCV type. 
4) Partial Sequence of the Putative Core Region of HCV. 
The region encoding the putative core protein is comparatively well 
conserved in its nucleotide sequence between known type 1 variants, 
showing nucleotide and amino acid sequence similarities of 90-98% and 
98-99% respectively (11, 24). Part of the core region from the blood donor 
Eb1, who has type 3 sequences in other regions analysed was amplified with 
primers 410 (SEQ ID NO: 26) and 406 (SEQ ID NO: 27) and compared with 
previously published type 1 sequences (FIGS. 7, 8; table 3). This analysis 
confirms that the type 3 sequence (SEQ ID NO: 15) was distinct from those 
of type 1, and again there was a prominent subdivision of type 1 sequences 
into Japanese (HCV-J, HCV-BK, HC-J4, JH and J7) and USA/European (HCV-1, 
H77, H90, GM1, GM2) sequences. As was found in NS-3, very little amino 
acid sequence variation is found in the core regions of type 1 sequences; 
almost all of the nucleotide differences between the two groups are at 
"silent" sites. By contrast, the type 3 sequence (SEQ ID NO: 15) shows 7-8 
amino acid substitutions on comparison with type 1 sequences. 
TABLE 1 
__________________________________________________________________________ 
SEQUENCES AND SOURCES OF PRIMERS USED FOR AMPLIFICATION OF HCV GENOME. 
Position 
Name Region 
of 5' base.sup.a 
Sense.sup.b 
Sequences 5'-3' Ref. 
__________________________________________________________________________ 
209 5'NCR 
8 - ATACTCGAGGTGCACGGTCTACGAGACCT SEQ ID 
(12)2 
211 5'NCR 
-29 - CACTCTCGAGCACCCTATCAGGCAGT SEQ ID NO:23 
(12) 
939 5'NCR 
-297 + CTGTGAGGAACTACTGTCTT SEQ ID NO:24 
(25) 
940 5'NCR 
-279 + TTCACGCAGAAAGCGTCTAG SEQ ID NO:25 
(25) 
410 CORE 
410 - ATGTACCCCATGAGGTCGGC SEQ ID NO:26 
406 CORE 
-21 + AGGTCTCGTAGACCGTGCATCATGAGCAC SEQ ID NO:27 
288 NS-3 
4951 - CCGGCATGCATGTCATGATGTAT SEQ ID NO:28 
(31) 
290 NS-3 
4932 - GTATTTGGTGACTGGGTGCGTC SEQ ID NO:29 
(31) 
208 NS-3 
4662 + TCTTGAATTTTGGGAGGGCGTCTT SEQ ID NO:30 
207 NS-3 
4699 + CATATAGATGCCCACTTCCTATC SEQ ID NO:31 
007 NS-4 
5293 - AACTCGAGTATCCCACTGATGAAGTTCCACAT SEQ ID NO:32 
220 NS-4 
5278 - CACATGTGCTTCGCCCAGAA SEQ ID NO:33 
HCV type 3:.paragraph. 
221 NS-4 
4858 + GGACCTACGCCCCTTCTATA SEQ ID NO:34 
008 NS-4 
4878 + TCGGTTGGGGCCTGTCCAAAATG SEQ ID NO:35 
HCV type 2: 
281 NS-4 
4858 + GGTCCCACCCCTCTCCTGTA SEQ ID NO:36 
509 NS-4 
4878 + CCGCTTGGGTTCCGTTACCAACG SEQ ID NO:37 
HCV type 1: 
253 NS-4 
4858 + GGGCCAACACCCCTGCTATA SEQ ID NO:38 
196 NS-4 
4878 + CAGACTGGGCGCCGTTCAGAATG SEQ ID NO:39 
242 NS-5 
8304 - GGCGGAATTCCTGGTCATAGCCTCCGTGAA SEQ ID 
(7)40 
555 NS-5 
8227 - CCACGACTAGATCATCTCCG SEQ ID NO:41 
243 NS-5 
7904 + TGGGGATCCCGTATGATACCCGCTGCTTTGA SEQ ID 
(7)42 
554 NS-5 
7935 + CTCAACCGTCACTGAACAGGACAT SEQ ID NO:43 
__________________________________________________________________________ 
.sup.a Position of 5' base relative to HCV genomic sequence in ref. no. 
(4) 
.sup.b Orientation of primer sequence (+: sense: -: antisense) 
.dagger-dbl.Abbreviations: A: adenine, c: cytidine: G: guanidine, T: 
thymidine. 
.paragraph.Separate sense primers required to enable amplification of eac 
HCV type. 
TABLE 2 
__________________________________________________________________________ 
SOURCE AND CITATION OF PREVIOUSLY 
PUBLISHED HCV SEQUENCES USED IN THIS STUDY 
Geographical 
No. Type 
Abbreviation 
Source 
Reference Ref. No. 
__________________________________________________________________________ 
1 1 HCV-1 U.S.A. 
Choo et al., 1991 
(4) 
2 1 Pt-1 Japan Nakao et al., 1991 
(23) 
Enomoto et al., 1990 
(7) 
3,4 1 H77. H90 
U.S.A. 
Ogata et al., 1991 
(24) 
5,6 1 GM-1, GM-2 
Germany 
Fuchs et al., 1991 
(11) 
7 1 J1 Japan Han et al., 1991 
(13) 
8 1 A1 Australia 
Han et al., 1991 
(13) 
9 1 S1 S. Africa 
Han et al., 1991 
(13) 
10 1 T1 Taiwan 
Han et al., 1991 
(13) 
11 1 U18/I24 
U.S.A./Italy 
Han et al., 1991 
(13) 
12 1 HCV-J Japan Kato et al., 1990 
(16) 
13 1 HCV-BK Japan Takamizawa et al., 1991 
(33) 
14, 15 
1 HC-J1,-J4 
Japan Okamoto et al., 1990 
(26) 
16-20 
1 K1, Kl- 1-4 
Japan Enomoto et al., 1990 
(7) 
21 1 JH Japan Kubo et al., 1990 
(17) 
22 1 J7 Japan Takeuchi et al., 1990 
(34) 
23-26 
2 K2a, K2a-1. 
Japan Nakao et al., 1991 
(23) 
K2b, K2b-1 Enomoto et al., 1990 
(7) 
27 ? Clone A 
Japan Tsukiyama-Kohara, 1991 
(35) 
__________________________________________________________________________ 
TABLE 3 
______________________________________ 
NUCLEOTIDE DISTANCES BETWEEN THE THREE HCV TYPES 
IN FOUR REGIONS OF THE GENOME. 
REGION TYPES (n.sup.a) 
1a 1b 2a 2b 3 
______________________________________ 
5'NCR 1 (20) 0.0163 n/a.sup.b 
2 (6) 0.0869 n/a 0.0214 
3 (8) 0.0948 n/a 0.1331 
n/a 0.0123 
CORE 1a (6) 0.0358 
1b (5) 0.0855 0.0227 
3 (1) 0.1801 0.1511 
n/d.sup.c 
n/d 0.0000 
NS-3 1a (34) 0.0699 
1b (3) 0.2270 0.0535 
3 (4) 0.3689 0.4279 
n/d n/d 0.0460 
NS-5 1a (4) 0.0743 
1b (7) 0.2477 0.0372 
2a (2) 0.6092 0.6206 
0.0612 
2b (3) 0.5214 0.5732 
0.2252 
0.0655 
3 (4) 0.4754 0.4890 
0.5983 
0.5299 
0.0322 
______________________________________ 
.sup.a number of sequences analysed 
.sup.b n/a: not applicable 
.sup.c n/d: not done 
II). SEROLOGICAL REACTIVITY OF BLOOD DONORS INFECTED WITH THREE DIFFERENT 
TYPES OF HEPATITIS C VIRUS. 
HCV sequences were amplified in the 5'non-coding region (5'NCR), core, NS-3 
and NS-5 regions from blood donors, haemophiliacs and intravenous drug 
abusers. 
Blood donations that were repeatedly reactive on screening with Abbott 2nd 
generation enzyme immunoassay (EIA) and positive or indeterminate by Ortho 
recombinant immunoblot assay (RIBA) were amplified by primers in the 5'NCR 
(reference 10). The first fourteen PCR-positive blood donations (where PCR 
was used to amplify and thus detect HCV RNA present in the blood) were 
then typed by sequence analysis of the amplified region, and compared with 
their serological reactivity to a range of structural and non-structural 
peptides in two 1st generation EIAs (Ortho HCV ELISA; Abbott HCV EIA) and 
two RIBA assays (Ortho RIBA and Innogenertics LIA; Table 4). The five 
donations containing HCV type 1 sequences were positive in both EIAS, 
reacted with all antigens in the Ortho RIBA assay, and were broadly 
reactive in the LIA. However, all but two of the sera from donors with 
type 2 and 3 infections were completely negative an anti-C100 EIA 
screening and failed to react with 5-1-1, C100 (RIBA) and NS4 (LIA). 
Furthermore, some carriers of HCV type 3 variants reacted poorly with the 
C33 (NS-3) peptide in the Ortho RIBA, and yielded two "indeterminate" 
results (donor nos. 11 and 13). 
Thus, current tests using Ortho RIBA and (to a lesser extent) Innogenetics 
LIA tests are unable to reliably detect HCV-2 and HCV-3 genotypes. For 
reliable testing for all HCV types, antigens from 5-1-1, C100 and NS4 for 
each of the three types of HCV should preferably be included in the panel 
of antigens. 
TABLE 4 
__________________________________________________________________________ 
SEROLOGICAL REACTIVITY OF SERA FROM BLOOD DONORS 
INFECTED WITH THREE TYPES OF HEPATITIS C VIRUS 
anti- 
Donor 
HCV C100 
Ortho RIBA Innogenetics LIA 
Number 
genotype 
O* 
A.dagger. 
5-1-1 
C100 
C33 
C22 
NS4 
NS5 
C1.dagger-dbl. 
C2 
C3 
C4 
__________________________________________________________________________ 
E-b13 
1 + + 3.sctn. 
4 4 4 2.sctn. 
3 1 2 1 1 
E-b15 + + 4 4 4 4 2 3 3 2 2 1 
E-b16 + + 4 4 4 4 2 3 2 3 3 -- 
E-b17 + + 4 4 4 4 3 3 3 2 1 1 
E-b18 + + 4 4 4 4 3 -- 2 1 1 -- 
E-b9 
2 + + -- 1 3 4 -- -- 3 1 1 3 
E-b10 - - -- -- 4 4 -- 3 2 2 2 -- 
E-b11 - - -- -- 4 4 -- 3 4 2 2 3 
E-b12 - - -- -- 4 4 -- 1 3 1 2 2 
E-b1 
3 - - -- -- -- 4 -- 1 3 1 -- 
3 
E-b2 - - -- -- 4 4 -- 2 1 1 1 2 
E-b3 + + -- -- 2 4 2 2 1 2 2 1 
E-b5 - - -- -- 2 4 -- -- 3 1 2 3 
E-b7 - - -- -- -- 4 -- 2 3 1 1 4 
__________________________________________________________________________ 
*Ortho HCV ELISA (Recombinant C1003) 
.dagger.Abbott HCV ELA (Hepatitis C Recombinant DNA Antigen) 
.dagger-dbl.Core oligopeptides, 1-4 
.sctn.Bands scored (negative) to 4 (strong positive) according to 
manufacturers instructions. 
T III. MAPPING OF ANTIGENIC DETERMINANTS IN NS-4 
Introduction 
With an overall aim of improving serological screening assays, we have 
obtained sequence data from the antigenic region of region corresponding 
to c100-3 for types 2 and 3. This information was used to epitope map the 
region, to define additional immunoreactive peptides that could be used to 
improve serological anti-HCV assays. 
Methods: 
PCR and sequencing. Plasma samples from Scottish blood donors yielding 
repeatedly reactive donations on 2nd generation anti-HCV screening (Abbott 
or Ortho), and which were confirmed or indeterminate on confirmatory 
testing by RIBA (Chiron) were referred to the Department of Medical 
Microbiology from the Scottish National Blood Transfusion Service 
Microbiology Reference Laboratory. HCV RNA within the plasma samples was 
extracted and amplified with primers in the 5'NCR as described previously 
(Chan et al., 1992). HCV was typed by sequence analysis of the amplified 
DNA as described previously (Simmonds et al., 1990) and by RFLP analysis. 
Five samples from different donors infected with HCV type 3 (nos. 40, 38, 
36, 26 and 1787), four infected with type 2 (nos. 31, 59, 940 and 810) and 
five with type 1 infection (nos. 16, 42, 77, 1801 and 1825) were amplified 
with primers corresponding to sense and anti-sense sequences (SEQ ID NO: 
32 through 39) spanning the antigenic region of NS-4 (table 1). Nucleotide 
sequences obtained from the amplified DNA were compared and used to define 
consensus sequences for each HCV type. In-frame translation of the 
nucleotide sequences yielded an uninterrupted consensus amino acid 
sequence that was used to define a series of overlapping oligopeptides for 
epitope mapping. 
Epitope mapping and determination of antibody specificities 
Overlapping synthetic peptides were synthesised on polypropylene pins using 
kits commercially available from Cambridge Research Biochemicals Ltd. The 
principle of the addition reactions is described in refs (Geysen et al., 
1984; Geysen et al., 1985). Antibody reactions were carried out on pins 
disrupted by sonication (30 minutes) in 1% sodium dodecyl sulphate, 0.1% 
2-mercaptoethanol, 0.1M sodium dihydrogen orthophosphate. Pins were 
pre-coated in 1% ovalbumin, 1% bovine serum albumin, 0.1% Tween-20 in 
phosphate buffered saline (PBS) for one hour at room temperature. Serum or 
plasma was diluted 1.40 in PBS+0.1% Tween-20 (PBST) and incubated with the 
blocked pins at 4.degree. C. for 18 hours. After washing in 4 changes of 
PBST (10 minutes at room temperature, with agitation), bound antibody was 
detected by incubation in a 1/20000 dilution of affinity isolated 
anti-human IgG, peroxidase conjugate (Sigma) for one hour at room 
temperature. Following washing (4 changes in PBST), pins were incubated in 
a 0.05% solution of azino-di-3-ethyl-benzthiazodinsulphonate in 0.1M 
sodium phosphate/sodium citrate buffer (pH 4.0) containing 0.03% hydrogen 
peroxide for 20 minutes. Optical densities were read at 410 nm. 
RESULTS 
HCV RNA in plasma samples from five donors infected with HCV type 3 by 
sequence analysis of the 5'NCR, and by RFLP were amplified in the NS-4 
region using primers (SEQ ID NO: 32 through 39) listed in Table 1. Because 
of the high degree of sequence variability in this region, it was 
necessary to use separate sense primers (SEQ ID NO: 34 through 39) for the 
amplification of different HCV types. However, the anti-sense primers (SEQ 
ID NO: 32 and 33) were in a highly conserved region and could be used for 
amplification of all three types. Sequence analysis was carried cut as 
previously described. This gave a continuous sequence from position 4911 
to 5271 (numbered as in Choo et al., 1991) (HCV-3--SEQ ID NO: 16) (FIG. 
9a). Little sequence variability (highlighted) was observed between the 
four different donors in this region. 
The nucleotide sequences were used to deduce the sequence of the encoded 
peptide (FIG. 9b). The putative protein contains mainly hydrophillic 
residues but no potential sites for N-linked glycosylation. Amino acid 
sequence variability with HCV type 3 was confined to only five residues 
(SEQ ID NO: 17) (FIG. 9b). However, this region differed considerably from 
the amino acid sequences of other blood donors infected with HCV types 1 
and 2 (T16, 42, 77, 1801, 1825, 351, 940 and 810; FIG. 10a). Sequence 
comparison between the major HCV types from residues 1679 to 1769 reveals 
three regions of considerable amino acid sequence variability. Most of the 
observed differences between types involve non-synonymous amino acid 
substitutions, particularly alternation of acidic and basic residues in 
the hydrophillic regions. These changes would be expected to profoundly 
alter the overall conformation of the protein, and its antigenicity. 
The consensus amino acid sequences in this region of types 1, 2 and 3 (SEQ 
ID NO: 17) (FIG. 10b) were used to define three series of 82 nonameric 
oligopeptides (spanning residues 42 to 128 of (SEQ ID NO: 17) overlapping 
by eight of the nine residues with those before and after in the series 
(FIG. 11a-c). These were synthesised on a 12.times.8 arrays of 
polypropylene pins as described in Methods. Antibody reactivity to the 
immobilised antigens on the pins was determined by indirect ELISA, using 
an overnight incubation with a 1/40 dilution of test serum overnight at 
4.degree. C., followed by washing, and detection with an anti-human 
IgG-peroxidase conjugate and appropriate substrate (see Methods). 
Reactivity of an anti-HCV negative, PCR-negative donor, with no known risk 
factors for HCV infection with the three series of peptides was 
determined. No significant reactivity is shown with any of the HCV-encoded 
oligopeptides. Reactivity of sera from three donors infected with HCV type 
3 (derived from residues 42 to 128 of (SEQ ID NO: 17) to each of the 
oligopeptides is shown in FIGS. 12a-12c. All three sera reacted with 
peptides ranging from No. 13 (sequence KVPDKE amino acids 54 to 62 in 
(SEQ ID NO: 17; FIG. 7) to No. 22 (sequence VLYQQYDEM; residues 63 to 71 
in (SEQ ID NO: 17) in the first antigenic region, although the precise 
peptides recognised varied slightly between individuals. All three sera 
reacted to varying extents with a second antigenic region, lying in the 
range from oligopeptides 32 to 42 (of sequence ECSQAAPYI, residues 73 to 
81 of (SEQ ID NO: 17, to QAQVIAHQF, residues 83 to 91 of (SEQ ID NO: 17. 
Weaker and more variable reactivity was observed to peptides 48 (residues 
88 to 96 of (SEQ ID NO: 17) to 53 (residues 94 to 102 of (SEQ ID NO: 17). 
Finally, significant reactivity was also observed to single 
oligonucleotides 2 (residues 43 to 51 of (SEQ ID NO: 17) (2 of 3 samples), 
61 (residues 102-110 of (SEQ ID NO: 17) (2 of 3), 66 (residues 107 to 115 
of (SEQ ID NO: 17) (3 of 3), 73 (residues 114 to 122 of (SEQ ID NO: 17) (3 
of 3) and 80 (residues 121 to 129 of (SEQ ID NO: 17) (2 of 3). 
The sequences of the major antigenic regions of HCV type 3 differ 
considerably from those encoded by any of the type 1 or type 2 variants. 
The region bounded by peptides 13 to 22 (SEQ ID NO: 1) shows average 
homologies of 50% with HCV type 2 (SEQ ID NO: 3 and 4) variants and 67% 
with type 1 (SEQ ID NO: 7 and 8). Between peptides 32 to 42 (SEQ ID NO: 
2), there are homologies of 39% with type 2 (SEQ ID NO: 5 and 6) and 58% 
with type 1 (SEQ ID NO: 10 and 11) variants. Thus, although similar 
regions of each NS-4 sequence are antigenic, the actual epitopes differ 
considerably between HCV types. 
Discussion 
The NS-4 region of HCV type 3 (SEQ ID NO: 16 and 17) shows considerable 
sequence divergence from other variants of HCV, that exceeds that found in 
the core, NS-3 or NS-5 regions previously analysed (Chan et al, 1992). The 
function of the protein encoded by this region of the HCV genome is 
unknown, and the consequences of this variability on virus replication and 
pathogenesis are unknown. The function of the NS-4 region in flaviviruses 
and pestiviruses is also poorly defined. 
The degree of amino acid sequence variability, and the nature of the amino 
acid substitutions indicate that the major sites of antibody reactivity 
are also those of antigenic variability. This undoubtedly underlies the 
restricted cross-reactivity of HCV type 1 NS-4 encoded antigens with sera 
from individuals infected with different HCV types. Serological diagnosis 
of infection is currently based entirely on recombinant or synthetic 
oligopeptide sequences derived ultimately from HCV type 1 sequences (Choo 
et al., 1991). The serological response to infection is often very 
restricted in its initial stages, with antibody to only one of the 
recombinant antigens used for screening. Not only does this present 
difficulties with supplementary antibody tests, where reactivity to two 
HCV-encoded antigens is required for confirmation, but can lead to an 
increased probability of failing to detect early infection with HCV types 
2 and 3. 
Table 7 relates HCV typing determined by PCR, using type-specific sense 
primers (SEQ ID NO: 44 through 49) and the nontype-specific anti-sense 
primers (SEQ ID NO: 22 and 23) (Table 6), to results obtained using 
type-specific antigens (TSA) and shows good correlation for HCV1-3 types. 
TABLE 5 
__________________________________________________________________________ 
SEQUENCES OF NS-4 ENCODED ANTIGENS FOR (A) IMPROVED 
SEROLOGICAL DIAGNOSIS, AND (B) FOR SEROLOGICAL 
DISCRIMINATION OF INFECTION WITH DIFFERENT HCV TYPES 
__________________________________________________________________________ 
A) 
Type 
Region 1 (1691-1708)* 
Region 2 (1710-1728) 
__________________________________________________________________________ 
3 KVPDKEVLYQQYDEM.dagger.SEQ ID NO:1 
ECSQAAPYIEQAQVIAHQF SEQ ID NO:2 
2.dagger-dbl. 
RVVVTPDKEILYEAFDEM SEQ ID NO:3 
ECASKAALIEEGQRMAEML SEQ ID NO:5 
RAVIAPDKEVLYEAFDEM SEQ ID NO:4 
ECASRAALIEEGQRIAEML SEQ ID NO:6 
__________________________________________________________________________ 
B) 
Type 
Region 1 (1691-1708) 
Region 2 (1710-1728) 
__________________________________________________________________________ 
3 KVPDKEVLYQQYDEM SEQ ID NO:1 
ECSQAAPYIEQAQVIAHQF SEQ ID NO:2 
2 RVVVTPDKEILYEAFDEM SEQ ID NO:3 
ECASKAALIEEGQRMAEML SEQ ID NO:5 
RAVIAPDKEVLYEAFDEM SEQ ID NO:4 
ECASRAALIEEGQRIAEML SEQ ID NO:6 
1 KPAIIPDREVLYREFDEM SEQ ID NO:7 
ECSQHLPYIEGMLAEQF SEQ ID NO:10 
KPAVIPDREVLYREFDEM SEQ ID NO:8 
ECSQHLPYIEGALAEQF SEQ ID NO:11 
__________________________________________________________________________ 
*Amino acid positions numbered as in Choo et al., (1991). 
.dagger.Amino acid codes: A: alanine: R: arginine: N: asparagine: D: 
aspartic acid: C: cysteine: Q: glutamine: E: glutamic acid: G: glycine: H 
histidine: I: isoleucine: L: leucine: K: lysine: M: methionine: F: 
phenylalanine: P: proline: S: serine: T: threonine: W: tryptophan: Y: 
tyrosine: V: valine. 
.dagger-dbl.Alternative peptides, where there is variability within an HC 
type.. 
TABLE 6 
__________________________________________________________________________ 
SEQUENCES OF OLIGONUCLEOTIDES SUITABLE FOR DIRECT DETECTION OF 
HCV TYPE 3 IN CLINICAL SPECIMENS BY POLYMERASE CHAIN REACTION 
Position 
Name Region 
of 5' base* 
Pol..dagger. 
Sequences 5'-3'.dagger-dbl. 
__________________________________________________________________________ 
007 NS-4 
5293 - AACTCGAGTATCCCACTGATGAAGTTCCACAT SEQ ID NO:32 
220 NS-4 
5278 - CACATGTGCTTCGCCCAGAA SEQ ID NO:33 
Type 3:.paragraph. 
TS-3a 
NS-4 
5140 + GCCGCCCCATATATCGAACA SEQ ID NO:44 
TS-3b 
NS-4 
5161 + GCTCAGGTAATAGCCCACCA SEQ ID NO:45 
Type 2: 
TS-2a 
NS-4 
5140 + AAAGCCGCCCTCATTGAGGA SEQ ID NO:46 
TS-2b 
NS-4 
5161 + GGGCAGCGGATGGCGGAGAT SEQ ID NO:47 
Type 1: 
TS-1a 
NS-4 
5140 + CACTTACCGTACATCGAGCA SEQ ID NO:48 
TS-1b 
NS-4 
5161 + GGGATGATGCTCGCCGAGCA SEQ ID NO:49 
__________________________________________________________________________ 
*Position of 5' base relative to HCV genomic sequence in Choo et al., 
(1991). 
.dagger.Orientation of primer sequence (+: sense: -: antisense) 
.dagger-dbl.Abbreviations: A: adenine, C: cytidine: G: guanidine, T: 
thymidine. 
.paragraph.Typespecific sense primers for amplification of HCV types 3, 2 
and 1 variants. 
TABLE 7 
______________________________________ 
COMISON OF SEROLOGICAL 
TYPING BY HCV-TSA WITH PCR 
Number TYPE-SPECIFIC ANTIBODY 
PCR.sup.a 
tested 1 2 3 1 + 2 
1 + 3 
2 + 3 
NTS.sup.b 
NR.sup.c 
______________________________________ 
1 57 63 -- -- -- 1 -- 3 3 
2 12 -- 11 -- -- -- 1 1 0 
3 47 1 -- 45 -- 2 -- 4 4 
Haem.sup.d 
27 11 -- 4 1 4 -- 3 4 
______________________________________ 
.sup.a Genotype of HCV sequences amplified by PCR and typed by RFLP 
(McOmish et al 1992) 
.sup.b NTS: No typespecific antibody detected 
.sup.c NR: nonreactive with NS4 peptides 
.sup.d Samples from HCVinfected haemophiliacs. untyped by PCR. 
T IV IDENTIFICATION OF HCV TYPE-4 
Introduction 
Investigations were carried out on sequence variations in the 5' non-coding 
region (5'NCR) of HCV samples from a variety of worldwide geographical 
locations (FIG. 13), and also in the core region (FIGS. 15A and 15B). 
Phylogenetic analysis (FIGS. 14 and 16) revealed a new distinct HCV type 
which we refer to herein as HCV-4. 
Methods 
Samples. RNA was extracted from plasma samples that were repeatedly 
reactive on second generation screening assays for HCV, and which were 
either confirmed (significant reactivity with two or more antigens in the 
Chiron recombinant immunoblot assay; Chiron Corporation, Emeryville, 
Calif., USA) or indeterminate (reactive with only one antigen) from blood 
donors and patients with NANBH. Most of the samples containing sequences 
that differed substantially fcm known HCV types came from Egypt (EG 1-33). 
Others came from Holland (NL-26), Hong Kong (HK 1-4), Iraq (IQ-48) and XX 
(xx-(6). 
Sequence determination. HCV sequences were reverse transcribed and 
amplified with primers matching conserved regions in the 5'NCR as 
previously described 1!. For analysis of the core region, RNA was reverse 
transcribed using a primer of sequence CA(T/C)GT(A/G)AGGGTATCGATGAC (SEQ 
ID NO: 50) (5' base: xxx, numbered as in 20!). CDNA was amplified using 
this primer and a primer in the 5'NCR of sequence ACTGCCTGATAGGGTGCTTGCGAG 
(SEQ ID NO: 51) (5' base: -54). The second PCR used primers of sequences 
AGGTCTCGTAGACCGTGCATCATG (SEQ ID NO: 52) (5' base: -21) and 
TTGCG(G/T/C)GACCT(A/T)CGCCGGGGGTC (SEQ ID NO: 53) (5' base: xxx). 
Amplified DNA in both regions was directly sequenced as described 
previously (reference 1a). 
Sequence analysis. Sequences were aligned using the CLUSTAL program in the 
University of Wisconsin GCG package (reference 6). Phylogenetic trees were 
constructed by the DNAML program in the PHYLIP package of Felsenstein 
(version 3.4, June 1991; (reference 9), using the global option. RNA 
secondary structures in the 5'NCR of 4 representative HCV variants (refs) 
were predicted using the program FOLD. Three predictions were made from 
each sequence between nucleotides -341 to -1, -341 to +300, and -341 to 
+900 to allow for possible long range interactions. Comparison of the 
predicted conformations for each sequence over the different lengths 
showed that only relatively small scale features, such as the stem/loop 
analysed in the Results were at all conserved (data not shown). 
All sequences reported in this part have been submitted to GenBank. 
Results 
Divergent 5'NCR sequences (SEQ ID NO: 58). Several sequences in the 5'NC 
region detected in samples of blood donors from Saudi Arabia, Holland and 
Hong Kong, and from NANBH patients in Iraq and xxx differed substantially 
from those found in Scottish blood donors-and those reported elsewhere 
(FIG. 13). Instead of showing the well characterised nucleotide 
substitutions that distinguish HCV types 1, 2 and 3 from each other, a new 
set of sequence differences were observed in the new variants that 
appeared to place them outside the existing system of virus 
classification. This can be more simply represented by reconstructing a 
phylogeny of the sequences and presenting the results as an evolutionary 
tree (FIG. 14). This analysis confirms that sequences 1-10 cluster 
separately from the variants previously typed as 1, 2 and 3 (SEQ ID NO: 
12). For convenience we will refer to sequences within this new group as 
HCV type 4 (SEQ ID NO: 18). Mean distances within type 4 and between type 
4 and the other HCV types in the 5'NCR were comparable to those previously 
described for type 1-3. Although sequences within type 4 (SEQ ID NO: 18) 
are relatively closely grouped, sequences 11, 12 and 13 differ 
considerably from any of the known types. 
Using this phylogenetic tree, it can be seen that the majority of 
previously Published 5'NCR sequences can be readily identified as types 1, 
2 or 3 (SEQ ID NO: 12). Furthermore, almost all of the sequences from 
Zaire (shown as hollow squares) cluster closely within type 4, suggesting 
a wider distribution in Africa. However, a further complication is that 
three identical sequences obtained from South African patients appeared 
distinct from both the type 1 and the type 4 group, and may represent yet 
another HCV type. 
RNA from three representative type 4 variants (Eg 29, 33, 21; corresponding 
to 5'NCR sequences nos. 1-3) was amplified using primers in the core 
region of HCV polyprotein. All three sequences differed considerably at 
both the nucleotide (SEQ ID NO: 20) and amino acid (SEQ ID NO: 21) level 
from HCV types 1, 2 and 3 (SEQ ID NO: 19 and residues 1 to 89 of SEQ ID 
NO: 15, respectively) (FIG. 15A/B). Phylogenetic analysis of these 
sequences and those previously analysed indicated that they formed a 
separate, relatively homogeneous group distinct from the other types (FIG. 
16). Reconstructed nucleotide distances between type 4 (SEQ ID NO: 20) and 
types 1, 2 and 3 (SEQ ID NO: 19) were comparable to those that exist 
between the three known HCV types of HCV. Although most of the nucleotide 
sequence differences were silent, there were between 4 and 9 amino acid 
differences between the new variants (SEQ ID NO: 21) and other types. 
T V HCV TYPING 
Introduction 
In view of the sequence variations between HCV types 1, 2, 3 and 4 
differences in restriction enzyme cleavage sites exist, leading to 
different endonuclease cleavage patterns. This technique was used to 
identify HCV genotypes in blood samples from a variety of sources 
worldwide. 
(A) Typing of HCV1-3 
METHODS 
Serum Samples: Samples from blood donors in six countries, Scotland, 
Finland, Netherlands, Hong Kong and Australia and Japan, were available 
from routine 2nd Generation anti-HCV ELISA screening (Ortho or Abbott). 
Donor samples that were repeatedly reactive in the above tests were 
further investigated using a supplementary test (Ortho RIBA: Finland, 
Netherlands, Australia, Egypt, Abbott Matrix: Hong Kong) or samples were 
titred for anti-HCV by ELISA (Japan). Samples that were positive 
(significant reactivity with two or more HCV antigens (1+ to 4+) or 
indeterminate (reactivity with one antigen only) in the RIBA test or had a 
titer of &gt;X 4096 by ELISA (Japan only) were tested for viral RNA by 
Polymerase Chain Reaction (PCR). 
RNA PCR: PCR for the detection of HCV RNA was carried out as previously 
described by Chan et al (reference 1a) using primers in the 5'non-coding 
region (5'NCR) in a nested PCR, with primers 209 (SEQ ID NO: 22)/939 (SEQ 
ID NO: 24) and 211 (SEQ ID NO: 24)/940 (SEQ ID NO: 23) (SEQ ID NO: 23) (5) 
(SEQ ID NO: 25) in first and second reactions respectively. 
HCV TYPING 
The existence of relatively conserved patterns of substitutions in the 
5'NCR that are characteristic of different HCV types provide useful 
signature sequences for identification of HCV genotypes. Having compared 
large numbers of different HCV type 1, 2 and 3 sequences, we developed a 
method that differentiated HCV types 1-3 by restriction endonuclease 
cleavage of amplified DNA. However, the 19 type 4 sequences would appear 
as type 1 (electrophoretic types Aa and Ab), and for concurrent studies it 
has been necessary to modify the conditions to identify the new HCV type. 
All type 4 sequences showed a T.fwdarw.C change at position -167 (position 
78 in SEQ ID NO: 18) that creates a novel Hinfl site that is absent in all 
type 1 (and type 2) sequences. In combination with ScrFI, and HaeIII/RsaI, 
it has now proved possible to identify the new type reliably in numerous 
countries in the Middle East and elsewhere. 
RESULTS 
The results are summarised in Table 8 for HCV types 1, 2 and 3. The 
Egyptian samples gave aberrant restriction patterns on the single ScrFI 
digest and were identified as type 4. 
TABLE 8 
______________________________________ 
PREVALENCE OF HCV TYPES IN DIFFERENT COUNTRIES 
HCV TYPES (%) 
COUNTRY HCV-1 HCV-2 HCV-3 
______________________________________ 
Scotland 86 (51%) 21 (13%) 60 (36%) 
Finland 3 (25%) 5 (42%) 4 (33%) 
Netherlands 18 (60%) 7 (23%) 5 (17%) 
Hong Kong 22 (63%) 0 (0%) 0 (0%) 
Australia 13 (57%) 3 (13%) 7 (30%) 
Japan 31 (77%) 9 (23%) 0 (0%) 
Egypt 0 (0%) 0 (0%) 0 (0%) 
______________________________________ 
(B) MODIFICATION OF PCR-BASED TYPING ASSAY TO DETECT INFECTION WITH HCV 
TYPE 4 IN CLINICAL SPECIMENS 
Methods 
Extraction of RNA. RNA was extracted from 100 ul aliquots of plasma of 
non-A, non-B patients by addition of 1 ml RNAzol solution (2M guanidinium 
thiocyanate, 12.5 mM sodium citrate pH7.0!, 0.25% w/v N-lauroylsarcosine, 
0.05M 2-mercaptoethanol, 100 mM sodium acetate pH 4.0!, 50% w/v water 
saturated phenol) as previously described (Chomczynski et al. 1987), and 
mixed until precipitate dissolved. After addition of 100 ul chloroform, 
each sample was spun for 5 minutes at 14000.times.g and the aqueous phase 
re-extracted with 0.5 ml chloroform. RNA was precipitated by addition of 
an equal volume of isopropanol and incubation at -20.degree. C. for at 
least 1 hour. An RNA pellet was produced by centrifugation at 
14000.times.g for 15 minutes at 4.degree. C., washed in 1 ml 70% cold 
ethanol solution, dried and resuspended in 20 ul diethylpyrocarbonate 
treated distilled water. Of the 100 directly extracted samples, a total of 
19 were PCR-negative (see below). Two ml volumes of the negative samples 
were ultracentrifuged at 200 000.times.g for 2 hours and the pellet 
re-extracted as described above. Extraction from the larger volume of 
plasma yielded an additional 3 positive samples (numbers 66, 80, 85). 
PCR and typing. RNA was reverse transcribed with primer 940 (SEQ ID NO: 25) 
and cDNA amplified in a two stage nested PCR reaction with primers 940 
(SEQ ID NO: 25)/939 (SEQ ID NO: 24), followed by 209 (SEQ ID NO: 22)/211 
(SEQ ID NO: 23) as previously described (Chan et al. 1992). PCR product 
was radiolabelled with .sup.35 S!-dATP analysed by restriction 
endonuclease cleavage (McOmish et al. Transfusion, 32:no.11 1992). Samples 
were cleaved with ScrFI and a combination of HaeIII/RsaI in two separate 
reactions to identify HCV types 1/4, 2, 3. FIG. 17 shows endonuclease 
cleavage patterns. HCV types 1 and 4 were differentiated by a third 
reaction with Hinfl (see Results). Two samples yielded restriction 
patterns that were different from those of the four known types of HCV and 
were analysed further by direct sequence analysis of the amplified DNA 
(Chan et al. 1992). These two samples contained 5'NCR sequences distinct 
from those of known HCV types and currently remain unclassified. 
RESULTS 
Modification of RFLP method to identify HCV type 4. 
Previous sequence analysis in the 5'NCR of HCV amplified from plasma of 
Egyptian blood donors revealed a relatively homogeneous group of novel 
sequence variants in both the 5'NCR (SEQ ID NO: 18) and core (SEQ ID NO: 
20 and 21) region which were as distinct from HCV types 1, 2 and 3 (SEQ ID 
NO: 19 and residues 1 to 89 of (SEQ ID NO: 15) as these latter types were 
from each other (see previous submission). This new group was designated 
as HCV type 4. 
Comparison of cleavage patterns of type 4 sequences with those of type RFLP 
analysis of the previously identified type 4 sequences produced a 
distribution of electropherotypes with ScrFI and HaeIII/RsaI similar to 
that HCV type 1 (Table 9). Type 1 sequences yielded 9 patterns of aA/B, 35 
of bA/B and 1 bC. With these enzymes alone, type 4 sequences were thus 
indistinguishable from type 1 (14 aA/B, 4 bA/B). However, type 1 and type 
4 sequences consistently differ in the number of HinfI sites. All 18 type 
4 sequences contain one or two potential cleavage sites (producing 
patterns band c; table 5) while none are found in any of the 45 type 1 
sequences analysed (pattern a). One of the type 4 sequences was further 
differentiated from type 1 and other HCV types by the loss of a 
restriction site for RsaI, leading to a new pattern of bands designated h 
(44, 172, 9, 26; first column, Table 9). Finally, a single sequence, EG-28 
lost two sites to produce bands of 216, 9, and 26 bps (pattern i; Table 
9). This sequence was distinct from that of any of the known HCV types 
(including type 4) and is shown in the table in the column labelled U 
(unclassified) 
Typing of study subjects. RNA was extracted from 100 samples of patients 
with NANB hepatitis and amplified with primers in the 5'NCR. Of these, 84 
were PCR positive, and enabled HCV typing to be carried out by RFLP. This 
was initially carried out with HaeIII/RsaI and ScrFl, and allowed the 
identification of 10 type 2 and 10 type 3 variants (Table 10). Samples 
showing electrophoretic patterns aA/B or bA/B were further analysed by 
cleavage with hinfl, yielding 38 samples with pattern a, thus identified 
as type 1, 22 with pattern b and 2 with pattern c, both identified as type 
4. Finally, two samples showed the unusual cleavage patterns h and i with 
HaeII/RsaI and pattern b with Hinfl, and were therefore directly 
sequenced. These two sequences were similar to each other but were unlike 
any of the known HCV types, and also distinct from EG-28, the other 
sequence showing pattern i with HaeIII/RsaI (Table 10). As they cannot be 
currently classified, they will be referred to as type U. 
TABLE 9 
______________________________________ 
PREDICTED CLEAVAGE PATTERNS OF PUBLISHED 
5'NCR SEQUENCES OF HCV TYPES 1, 2, 3 AND 4 WITH 
RSaI/HaeIII, ScrFI AND Hinfl 
Predicted cleavage pattern.sup.a 
HaeIII/ HCV type 
RsaI ScrFI Hinfl 1 2 3 4 U.sup.b 
______________________________________ 
a A/B a.sup.c 
9 -- -- -- -- 
b A/B a 35 -- -- -- -- 
b C a 1 -- -- -- -- 
a A/B b.sup.d 
-- -- -- 13 -- 
a A/B c.sup.e 
-- -- -- 1 -- 
b A/B b -- -- -- 4 -- 
c D a -- 5 -- -- -- 
d D a -- 1 -- -- -- 
d E a -- 2 -- -- -- 
e D a -- 1 -- -- -- 
e E a -- 1 -- -- -- 
f G b -- -- 14 -- -- 
f G c -- -- 1 -- -- 
g G b -- -- 8 -- -- 
h.sup.f A/B b -- -- -- 1 -- 
i.sup.g A/B b -- -- -- -- 1 
______________________________________ 
.sup.a Cleavage patterns designated for HaeIII/RsaI and ScrFI as describe 
previously (McOrnish et al. 1992). 
.sup.b Cleavage pattern of an HCV variant of undesigned type 
.sup.c Pattern a: uncleaved by HinfI 
.sup.d Pattern b: DNA cleaved to generate two fragments of sizes 107 and 
142 bps (in order 53') 
.sup.e Pattern c: DNA cleaved to generate three fragments of 56, 51 and 
142 bps 
.sup.f New cleavage pattern for HaeIII/Rsai designated h (bands of 44 bps 
172 bps, 9 bps, 26 bps) 
.sup.g New cleavage pattern for HaeIII/Rsai designated i (216 bps, 9 bps, 
26 bps) 
TABLE 10 
______________________________________ 
IDENTIFICATION OF HCV TYPES 1-4 IN 
STUDY SUBJECTS BY RFLP ANALYSIS OF 5'NCR 
SEQUENCES WITH RsaI/HaeIII, ScrFI AND Hinfl 
Observed cleavage pattern 
HaeIII/ Inferred HCV type 
RsaI ScrFI Hinfl 1 2 3 4 U.sup.a 
______________________________________ 
a A/B a 2 -- -- -- -- 
b A/B a 36 -- -- -- -- 
a A/B b -- -- -- 16 -- 
a A/B c -- -- -- 2 -- 
b A/B b -- -- -- 6 -- 
c D n.d. -- 7 -- -- -- 
d E n.d. -- 3 -- -- 
f G n.d. -- -- 7 -- -- 
g G n.d. -- -- 3 -- -- 
h A/B b -- -- -- -- 1 
i A/B b -- -- -- -- 1 
TOTALS 38 10 10 24 2 
______________________________________ 
.sup.a Two samples yielded unusual restriction patterns with HaeIII/RsaI 
(h.i). Sequence analysis of the 5'NCR placed them outside existing HCV 
classification (samples IQ48, EG96). 
T VI Expression and Assay etc. Techniques 
The present invention also provides expression vectors containing the DNA 
sequences as herein defined, which vectors being capable, in an 
appropriate host, of expressing the DNA sequence to produce the peptides 
as defined herein. 
The expression vector normally contains control elements of DNA that effect 
expression of the DNA sequence in an appropriate host. These elements may 
vary according to the host but usually include a promoter, ribosome 
binding site, translational start and stop sites, and a transcriptional 
termination site. Examples of such vectors include plasmids and viruses. 
Expression vectors of the present invention encompass both 
extrachromosomal vectors and vectors that are integrated into the host 
cell's chromosome. For use in E. coli, the expression vector may contain 
the DNA sequence of the present invention optionally as a fusion linked to 
either the 5'- or or 3'-end of the DNA sequence encoding, for example, 
B-galactosidase or to the 3'-end of the DNA sequence encoding, for 
example, the trp E gene. For use in the insect baculovirus (AcNPV) system, 
the DNA sequence is optionally fused to the polyhedrin coding sequence. 
The present invention also provides a host cell transformed with expression 
vectors as herein defined. 
Examples of host cells of use with the present invention include 
prokaryotic and eukaryotic cells, such as bacterial, yeast, mammalian and 
insect cells. Particular examples of such cells are E. coli, S. 
cerevisiae, P. pastoris. Chinese hamster ovary and mouse cells, and 
Spodoptera frugiperda and Tricoplusia ni. The choice of host cell may 
depend on a number of factors but, if post-translational modification of 
the HCV viral peptide is important, then an eukaryotic host would be 
preferred. 
The present invention also provides a process for preparing a peptide as 
defined herein which comprises isolating the DNA sequence, as herein 
defined, from the HCV genome, or synthesising DNA sequence encoding the 
peptides as defined herein, or generating a DNA sequence encoding the 
peptide, inserting the DNA sequence into an expression vector such that it 
is capable, in an appropriate host, of being expressed, transforming host 
cells with the expression vector, culturing the transformed host cells, 
and isolating the peptide. 
The DNA sequence encoding the peptide may be synthesised using standard 
procedures (Gait, Oligonucleotide Synthesis: A Practical Approach, 1984, 
Oxford, IRL Press). 
The desired DNA sequence obtained as described above may be inserted into 
an expression vector using known and standard techniques. The expression 
vector is normally cut using restriction enzymes and the DNA sequence 
inserted using blunt-end or staggered-end ligation. The cut is usually 
made at a restriction site in a convenient position in the expression 
vector such that, once inserted, the DNA sequences are under the control 
of the functional elements of DNA that effect its expression. 
Transformation of an host cell may be carried out using standard 
techniques. Some phenotypic marker is usually employed to distinguish 
between the transformants that have successfully taken up the expression 
vector and those that have not. Culturing of the transformed host cell and 
isolation of the peptide as required may also be carried out using 
standard techniques. 
The peptides of the present invention may be prepared by synthetic methods 
or by recombinant DNA technology. The peptides are preferably synthesized 
using automatic synthesizers. 
Antibody specific to a peptide of the present invention can be raised using 
the peptide. The antibody may be polyclonal or monoclonal. The antibody 
may be used in quality control testing of batches of the peptides; 
purification of a peptide or viral lysate; epitope mapping; when labelled, 
as a conjugate in a competitive type assay, for antibody detection; and in 
antigen detection assays. 
Polyclonal antibody against a peptide of the present invention may be 
obtained by injecting a peptide, optionally coupled to a carrier to 
promote an immune response, into a mammalian host, such as a mouse, rat, 
sheep or rabbit, and recovering the antibody thus produced. The peptide is 
generally administered in the form of an injectable formulation in which 
the peptide is admixed with a physiologically acceptable diluent. 
Adjuvants, such as Freund's complete adjuvant (FCA) or Freund's incomplete 
adjuvant (FIA), may be included in the formulation. The formulation is 
normally injected into the host over a suitable period of time, plasma 
samples being taken at appropriate intervals for assay for anti-HCV viral 
antibody. When an appropriate level of activity is obtained, the host is 
bled. Antibody is then extracted and purified from the blood plasma using 
standard procedures, for example, by protein A or ion-exchange 
chromatography. 
Monoclonal antibody against a peptide of the present invention may be 
obtained by fusing cells of an immortalising cell line with cells which 
produce antibody against the viral or topographically related peptide, and 
culturing the fused immortalised cell line. Typically, a non-human 
mammalian host, such as a mouse or rat, is inoculated with the peptide. 
After sufficient time has elapsed for the host to mount an antibody 
response, antibody producing cells, such as the splenocytes, are removed. 
Cells of an immortalising cell line, such as a mouse or rat myeloma cell 
line, are fused with the antibody producing cells and the resulting 
fusions screened to identify a cell line, such as a hybridoma, that 
secretes the desired monoclonal antibody. The fused cell line may be 
cultured and the monoclonal antibody purified from the culture media in a 
similar manner to the purification of polyclonal antibody. 
Diagnostic assays based upon the present invention may be used to determine 
the presence or absence of HCV infection. They may also be used to monitor 
treatment of such infection, for example in interferon therapy. 
In an assay for the diagnosis of viral infection, there are basically three 
distinct approaches that can be adopted involving the detection of viral 
nucleic acid, viral antigen or viral antibody. Viral nucleic acid is 
generally regarded as the best indicator of the presence of the virus 
itself and would identify materials likely to be infectious. However, the 
detection of nucleic acid is not usually as straightforward as the 
detection of antigens or antibodies since the level of target can be very 
low. Viral antigen is used as a marker for the presence of virus and as an 
indicator of infectivity. Depending upon the virus, the amount of antigen 
present in a sample can be very low and difficult to detect. Antibody 
detection is relatively straightforward because, in effect, the host 
immune system is amplifying the response to an infection by producing 
large amounts of circulating antibody. The nature of the antibody response 
can often be clinically useful, for example IgM rather than IgG class 
antibodies are indicative of a recent infection, or the response to a 
particular viral antigen may be associated with clearance of the virus. 
Thus the exact approach adopted for the diagnosis of a viral infection 
depends upon the particular circumstances and the information sought. In 
the case of HCV, a diagnostic assay may embody any one of these three 
approaches. 
In an assay for the diagnosis of HCV involving detection of viral nucleic 
acid, the method may comprise hybridising viral RNA present in a test 
sample, or cDNA synthesised from such viral RNA, with a DNA sequence 
corresponding to the nucleotide sequences of the present invention or 
encoding a peptide of the invention, and screening the resulting nucleic 
acid hybrids to identify any HCV viral nucleic acid. The application of 
this method is usually restricted to a test sample of an appropriate 
tissue, such as a liver biopsy, in which the viral RNA is likely to be 
present at a high level. The DNA sequence corresponding to a nucleotide 
sequence of the present invention or encoding a peptide of the invention 
may take the farm of an oligonucleotide or a CDNA sequence optionally 
contained within a plasmid. Screening of the nucleic acid hybrids is 
preferably carried out by using a labelled DNA sequence. Preferably the 
peptide of the present invention is part of an oligonucleotide wherein the 
label is situated at a sufficient distance from the peptide so that 
binding of the peptide to the viral nucleic acid is not interfered with by 
virtue of the label being too close to the binding site. One or more 
additional rounds of screening of one kind or another may be carried out 
to characterise further the hybrids and thus identify any HCV viral 
nucleic acid. The steps of hybridisation and screening are carried out in 
accordance with procedures known in the art. 
The present invention also provides a test kit for the detection of HCV 
viral nucleic acid, which comprises 
i) a labelled oligonucleotide comprising a DNA sequence of the present 
invention or encoding a peptide of the present invention; and 
ii) washing solutions, reaction buffers and a substrate, if the label is an 
enzyme. 
Advantageously, the test kit also contains a positive control sample to 
facilitate in the identification of viral nucleic acid. 
In an assay for the diagnosis of HCV involving detection of viral antigen 
or antibody, the method may comprise contacting a test sample with a 
peptide of the present invention or a polyclonal or monoclonal antibody 
against the peptide and determining whether there is any antigen-antibody 
binding contained within the test sample. For this purpose, a test kit may 
be provided comprising a peptide, as defined herein, or a polyclonal or 
monoclonal antibody thereto and means for determining whether there is any 
binding with antibody or antigen respectively contained in the test 
sample. The test sample may be taken from any of the appropriate tissues 
and physiological fluids mentioned above for the detection of viral 
nucleic acid. If a physiological fluid is obtained, it may optionally be 
concentrated for any viral antigen or antibody present. 
A variety of assay formats may be employed. The peptide can be used to 
capture selectively antibody against HCV from solution, to label 
selectively the antibody already captured, or both to capture and label 
the antibody. In addition, the peptide may be used in a variety of 
homogeneous assay formats in which the antibody reactive with the peptide 
is detected in solution with no separation of phases. 
The types of assay in which the peptide is used to capture antibody from 
solution involve immobilization of the peptide on to a solid surface. This 
surface should be capable of being washed in some way. Examples of 
suitable surfaces include polymers or various types (moulded into 
microtitre wells; beads; dipsticks of various types; aspiration tips; 
electrodes; and optical devices), particles (for example latex; stabilized 
red blood cells; bacterial or fungal cells; snores; gold or other metallic 
or metal-containing sols; and proteinaceous colloids) with the usual size 
of the particle being from 0.02 to 5 microns, membranes (for example of 
nitrocellulose; paper; cellulose acetate; and high porosity/high surface 
area membranes of an organic or inorganic material). 
The attachment of the peptide to the surface can be by passive adsorption 
from a solution of optimum composition which may include surfactants, 
solvents, salts and/or chaotropes; or by active chemical bonding. Active 
bonding may be through a variety of reactive or activatible functional 
groups which may be exposed on the surface (for example condensing agents; 
active acid esters, halides and anhydrides; amino, hydroxyl, or carboxyl 
groups; sulphydryl groups; carbonyl groups; diazo groups; or unsaturated 
groups). Optionally, the active bonding may be through a protein (itself 
attached to the surface passively or through active bonding), such as 
albumin or casein, to which the viral peptide may be chemically bonded by 
any of a variety of methods. The use of a protein in this way may confer 
advantages because of isoelectric point, charge, hydrophilicity or other 
physico-chemical property. The viral peptide may also be attached to the 
surface (usually but not necessarily a membrane) following electrophoretic 
separation of a reaction mixture, such as immunoprecipitation. 
After contacting (reacting) the surface bearing the peptide with a test 
sample, allowing time for reaction, and, where necessary, removing the 
excess of the sample by any of a variety of means, (such as washing, 
centrifugation, filtration, magnetism or capillary action) the captured 
antibody is detected by any means which will give a detectable signal. For 
example, this may be achieved by use of labelled molecule or particle as 
described above which will react with the captured antibody (for example 
protein A or protein G and the like; anti-species or 
anti-immunoglobulin-sub-type: rheumatoid factor: or antibody to the 
peptide, used in a competitive or blocking fashion), or any molecule 
containing an epitope contained in the peptide. 
The detectable signal may be optical or radioactive or physico-chemical and 
may be provided directly by labelling the molecule or particle with, for 
example, a dye, radiolabel, electroactive species, magnetically resonant 
species or fluorochore, or indirectly by labelling the molecule or 
particle with an enzyme itself capable of giving rise to a measurable 
change of any sort. Alternatively the detectable signal may be obtained 
using, for example, agglutination, or through a diffraction or 
birefringent effect if the surface is in the form of particles. 
Assays in which a peptide itself is used to label an already captured 
antibody require some form of labelling of the peptide which will allow it 
to be detected. The labelling may be direct by chemically or passively 
attaching for example a radio label, magnetic resonant species, particle 
or enzyme label to the peptide; or indirect by attaching any for of label 
to a molecule which will itself react with the peptide. The chemistry of 
bonding a label to the peptide can be directly through a moiety already 
present in the peptide, such as an amino group, or through an intermediate 
moiety, such as a maleimide group. Capture of the antibody may be on any 
of the surfaces already mentioned by any reagent including passive or 
activated adsorption which will result in specific antibody or immune 
complexes being bound. In particular, capture of the antibody could be by 
anti-species or anti-immunoglobulin-sub-type, by rheumatoid factor, 
proteins A, G and the like, or by any molecule containing an epitope 
contained in the peptide. 
The labelled peptide may be used in a competitive binding fashion in which 
its binding to any specific molecule on any of the surfaces exemplified 
above is blocked by antigen in the sample. Alternatively, it may be used 
in a non-competitive fashion in which antigen in the sample is bound 
specifically or non-specifically to any of the surfaces above and is also 
bound to a specific bi- or poly-valent molecule (e.g. an antibody) with 
the remaining valencies being used to capture the labelled peptide. 
Often in homogeneous assays the peptide and an antibody are separately 
labelled so that, when the antibody reacts with the recombinant peptide in 
free solution, the two labels interact to allow, for example, 
non-radiative transfer of energy captured by one label to the other label 
with appropriate detection of the excited second label or quenched first 
label (e.g. by fluorimetry, magnetic resonance or enzyme measurement). 
Addition of either viral peptide or antibody in a sample results in 
restriction of the interaction of the labelled pair and thus in a 
different level of signal in the detector. 
A suitable assay format for detecting HCV antibody is the direct sandwich 
enzyme immunoassay (EIA) format. A peptide is coated onto microtitre 
wells. A test sample and a peptide to which an enzyme is coupled are added 
simultaneously. Any HCV antibody present in the test sample binds both to 
the peptide coating the well and to the enzyme-coupled peptide. Typically, 
the same peptide are used on both sides of the sandwich. After washing, 
bound enzyme is detected using a specific substrate involving a colour 
change. A test kit for use in such an EIA comprises: 
(1) a peptide, as herein defined labelled with an enzyme; 
(2) a substrate for the enzyme; 
(3) means providing a surface on which a peptide is immobilised; and 
(4) optionally, washing solutions and/or buffers. 
It is also possible to use IgG/IgM antibody capture ELISA wherein an 
antihuman antibody is coated onto microtiter wells, a test sample is added 
to the well. Any IgG or IgM antibody present in the test sample will then 
bind to the anti-human antibody. A peptide of the present invention, which 
has been labelled, is added to the well and the peptide will bind to any 
IgG or IgM antibody which has resulted due to infection by HCV. The IgG or 
IgM antibody can be visualized by virtue of the label on the peptide. 
It can thus be seen that the peptides of the present invention may be used 
for the detection of HCV infection in many formats, namely as free 
peptides, in assays including classic ELISA, competition ELISA, membrane 
bound EIA and immunoprecipitation. Peptide conjugates may be used in 
amplified assays and IgG/IgM antibody capture ELISA. 
An assay of the present invention may be used, for example, for screening 
donated blood or for clinical purposes, for example, in the detection and 
monitoring of HCV infections. For screening purposes, the preferred assay 
formats are those that can be automated, in particular, the microtitre 
plate format and the bead format. For clinical purposes, in addition to 
such formats, those suitable for smaller-scale or for single use, for 
example, latex assays, may also be used. For confirmatory assays in 
screening procedures, antigens may be presented on a strip suitable for 
use in Western or other immunoblotting tests. 
As indicated above, assays used currently to detect the presence of 
anti-HCV antibodies in test samples, particularly in screening donated 
blood, utilise antigenic peptides obtained from HIV type 1 only and, as 
demonstrated herein, such antigens do not reliably detect other HCV 
genotypes. Accordingly, it is clearly desirable to supplement testing for 
HIV-1 with testing for all other genotypes, for example, types 2, 3 and 4, 
and also any further genotypes that may be discovered. 
To test for a spectrum of genotypes, there may be provided a series of 
assay means each comprising one or more antigenic peptides from one 
genotype of HCV, for example, a series of wells in a microtitre plate, or 
an equivalent series using the bead format. Such an assay format may be 
used to determine the genotype of HCV present in a sample. Alternatively, 
or in addition, an assay means may comprise antigenic peptides from more 
than one genotype, for example, a microwell or bead may be coated with 
peptides from more than one genotype. 
It has been found advantageous to use more than one HCV antigen for 
testing, in particular, a combination comprising at least one antigenic 
peptide derived from the structural region of the genome and at least one 
antigenic peptide derived from the non-structural region, especially a 
combination of a core antigen and at least one antigen selected from the 
NS3, NS4 and NS5 regions. The wells or beads may be coated with the 
antigens individually. It has been found advantageous, however, to fuse 
two or more antigenic peptides as a single polypeptide, preferably as a 
recombinant fusion polypeptide. Advantages of such an approach are that 
the individual antigens can be combined in a fixed, predetermined ratio 
(usually equimolar) and that only a single polypeptide needs to be 
produced, purified and characterised. One or more such fusion polypeptides 
may be used in an assay, if desired in addition to one or more unfused 
peptides. It will be appreciated that there are many possible combinations 
of antigens in a fusion polypeptide, for example, a fusion polypeptide may 
comprise a desired range of antigens from one serotype only, or may 
comprise antigens from more than one serotype. The antigenic peptides from 
serotypes 2, 3 and 4 are preferably those described herein. 
To obtain a polypeptide comprising multiple peptide antigens, it is 
preferred to fuse the individual coding sequences into a single open 
reading frame. The fusion should, of course, be carried out in such a 
manner that the antigenic activity of each component peptide is not 
significantly compromised by its position relative to another peptide. 
Particular regard should of course be had for the nature of the sequences 
at the actual junction between the peptides. The resulting coding sequence 
can be expressed, for example, as described above in relation to 
recombinant peptides in general. The methods by which such a fusion 
polypeptide can be obtained are known in the art, and the production of a 
recombinant fusion polypeptide comprising multiple antigens of a strain of 
HCV type 1 is described in GB-A-2 239 245 immunoprecipitation. Peptide 
conjugates may be used in amplified assays and IgG/IgM antibody capture 
ELISA. 
The peptide of the present invention may be incorporated into a vaccine 
formulation for inducing immunity to HCV in man. For this purpose the 
peptide may be presented in association with a pharmaceutically acceptable 
carrier. 
For use in a vaccine formulation, the peptide may optionally be presented 
as part of an hepatitis B core fusion particle, as described in Clarke et 
al (Nature, 1987, 330, 381-384), or a polylysine based polymer, as 
described in Tam (PNAS, 1988, 85, 5409-5413). Alternatively, the peptide 
may optionally be attached to a particulate structure, such as liposomes 
or ISCOMS. 
Pharmaceutically acceptable carriers include liquid media suitable for use 
as vehicles to introduce the peptide into a patient. An example of such 
liquid media is saline solution. The peptide may be dissolved or suspended 
as a solid in the carrier. 
The vaccine formulation may also contain an adjuvant for stimulating the 
immune response and thereby enhancing the effect of the vaccine. Examples 
of adjuvants include aluminium hydroxide and aluminium phosphate. 
The vaccine formulation may contain a final concentration of peptide in the 
range from 0.01 to 5 mg/ml, preferably from 0.03 to 2 mg/ml. The vaccine 
formulation may be incorporated into a sterile container, which is then 
sealed and stored at a low temperature, for example 4.degree. C., or may 
be freeze-dried. 
In order to induce immunity in man to HCV, one or more doses of the vaccine 
formulation may be administered. Each dose may be 0.1 to 2 ml, preferably 
0.2 to 1 ml. A method for inducing immunity to HCV in man, comprises the 
administration of an effective amount of a vaccine formulation, as 
hereinbefore defined. 
The present invention also provides the use of a peptide as herein defined 
in the preparation of a vaccine for use in the induction of immunity to 
HCV in man. 
Vaccines of the present invention may be administered by any convenient 
method for the administration of vaccines including oral and parenteral 
(e.g. intravenous, subcutaneous or intramuscular) injection. The treatment 
may consist of a single dose of vaccine or a plurality of doses over a 
period of time. 
LITERATURE CITED 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 53 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LysProAlaLeuValProAspLysGluValLeuTyrGlnGlnTyrAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GluCysSerGlnAlaAlaProTyrIleGluGlnAlaGlnValIleAla 
151015 
HisGlnPhe 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ArgValValValThrProAspLysGluIleLeuTyrGluAlaPheAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ArgAlaValIleAlaProAspLysGluValLeuTyrGluAlaPheAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GluCysAlaSerLysAlaAlaLeuIleGluGluGlyGlnArgMetAla 
151015 
GluMetLeu 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GluCysAlaSerArgAlaAlaLeuIleGluGluGlyGlnArgIleAla 
151015 
GluMetLeu 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LysProAlaIleIleProAspArgGluValLeuTyrArgGluPheAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino-acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LysProAlaValIleProAspArgGluValLeuTyrArgGluPheAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ArgProAlaValXaaProAspArgGluValLeuTyrGlnGluPheAsp 
151015 
GluMet 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GluCysSerGlnHisLeuProTyrIleGluGlyMetLeuAlaGluGln 
151010 
Phe 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GluCysSerGlnHisLeuProTyrIleGluGlyAlaLeuAlaGluGln 
151010 
Phe 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 194 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
NNNNNNNNNNCGAGTGTCGTGCAGCCTCCAGGMCYCCCCCTCCCGGGAGAGCCATAGTGG60 
TCTGCGGAACCGGTGAGTACACCGGAATCGCTGGGGTGACCGGGTCCTTTCTTGGARCAA120 
CCCGCTCAATACCCAGAAATTTGGGCGTGCCCCCGCRAGAYCACTAGCCGAGTAGTGTTG180 
GGTCGCGAAAGGCC194 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 85 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: yes 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaGluIleTyrGlnCysCys 
151015 
AsnLeuGluProGluAlaArgLysValIleSerSerLeuThrGluArg 
202530 
LeuTyrCysGlyGlyProMetPheAsnSerLysGlyAlaGlnCysGly 
354045 
TyrArgArgCysArgAlaSerGlyValLeuProThrSerPheGlyAsn 
505560 
ThrIleThrCysTyrIleLysAlaThrAlaAlaCysXaaAlaAlaGly 
65707580 
LeuArgAsnProAsp 
85 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: yes 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LysGlnGlnGlyLeuAsnPheSerTyrLeuXaaAlaTyrGlnAlaThr 
151015 
ValCysAlaArgAlaGlnAlaXaaProProSerTrpAspGluXaaTrp 
202530 
LysCysLeuValArgLeuLysProThrLeuHisGlyProThrProLeu 
354045 
LeuTyrArgLeuGlyProValGlnAsn 
5055 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 124 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: yes 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AlaLysProGlnArgLysThrLysArgAsnThrIleArgArgProGln 
151015 
AspValLysPheProGlyGlyGlyGlnIleValGlyGlyValTyrVal 
202530 
LeuProArgArgGlyProArgLeuGlyValCysAlaThrArgLysThr 
354045 
SerGluArgSerGlnProArgGlyArgArgGlnProIleProLysAla 
505560 
ArgArgSerGluGlyArgSerTrpAlaGlnProGlyTyrProTrpPro 
65707580 
LeuTyrGlyAsnGluGlyCysGlyTrpAlaGlyTrpLeuLeuSerPro 
859095 
ArgGlySerArgProSerTrpGlyProAsnAspProArgArgArgSer 
100105110 
ArgAsnLeuGlyLysValIleAspThrLeuThrTrp 
115120 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 367 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
NNNNCACCCYRUCRCRAAAUACVUCAUGGCAUGYAUGUCAGCUGAUCUGGAAGUAACCAC60 
CAGCACCUGGGUGUUGCUUGGAGGRGUCCUCGCKGCCCUAGCGGCCUACUGCUUGUCAGU120 
CGGCUGCGUUGUGAUUGUGGGYCAUAUUGAGCUGGGRGGCAAGCCVGCAMUCGUUCCAGA180 
CAARGARGUGUUGUAUCAACAAUACGAUGAGAUGGAGGAGUGCUCGCAAGCYGCCCCAUA240 
UAUCGAACAAGCUCARGURAUAGCCCACCAGUUCAAGGAGAAAGUCCUUGGRUUGCUGCA300 
GCGRGCCACCCAACAACARGCUGUYAUUGAGCCMAUAGUAGCUACCAACUGGCAAAANNN360 
NNNNNNN367 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 128 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: yes 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
XaaHisProXaaXaaLysTyrXaaMetAlaCysMetSerAlaAspLeu 
151015 
GluValThrThrSerThrTrpValLeuLeuGlyGlyValLeuAlaAla 
202530 
LeuAlaAlaTyrCysLeuSerValGlyCysValValIleValGlyHis 
354045 
IleGluLeuGlyGlyLysProAlaXaaValProAspLysGluValLeu 
505560 
TyrGlnGlnTyrAspGluMetGluGluCysSerGlnAlaAlaProTyr 
65707580 
IleGluGlnAlaGlnValIleAlaHisGlnPheLysGluLysValLeu 
859095 
GlyLeuLeuGlnArgAlaThrGlnGlnGlnAlaValIleGluProIle 
100105110 
ValAlaThrAsnTrpGlnXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa 
115120125 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 177 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
TGAGTGTYGTRCAGCCTCCAGGAYYCCCCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN60 
CGGTGAGTWCACCGGAATCGCCGGGAYGACCGGGTCCTTTCTTGGANNHWACCCGCTCMA120 
TGCCCGGAAATNNNNNNNNNNNNNNGCRAGACYGCTAGCCGAGTAGTGTTGGGTCGC177 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 240 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AAACCAAAAGAAACACCATCCGTCGCCCACAGGACGTCAAGTTCCCGGGTGGCGGACAGA60 
TCGTTGGTGGAGTATACGTGTTGCCGCGCAGGGGCCCACGATTGGGTGTGTGCGCGACGC120 
GTAAAACTTCTGAACGGTCACAGCCTCGCGGACGACGACAGCCTATCCCCAAAGCGCGTC180 
GGAGCGAAGGCAGGTCCTGGGCTCAGCCCGGGTACCCGTGGCCCCTCTATGGTAACGAGG240 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 240 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
NNNNNNNNNNTAACACCAACCGCCGCCCCAYGGACGTTAAGTTCCCGGGTGGTGGYCAGA60 
TCGTTGGCGGAGTTTACTTGTTGCCGCGCAGGGGCCCYMGKTTGGGTGTGCGCGCGACTS120 
GRAAGACTTCGGAGCGGTCGCAACCTCGTGGGAGACGYCARCCTATCCCMAAGGCGCGTC180 
GATCCGAGGGAAGGTCCTGGGCACARCCAGGATWTCCATGNNNNNNNNNNNNNNNNNNNN240 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: yes 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
XaaXaaXaaXaaLysArgAsnThrAsnArgArgProXaaAspValLys 
151015 
PheProGlyGlyGlyGlnIleValGlyGlyValTyrLeuLeuProArg 
202530 
ArgGlyProArgLeuGlyValArgAlaThrXaaLysThrSerGluArg 
354045 
SerGlnProArgGlyArgArgGlnProIleProLysAlaArgArgSer 
505560 
GluGlyArgSerTrpAlaGlnProGlyXaaProTrpProLeuTyrXaa 
65707580 
XaaXaa 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
ATACTCGAGGTGCACGGTCTACGAGACCT29 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CACTCTCGAGCACCCTATCAGGCAGT26 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CTGTGAGGAACTACTGTCTT20 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
TTCACGCAGAAAGCGTCTAG20 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
ATGTACCCCATGAGGTCGGC20 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
AGGTCTCGTAGACCGTGCATCATGAGCAC29 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CCGGCATGCATGTCATGATGTAT23 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GTATTTGGTGACTGGGTGCGTC22 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
TCTTGAATTTTGGGAGGGCGTCTT24 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
CATATAGATGCCCACTTCCTATC23 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
AACTCGAGTATCCCACTGATGAAGTTCCACAT32 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
CACATGTGCTTCGCCCAGAA20 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
GGACCTACGCCCCTTCTATA20 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
TCGGTTGGGGCCTGTCCAAAATG23 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GGTCCCACCCCTCTCCTGTA20 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
CCGCTTGGGTTCCGTTACCAACG23 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
GGGCCAACACCCCTGCTATA20 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
CAGACTGGGCGCCGTTCAGAATG23 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
GGCGGAATTCCTGGTCATAGCCTCCGTGAA30 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
CCACGACTAGATCATCTCCG20 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
TGGGGATCCCGTATGATACCCGCTGCTTTGA31 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
CTCAACCGTCACTGAACAGGACAT24 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
GCCGCCCCATATATCGAACA20 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
GCTCAGGTAATAGCCCACCA20 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
AAAGCCGCCCTCATTGAGGA20 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
GGGCAGCGGATGGCGGAGAT20 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
CACTTACCGTACATCGAGCA20 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide"' 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GGGATGATGCTCGCCGAGCA20 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
CAYGTRAGGGTATCGATGAC20 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
ACTGCCTGATAGGGTGCTTGCGAG24 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
AGGTCTCGTAGACCGTGCATCATG24 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "synthetic DNA 
oligonucleotide" 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis-C virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
TTGCGBGACCTWCGCCGGGGGTC23 
__________________________________________________________________________