Note: Descriptions are shown in the official language in which they were submitted.
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
NON-A-NON- E HEPATITIS VIRUS HAVING A TRANSLATABLE CORE REGION,
REAGENTS AND METHODS FOR THEIR USE
The invention concerns nonA-nonE hepatitis virus
(GBV hepatitis virus) having a translatable 5' core
region, polynucleotides, peptides, antibodies,
diagnostics and reagents thereof and methods for their
use.
Simons J.N. et al. (Nature Medicine, 1, 564, l995)
revealed a viral genome, Flavivirus-like, named GBV-C,
in a small percentage of nonA-nonE hepatitis affected
patient sera. The genome has some homology with genomes
from two virus, GBV-A a GBV-B, identified in tamarins
(Simons J.N. et al., Proc. Natl. Acad. Sci. USA, 92,
340l, I995). Said virus represent a phylogenetic separate
group, not comprising hepatitis C virus (HCV).
PCT patent application N. W095/21922, in the name of
Abbott Lab., shows a partial nucleotide sequence of a
GHV-C isolate. A method to reveal antibodies against GBV
is claimed (claim 44, p. 6l8); however no enabling
disclosure for such method able to detect with a
statistic significative percentage positive samples from
nonA-nonE patients is provided. PCT patent application N.
W095/21922 does not disclose aminoacid sequences to be
effectively used to design diagnostic peptides. As a
matter of fact, Figure 39 shows a panel of proteins,
having an around 30d aminoacid minimal lenght. Only 2 out
512 sera from drug-addicted subjects result to be
positive for these proteins in an ELISA assay. Moreover
at p. 168, table 23, ELISA assays on nonA-nonE patients
result to be positive in very few samples, thus rendering
the assay not workable: 0/89 Japan subjects; 0/67 Greece
subjects; 6/72 USA (set M) subjects; 1/64 USA (set T)
subjects; 3/62 USA (set 1/3) subjects; 18/32 Egypt
subjects. At p. 169, table 24, only 21 out 303 nonA-nonE
patients are positive with an ELISA assay using peptides
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
2
sequence. It is evident that said assay may not be used
neither for a screening of healthy subjects, nor for
diagnosis.
PCT patent application N. W095/32291, in the name of
Genelabs, identifies by ~epitope mapping different
antigenic regions of the GBV polyprotein. However no
peptides or a combination thereof for a diagnostic assay
are disclosed.
EP patent application N. 73660l, in the name of
Abbott Lab., discloses 88 nucleotides upstream of the
sequence shown in the PCT patent application N.
W095/21922. However no evidence is provided for an open
reading frame in this region able to code for a core-like
protein.
The nucleocapsid/El junction of the viral isolate
was identified by Leary, T. P., et al. 1996. J. Med.
Virol. 48:60-67. It is predicted to occur approximately
46 aminoacids downstream of the first methionine residue
of the ORF, while Linnen, J. et al. 1996. Science 27
1:505-508 have also identified an isolate in which the
putative capsid protein contains 83 aminoacid residues.
The apparent main difference among these two isolates is
one base deletion causing different initiation sites.
Thus, in comparison with other Flaviviridae, in which the
lenght of the capsid protein varies from 114 aminoacids
in dengue fever virus to 175 residues in HCV, the GBV-C
nucleocapsid appears too short to be functional.
The presence of core-like proteins in GBV-C has also
been challenged by recent translation studies
demonstrating that the site of translation initiation of
the viral polyprotein is immediately upstream of the
putative signal sequence for the El glycoprotein (Simons,
J. N., et al. 1996. J. Virol. 70:6126-6135).
Although it was originally suggested that the
apparent lack of a functional core protein could be
ascribed to artefacts of the sequencing process, due to
CA 022S1745 1998-10-14
WO 97/39129 PCT/IT96/00248
3
compressions caused by the high GC contents (teary et al.
supra), it is now clear that these sequences represent
the true status of GBV-C in the numerous isolates that
have been characterized (Simons et al. 1996, supra).
It has been proposed, therefore, that the NH,-
terminal end of the GBV-C polyprotein is a truncated
derivative of an ancestral capsid protein and that GBV-C
requires a helper virus to replicate (Heringlake, S . , H.
L. Tillmann, and M. P. Manns. 1996. J. Hepatol. 25:239-
247). Alternatively, since a limited number of virus
clones from each isolate has been examined so far, it is
possible that, after infection, a large population of
single base deletion mutants arise and these mutants
would have the highest probability of being examined.
The authors report the characterization of a GBV-C
isolate, obtained from a HIV and HCV positive patient, in
which translation can initiate at an AUG codon located
273 nucleotides upstream of the previously identified
initiator site (Simons et al. 1996, supra), leading to a
potentially functional nucleocapsid core protein of 107
aminoacids. This phenotype remained unvaried over the one
year monitoring of the patient, suggesting therefore that
stable replication competent GBV-C isolates may exist in
nature.
The authors of the instant invention have also
identified NS3 peptides of nonAnonE associated virus and
has set up an assay to detect antibodies able to
recognize epitopes comprised in said peptides. Said
antibodies are detected in chronic and acute hepatitis
affected subjects, classified as nonA-nonE for the
lacking ef known virus related markers, in a high
percentage of studied subjects (around 300). Surprisingly
the assay is able to detect said antibodies also in
subjects which are affected by other pathologies, as
hepatitis A, hepatitis B, hepatitis C, hepatitis Delta,
CA 02251745 1998-10-14
~.I~J~, ~j~~i" v~~,,-dStr:..~.L~e:-~ S.", ~"'~~..~'y dU.~CI:TL~,'_:i.~
i.'_~S~dSeS
(LES, s'~NA+) r Show~rig 3 ~''iatwCrn~:'z1p ~r=G:_ v:E''.' ;;~~
The assay is per formed preferentia? 1;: t~~ react' ng a
treated bzolcgzca~. samp~.e, as serum, ~i.t=~. Je~.ected
peptides to ge4 an eiFectve and d'agnos=is method.
Antigen~anti.bodyes complexes are revealed in an effective
ar.d reprcducir' a way by means of one of assays ~.-~cwr to
the expert .in the ficid, as ELIcA, RIB:, EIA,
i:nmunoblottin, atc.
1G y.~_~_ the foliow;;ny "~L~I vir~:s" means v' wa associated
t4 non.~~or.E ~:epa=i t::s; "izu~zunoloc~.cail~r r~omolcgcus
epi tope" means an ept =ope ::,at% irlg an a:~r.inca:.~.d sequence
w~ir_h d'_~fers 'nom th~ reFe=r'inCj v'~?ltd~L~2 Cv~ ~:Tln or m:J~~?
y
am~.ncac~.ds, but whic:~ i.s able to recc~nrze anc ~oi:.c. ~o
antibodies for the ~e''errng epitone, with 5u:os;.ar~~-ial' y
the sa~:.e binui::g a'fi :i ty.
It is a n object of the ~.;~~:er_ticr_ a ~oeptic'e
ab'e ~to s: eCif::cally bir:C tc no:~..~r_onE hepat_tis visas
a~~~~~oa,zas, having a =equer_ce cf a= least ~C,.
a~ inoaci s, said non.Anc.~.~ hepat. ti s virus 'oei
ng
characterized by hcs7~.Il~~ a trar.slatabie =~nc~i~.na_ core-
;ikA ~rorswn 'onger then ~~ arninr_acid res~d~~;es a:~d
by
CC="lpriSl::~ ~.-~ d~.'Ll._"lG3Cid Sac~u,eIlCE'.~.~ ~~aS~
hczrc'_cgeus to the secrlence of SEQ I~ ;do.
4:
25 '~SLrIPa.R'!PFULLTPT'IGFP,~PS, or an i.;unu:-:olcgically
ho:noloc~ous variant cr the peptidA. Prefer aiy the
peptide ~.as a 5equer~ce ccr~rrised in SEQ Iu Nc.4.
It is a Further object of tha i::vention
a
ccnposi~zor_ ccmpris~:~lQ at _east one o the peptides
of
the i nTrention. Prezerably said cor_p,csi ticn is adhered
Jn a SO1 ~ a ptla$e .
A.ccarding tc a preferred. embcdi.-~,A-:t
of the
in~.Antion said c:ornpcsition comprises a'~ least tt~rc
pepy~.~~ the irler.tion.
~.-'1"s ~ a.
AMEN~rp SH~.ET
r~ ~n" ~r,~. ,.: ~~ 7r, i ~Ln~ .~.; . ,nm _fty.. .,.~r_.'lrJ'', t7 ~,..~
_ . _. ~. , , _ . -.Z; r~. _ _ ~~ prr~-r nr~.
_ _..~.., ...,_.............,..~.~.._~.__ ... _...._....,a - ' ~ ~ : . _ ,.~
:j.. . I~: _ . ~ \ . ) ; ~
CA 02251745 1998-10-14
_ 5 _
F~azther object cf the invention is a
diagnostic ki: for nonRror.~, hepatitis carnprising as
specific reagent at least one peptide o~ Lha invention.
Preferably said kit comprises at least twe oei t~.des of
t'.:e invention.
Furtrer object o~ ~.he i nventicr is a rr.ethod
to detect ::cn.~, ron~ hepa=iris virus antibodies in a
sa.~.-~ple c..,rnprisi:~g the following steps:
- inc~,:,ating in conditions allow':.nc t_:e
for~!a~ion cf a.. artigen-aztibcdy co~:p~.e:c said
sample
with at least or.e peptide ;:f the inve:aion;
reveali-g saia cemp~.ex.
Tie izvsnti=n is new desl~r:.bed i n a non- i imi c'_ng .way
by reference tc the following figures wherein:
r
- riguro 1 renYeser_ts ~he ~r~.i:.eacic sect:enea o_ GDv
N53 region dod of used peptides, with their dEnomination:
A~nrnlnr~l ~:~rrT
IJILI'1'Jv. '
~~. ~ I _ ~mr ~~n~ "~I, ~:on~~ ,,.,;~_.~.,.: o .-m;i
n , i '.. . .. . , ~ ' ~ i f i , ,n _ . ~ ~ . a I r"1 ~1 ~ . .
- . . I , . , '. I y. , , ~ ' I , , i . v a 4
CA 02251745 1998-10-14
W~ 97/39l29 PCT/IT96/00248
6
- figure 2 represents a bar graphic of p3.1-p3.9
peptide reactivity in ELISA assay with sera from subjects
affected by different pathologies;
- figure 3a a 3b represent aligned aminoacid
sequences of GBV core region; first three from top: from
literature, others: isolated from the authors; "-" means
an identity with the U36380 sequence; "_" means an
identity with the U45966 sequence;
- figure 4a represents aligned aminoacid sequences
of a segment of GBV core region, from literature: the
first from the top from U45966 of Fig. 3a, others are
isolated from the authors; at the bottom the consensus
sequence is shown (SEQ ID No. 3);
- figure 4b represents a bar graphic of consensus
peptide of Fig. 4a reactivity in ELISA assay with sera
from subjects affected by different pathologies;
- figure 5 represents the nucleotide sequence
alignment of 10 GBV-C isolates obtained from 10 distinct
GBV-C positive patients, compared with the sequences of
the published isolates deposited in GenBank [GBV-C
isolate (teary et al., supra), PNF2161 isolate (Linnen et
al. supra) named GBV1 in the figure, and R10291 isolate
named GBV2 in the figure]. The sequences were analyzed
with the CLUSTAL alignment program. The alignment
includes the nucleotide sequence between AC1S and AS-CWP
primers (shadowed in grey), and comprises a partial
5'UTR, the putative core region and a partial E1 region
(corresponding to nt I12-733 of GBV-C, nt I29-752 of GHV1
and nt 56-680 of GBV2). The nucleotides are numbered only
for isolate 35 (identified as the isolate with the longer
ORF) and nucleotide +1 identifies~the nucleotide A of its
first putative start codon in frame with the long ORF.
Note that in the other isolates nucleotide insertions or
deletions are present. Gaps, introduced in the sequences
to preserve alignment, are represented by dashes. The
sequences between nucleotides 316 and 446, that are
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WO 97/39129 PCT/IT96/00248
7
conserved, are not shown for reason of space. The
nucleotides conserved among a11 the sequences are pointed
out by an asterisk. The ATG codons in frame with the long
ORF of each isolate are boxed;
- figure 6 represents the aminoacid sequence
alignment of the isolates described in Fig. 5 and deduced
from their nucleotide sequences. The aminoacid sequence
of isolate 35 is identified as 35/1, while the sequences
deduced from the two additional isolates derived from
distinct serum samples collected during the follow-up of
this patient are pointed out as 35/2 and 3S/3. The
sequences were analyzed with the CLUSTAL alignment
program. The sequence of the longer published ORF (GBV2)
is considered as the consensus sequence. Dashes denote
identity with the consensus sequence, whereas individual
variations are shown by the single aminoacid code. The
methionines in-frame with the long ORF are boxed and
pointed out with an arrow for each isolate. The areas
shadowed in grey mean the regions of high variability
detected in our isolates;
- figure 7 represents a SDS-gel pattern of
[3~S]cysteine-labelled in vitro translation products of
the constructs, containing the AC1S/AS-CWP amplified
region from the pointed out isolates. Positions of
molecular weight standards (in kilodaltons) are pointed
out. The last lane contains the negative control (no
RNA). The IVTT of pCR35/AC1S construct was performed with
and without capped mRNA;
- figure 8 represents the construction of the
deletion mutants of pCR35/AC1S, pCRl9/AC1S, pCR3064/AC1S
and pCR2095/AC1S vectors. In the figure, the strategy for
the construction of the pCR35/AC1S derivatives is
described. The location of the T7 promoter (in black),
the Bam H1 cleavage site and the position of the primers
(AC1S and AS-CWP) used for the construction of the parent
plasmid are shown. The numeration of the nucleotides is
CA 02251745 1998-10-14
WO 97I39129 PCT/IT96/00248
8
relative to the first putative start codon of the
sequence of isolate 35. The partial 5' non coding region
is white, the partial ORF is shadowed in grey and the
position of AUG codons are pointed out with the notation
Met. The positions of the sense primers (primers 1, 2, 3,
4 and 5) used with the antisense AS-CWP primer for the
construction of the deletion mutants are also shown. The
same strategy was followed for obtaining the pCRl9/AC1S,
pCR3064/AC1S and pCR2095/AC1S derivatives. The length of
the partial 5'UTR, of the partial ORF and the position of
the AUG codons of these isolates are shown in Fig. 5 and
6.
- figure 9 represents a SDS-gel pattern of
~35S~cysteine-labelled in vitro translation products
gLenerated by the O2, 43, 44 and 05 deletion mutants of
pCR35/AC1 (A), pCRl9/ACIS (B) and pCR2095/AC1S (C)
plasmids, obtained as described in Fig. 8. Positions of
molecular weight markers (in kilodaltons) are pointed
out. In panel (A)-the products of capped and uncapped
mRNAs derived from the pCR35/AC1S undeleted plasmid are
shown. A11 the other mRNAs used in these experiments were
capped transcripts. In the same panel the first lane
represents the product of translation of an unrelated
mRNA (UNR). In panel (B) the first lane represents the
product of translation of the pCR35/~, mutant, that was
used as control for the size of the product of isolate
19. In panel (C) the first lane contains the negative
control (no RNA) .
NS3 REGION
Analysis of GBV protein sequences
Protein sequences have been computer analyzed by
means of ANTIGEN program 6.85 (PCGene). ANTIGEN utilizes
Hopp and Wood algorithm to predict protein potentially
hydrophilic regions.
Peptide synthesis
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WO 97/39129 PCT/IT96/00248
9
Peptides have been synthesized with an automated
synthesizer MilliGen 9050 (Septrim). A11 of aminoacids
were ~noc a-amino protected and pre-activated at the
carboxy terminus pentafluorophenyl esters; only serine
and threonine were activated~as benzotriazine esters.
A11 of coupling reactions were performed with HOBT
0.33M/DMF; washings were made with DMF and the a-amino
group was released with 20o piperidine in DMF. Peptides
were released with from resin by treatment with a mixture
of TFA and scavengers which differ according to the
composition.
Cases
sera from 52 healthy donors controlled to be HBV-, HCV-
and HIV-;
sera from 27 acute nonAnonE hepatitis patients;
sera from 16 chronic nonAnonE hepatitis patients;
sera from 61 acute hepatitis B patients;
sera from 14 chronic hepatitis B patients;
sera from 15 acute hepatitis C patients;
sera from 15 chronic hepatitis C patients;
sera from 29 drug addicted HIV-, HCV+ patients;
sera from 31 HIV+ patients;
sera from 33 HIV+ piastrinopenic patients;
sera from 30 AIDS patients;
sera from 39 LES patients.
ELISA assay
The solid phase was prepared onto polivinylchloride
dishes (N unc), by using a peptide or a mixture thereof.
Peptides were dissolved separately in a mixture of 500
CH3CN/H20 and diluted to a single final concentration of
10 ug/ml (total peptide concentration of 20 ug/ml) in
carbonate buffer 50 mM, pH 9.6. 200 ul were added to each
well and incubated for 2 hrs at 37C. When a single
peptide was
used, the
final concentration
was 20 ~g/ml.
CA 02251745 1998-10-14
WO 97I39129 PCT/IT96100248
Following to peptide adsorption, solid phases were
saturated for 1 hr at 37~C with 300 ul/well of a BSA 2'
solution in Tris-HC1 0.1M pH 7.5.
200 ul/well of 1:21 diluted sera in a solution of
5 PBS containing 0.3 g/1 EDTA,. 2 g/1 BSA, 2 ml/1 Triton
x100 and 2 ml/1 Tween 20 were incubated 2 hrs at 37~C.
Following to 4 washes with PBS/Tween 20 0.1;, dishes
were treated with 100 ul/well of horseradish peroxidase
conjugated anti human Ig (Amersham). 1:30.000 diluted in
10 a PBS solution containing 0.04o Tween 20 and 20o non
immune goat serum.
Following to 4 washes with PBS/Tween 20 O.lo, l00
ul/well of chromogen/substrate were added (Sorin
Biomedica Diagnostics, DEIA KIT). The colorimetric
reaction was stopped after 30 min. by adding 200 ul/well
of sulphuric acid 1N. The optical density was read at 450
nm by referring to 630 nm.
CORE REGION
Reverse transcription (RT)-PCR
Analysis was performed with serum samples from 10
distinct subjects resulted positive for the presence of
GBV-C RNA with the RT-PCR and hybridization assay as
previously described (Fiordalisi, G., et al. J. Infect.
Dis. 174:l81-183). Four of them (patients identified with
the codes 35, 19, 47 and MG) were HIV+/HCV+ patients with
chronic liver disease; DM and 1159 were non A-E acute
hepatitis patients; BZ was a patient with hepatocellular
carcinoma; 3Q64 was a patient with fulminant non A-E
hepatitis; patient 2095 was an HCV+ subject with chronic
hepatitis; and finally DS39 was a healthy blood donor.
Total RNA was extracted from 100 ul of serum samples of
these subjects with TriPure reagent (Boehringer Mannheim
Germany) and subjected to RT with random hexanucleotides
using Superscript II (Life Tecnologies, Gaithersbul-g,
MD). The obtained cDNA was used as template for PCR
reaction with degenerated primers, designed in our
CA 02251745 1998-10-14
WO 97/39R9 PCT/1T96/00248
11
laboratory and derived from the sequences of GBVC and GBV
isolates deposited in Genbank database (Leary et al.,
Linnen et al. supra). The sequences of these primers,
located in the 5'-UTR and putative structural regions,
were:
AC1S sense primer:
5'AGG GTT SGW WGG TSG TAA ATC C 3' (SEQ ID No. S)
AS-CWP antisense primer:
5'CGC CTG RTA NAR NGG CCA RCA 3' (SEQ ID No.6)
wherein S = C or G; W = A or T; R = A or G; N = A or C or
G or T.
Each of the 45 cycles of PCR included 1 min at 94~C,
1 min at 52~C and 1 min at 72~C, with an elongation time
of 10 min in the last cycle. The sequence of the PCR
product obtained with these primers corresponds to nt 129
to 752 of GenBank accession no. U44402, the GBV-C isolate
with the longest core-like protein reported to date
(Linnen et al. supra).
Cloning and sequencing
The amplification products obtained with AC1S and
ASCWP primers were inserted into pCR2.1 vector
(Invitrogen, San Diego, CA) and the sequences of the
cloned inserts were obtained using the A.L.F. DNA
sequencer (Pharmacia Biotech, Uppsala, Sweden). The
sequenced plasmids were named pCR X/AC1S, where X
corresponded to the isolate derived from the 10 patients
considered in this study. Nucleotide and aminoacid
sequences were analyzed with the CLUSTAL alignment
program included in PC/GENE software package
(Intelligenetics Inc., Mountain Vlow, CA).
Construction of deletion mutants'
Mutants with deletions of different portions of the
5' region of pCR35/AC1S, pCRl9/AC1S, pCR3064/AC1S and
pCR2095/AC1S constructs were derived from these plasmids
by PCR amplification using synthetic primers. The
antisense primer was the AS-CWP primer described above,
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WO 97/39129 PCT/IT96/00248
12
while the sense primers, designed in our laboratory and
derived from the sequences of our GBV-C isolates, mapped
just upstream of the first 5 putative start codons on the
basis of the sequence of isolate 35 (Fig. 5 and 6), as
5 described in Fig. 8.
Primer 1: nt -43/-27
5'ACGTAAGCTT A GGT GTW GGY CCT ACC G 3' (SEQ ID. No.7)
Primer 2: nt +48/+64:
5'ACGTAAGCTT G TAC GGY CCA CGT CGC C 3' (SEQ ID. No.8);
Primer 3: nt +99/+117:
5'ACGTAAGCTT G CGA GTT GRC AAG GAC CAG 3'(SEQ ID. No.9);
Primer 4: nt +167/+Z83:
5'ACGTAAGCTT GR RVC GGG AAA TGC ATG 3'(SEQ ID. No.lO);
Primer 5: nt +239/+255:
15 5'ACGTAACCTT TG AGG GCG GGT GGC ATT 3'(SEQ ID. No.l1),
wherein underlined nucleotides correspond to specific
sequences of GBV-C, W = A or T; Y = C or T; R = A or G; V
- C or A or G. The position of the primers corresponded
to the nucleotide numbers obtained considering as +1 the
20 nucleotide A of the first ATG in the ORF of isolate 35
(see Fig. 5) . The products of PCR with 1, 2, 3, 4 and 5
sense primers and AS-CWP antisense primer were cloned in
pCR2.1 and named respectively pCRX/01, pCRX/02, pCRX/~3,
pCRX/~4 and pCRX/05, where X corresponded to the
25 identification code of the isolate (35, 19, 3064 and
2095y .
In vitro transcription and translation (IVTT)
Plasmids were linearized by digestion with BamHl and
transcribed in vitro with T7 RNA polymerase (mCAP mRNA
30 Capping kit, Stratagene, La Jolla, CA) according to the
manufacturer's instructions. In most cases, a m'GpppG cap
structure was incorporated at the 5' end of the RNA
transcripts. In vitro translation reactions were
performed with a rabbit reticulocyte lysate (RRL) system
35 (Amersham, Buckinghamshire, England), according to the
CA 02251745 1998-10-14
WO 97/391Z9
PCT/IT96/OOZ48
13
manufacturer's instructions. Reactions (25 ul) contained
20 U of rRNasin (Promega, Madison,WI), 20 uCi of ['SS]
cysteine (l.000 Ci/mmol, Amersham, England) and 1.5 ~g of
in vitro transcribed RNA. After incubation at 30~C for 1
hr, 2 ul aliquots were denatured for 5 min at 100~C in an
equal volume of 2 X sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) loading
buffer and analyzed on 18o SDS-polyacrylamide gels.
Protein gels were dried and exposed to X-ray film.
IO RESULTS
NS3 REGION
The analysed peptides are comprised in the NS3
region, from aa. 1346 to aa. 1550 (SEQ TD No.l), and are
shown in Figure 1.
All of peptides were adsorbed to the solid phase at
different concentrations and confronted with a panel of
sera from nonAnonE affected patients and healthy donors.
ELISA assays were performed with different
concentrations and adsorption conditions. Results show
that peptides of Table 1 react with sera with different
binding affinities. The reactivity may be increased by
previous adsorption to the solid phase of a mixture of
different peptides.
The G20T peptide has a decreased activity given to a
cysteine dimer at position 1467. The cysteine to serine
(G20T(C->S)) substitution solves this stability problem.
In order to get good reactivities solid phases
should be prepared either with a mixture of l0ug/ml S16V
and 10 ug/ml G20T(C->S) or with E21A peptide at 20 ug/ml.
To determine the cut-off reactivities of 200 sera
from healthy donors were tested and the following formula
was applied:
OD cut-off = average OD healthy donors + 8 s.d. (standard
deviation)
The value was for both of solid phases of 0.450
ODqsonm. A11 of samples with an OD>0.450 were considered
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
14
to be positive. Results from sera screening were reported
in Table 1.
Table 1
S16V+G20T(C-S) E21A
n. patientsa patients
patients+ + + +
HEALTHY 52 0 0 0 0
DONORS
HEP. acute 27 8 32 7 28
nAnE chronic 16 8 32 7 28
B acute 61 30 46, 17 26, 6
9
HEP. B 14 1 3,1 0 0
chronic
C acute 15 B 19,5 3 7,3
C 15 9 22,5 5 12,5
chronic
TD HIV-,HCV+ 29 17 32,7 19 36,5
HIV+ 31
HIV+ 33
piastrinopenic
AIDS 30
I,ES 3 9
Results obtained with p3.1-p3.9 peptides of Figure 1
are shown in Figure 2.
Data show trhat some of analyzed peptides are able
to specifically detect antibodies against epitopes of NS3
region of GBV virus.
CORE REGION
Identification of specific epitopes of "core" region
Aminoacid sequences (as in figure 3a a 3b) show
methionines which may be aligned with either the first or
the second methionine already described. The 35HIV clone
sequencing revealed a new aminoacid sequence, at the N
terminus region, starting with a methionine residue.
By clone sequencing aminoacid sequences of GBV virus
N-terminus were revealed. The "consensus" sequence is
shown in Figure 4a.
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
Figure 4b shows the reactivity obtained by assaying
different sera with the peptide having the consensus
sequence of Fig. 4a.
Peptide synthesis
5 Peptides were synthesized with an automated
synthesizer MilliGen 9050 (Septrim) as shown for peptide
for NS3 region.
Cases
sera from 20 healthy donors;
10 sera from 23 acute nonAnonE hepatitis patients;
sera from 10 chronic nonAnonE hepatitis patients;
sera from 22 acute hepatitis B patients;
sera from 33 drug addicted HIV-, HCV+ patients;
sera from 7 HIV+ patients;
15 sera from 17 HIV+ piastrinopenic patients;
sera from 47 Community Aquired HCV+ patients;
sera from 11 AIDS patients.
ELISA assay
Solid phase was prepared on polivinylchloride dishes
(Nunc) . l00 ul of a 20 ug/ml solution of G11C, D23C-VA,
M37C-LVA, D13G-TG and M27C-CTG peptides, in carbonate
buffer 50 mM, pH 9.6 were added to each well and
incubated for 2 hrs at 37~C.
Further to peptide adsorbtion solid phases were
saturated for 1 hr at 37~C with 300 ul/well of a BSA 20
solution in Tris-HC1 0.1M pH 7.5.
100 ul of 1:21 diluted serum in PBS containing 0.3
g/1 EDTA, 2 g/1 BSA, 2 m1/1 Triton x100 and 2 m1/1 Tween
20 were incubated into wells for 2 hrs at 37~C.
Following to 4 washes with PBS/Tween 20 0.1%, 100
ul/well of horseradish peroxidas'e conjugated goat anti
human Ig (Sorin Biomedica), diluted l:100 in a solution
of PBS containing 0.04% Tween 20 and 200 of preimmune
goat serum. were added to each well.
Following to 4 washes of PBS/Tween 20 0 . 1 o BSA, 100
ul of chromogen and substrate were added to each well
CA 02251745 1998-10-14
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16
(Sorin DEIA KIT). The colorimetric reaction was blocked
after 30 min with 200 ul/well of sulphuric acid 1N. The
OD reading was performed at 450 nm by referrng to OD at
630 nm.
Results show the presence of antibodies against
peptides of the "core" region of GBV virus.
Sequence varialbility of the 5' terminal region of
different GBV-C isolates
The GBV-C isolates so far analyzed have revealed
putative truncated core proteins of about 83 and 46 amino
acids in length (Linnen et al. supra, Simons et al. 1995
supra). These differences are due to nucleotide deletions
or insertions that introduce in the sequences a
frameshift causing the generation of distinct in-frame
starting codons. It is conceivable, therefore, that the
intrinsic genomic variability of this virus may
introduce, in some particular isolates, additional
mutations, insertions or deletions that could generate
potentially functional core proteins.
In order to evaluate this possibility, we analyzed
the nucleotide sequences, and their deduced translation
products. On the basis of this analysis, we found 4
different types of sequences. The sequences of isolates
19, DM, DS39, BZ, 1l59 and 47 encoded for a putative core
protein of approximately 83 aminoacids in length,
corresponding to the published sequence of R10291 isolate
(GenBank accession no. U45966) (Linnen et al. supra)
(GBV2 in Fig. 5 and 6). The most 5' and unique inframe
AUG codon of isolates 3064 and MG, on the other hand,
could initiate the translation of a putative core protein
of 46 aminoacids in length similar to the one potentially
encoded by the published sequences of GBV-C isolate
(GenBank accession no. U36380) (teary et aI. supra) and
PNF2161 isolate (Linnen et al. supra) (GenBank accession
no. U44402) (GBV-C and GBV1 in the Fig. 5 and 6). The
sequence of isolate 2095 was more peculiar since, due to
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96100248
17
one base deletion (Fig. 5), it contained only one
potential in-frame initiator AUG codon, downstream of the
putative start codon of GBV-C and HGVI and located 9
nucleotides only upstream of the sequence that specifies
the putative E1 signal sequence (Leary ET al. supra). The
mostly unexpected finding, however, was that the sequence
derived from the isolate 35 (35/1 in Fig. 5 and 6) could
potentially encode for a putative core protein of
approximately 106 aminoacids in length. This particular
feature was due to a point mutation that introduces an
additional in-frame AUG codon 69 nucleotides upstream of
the putative initiation codon of the HGV 2 reference
sequence.
In order to exclude the possibility of errors
(intrinsic to the polymerase chain reactions) or of
sequence misinterpretations (possibly due to the frequent
sequence compressions), we confirmed our results by
amplifying and sequencing several clones of each isolate
(data not shown). Moreover, in the case of patient 35, we
cloned and sequenced RT-PCR products derived from 2
additional sequential serum samples collected during the
one year follow-up of this patient (sequences 35/2 and
35/3 in Fig. 6). While this analysis confirmed the
presence of the upstream AUG codon in a11 the samples of
patient 35, it also established that this particular
isolate was stable over the time and that it was not
dominantly replaced by a population of mutants.,
Thus, our data provide the first evidence for the
existence of additional GBV-C sequences, potentially
leading to a core protein longer and not described in the
previously cDNA clones derived from the numerous viremic
patients analyzed so far (Leary et al., Simons et al.
1995, 1996, supra) .
Finally, our results establish that the putative
core proteins of our isolates are characterized by
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18
discrete regions of high variability, that are separated
by highly conserved motifs (Fig. 6).
Translation of isolates with distinct putative initiator
codons
5 The deduced translation products derived from the
nucleotide sequences of our isolates demonstrate that
these GBV-C sequences are very heterogeneous with respect
to the potential translation initiation site. Thus, in
order to understand the potential replicative competence
10 of these viral isolates, it was important to assess
whether initiation at any of these sites can indeed
occurr. This issue is particularly relevant since,
recently, Simons et al. (1996 supra) have provided
evidence suggesting that, in their GBV-C isolates,
15 initiation of translation occurred only immediately
upstream of the putative El signal sequence and that the
upstream in frame AUG seemed to be unable to direct the
synthesis of GBV-C polyprotein.
To determine the effect of the upstream in frame AUG
20 codons on translation of our viral isolates, mRNAs with
capped methylated (m'GpppG) 5' end, synthesized from
linearized pCRX/AC1S constructs, were examined for mRNA
activity in a cell-free translation system prepared from
rabbit reticulocytes. For this purpose we considered
25 isolates with distinct putative initiator codons.
Isolate 35 had 5 in-frame AUG codons in the putative
core region (Fig. 5 and 6) and therefore the pCR35/AC1S
construct (covering the partial 5'UTR, the putative core
and a portion of the E1 protein) can potentially mediate
30 the synthesis of different transcription products with
expected sizes of about 17, 14,11, 10 or 7 kDa. Figure 7
shows that efficient translation occurred and a
polypeptide with the apparent size of 17 kDa, compatible
with the activity of the first in-frame AUG, was observed
35 in a SDS-PAGE analysis. It is important to note that
efficient translation was also obtained with IVTT
CA 02251745 1998-10-14
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19
PCT/IT96/00248
reactions programmed with uncapped RNA derived from
pCR35/AC1S. Thus, these data strongly suggest that, at
least in this construct, the first available in-frame AUG
from the 5' end of the RNA can efficiently act as the
initiator codon. Furthermore, since the cap stmcture was
not determinant for efficient translation, the data also
suggest that, as recently proposed by Simons et al.
(Simons et al. 1996, supra), GBV-C RNA may not utilize
the classical ribosome scanning mechanism.
IO IVTT reaction mixtures were also programmed with
pCRDS39/AC1S, pCRDM/AC1S, pCRl9/AC1S, pCR3064/AC1S,
pCRMG/AC1S and pCR2095/AC1S constructs.
Isolates DS39, DM and 19 had the first in-frame AUG
corresponding to the second in-frame AUG of isolate 35
(Fig. 5 and 6). Moreover, isolate DS39 had also two
additional putative initiatior codons so that the
pCRDS39/AC1S construct could potentially direct the
synthesis of three possible proteins of 14, 10 and 7 kDa.
The pCRDM/ACIS construct, on the other hand, can
potentially initiate translation at 4 different AUG
codons, producing polypeptides of I4, II, 10 and 7 kDa.
Two of these plasmids, pCRDS39/AC1S and pCRl9/AC1S,
directed the translation of products of about 14 kDa as
detected by SDS-PAGE (Fig. 7). The size of these proteins
is compatible with the activity of the AUG sequence just
downstream of the start codon of the pCR35/AC1S
construct, suggesting that, also in these cases,
translation was initiated at the first available in-frame
AUG of the ORF. Since pCRl9/AC1S contained only one AUG
inframe with the long ORF, and since on the basis of the
nucleotide sequence we could also exclude the possibility
that the expressed proteins represented unrelated
products of translation directed by AUG codon located in
the other two frames, we conclude that the observed band
is the correct product of translation.
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
In contrast, no bands were detected in the reactions
programmed with the pCRDM/AC1S plasmid. Since this
construct contained the same potential in-frame initiator
AUG codon than pCR39/AC1S and pCRl9/AC1S, the results
5 rise the possibility that Sequences upstream of the
potential initiator codon may influence the translation
either positively or negatively. This possibility is also
reinforced by the fact that the plasmids derived from
isolates 3064 and MG (pCR3064/AC1S and pCRMG/AC1S), that
10 could potentially direct the synthesis of a band of 10
kDa, did not produce detectable quantities of specific
proteins, in accordance to the data of Simons et al.
(Simons et al. 1996 supra). Negative results were also
obtained with the pCR2095/AC1S construct, derived from
15 isolate 2095, that could potentially produce a protein of
about 7 kDa.
Analysis of deletion mutants
To examine in details the activity of the different
potential in-frame initiator AUG codons, to verify the
20 possibility of a regulatory role of the upstream
sequences and to unequivocally establish the size of the
obtained translation products, cell-free protein
synthesis experiments were performed with capped mRNA
transcribed by truncated forms of the 35, 19, 3064 and
2095 constructs described in Fig. 8. Figure 9A shows the
results obtained when IVTT reaction mixtures were
programmed with the 35 deletion mutant plasmids. In these
experiments, translation of the mRNA derived from
pCR35/AC1S yielded the major precursor band of 17 kDa as
well as 3 additional faint bands of 14, 11 and 7 kDa,
probably corresponding to translation products that
utilize the second, third and the fifth in-frame AUG as
initiator codons. A very similar pattern was obtained
with the pCR35/~1 construct. In vitro translation of
pCR35/02, pCR35/03, pCR35/~4 and pCR35/05 transcripts
CA 02251745 1998-10-14
WO 97/39129 PCT/IT96/00248
21
yielded bands of progressively lower molecular weights of
about 14, 11 and 7 kDa, implying therefore that, in this
isolate, a11 the in-frame AUG codons, with the exception
of the fourth AUG, might serve as potential site of
translation initiation. In the lane containing the
translation products of pCR35/02 mutant we found two
bands of 14 and 11 kDa corresponding to translation
products that utilized both the second and the third
available AUG initiation codons.
As expected, the analysis of the deletion mutants of
pCRl9/AC1S, that contains only one in-frame AUG, gave
different results (Fig. 9B). The 14 kDa bands obtained
with reactions programmed with pCRl9/AC1S, pCRl9/~1 and
pCRl9/~2 demonstrate that the first and unique in-frame
AUG is the site of translation initiation and that., at
least for this mutant, deletion of 5' UTR does not seem
to significantly influence the level of translation.
A quite unexpected finding of these experiments was
that the reactions programmed with some of the deletion
mutants of pCRl9/AC1S construct, that lacked the AUG
codon immediately upstream of the putative E1 signal
sequence, yielded a band with the same apparent molecular
mass of the 7 kDa protein detected in the reactions
programmed with pCR35/44 and pCR35/~5. Although we can
not exclude that this band may have resulted from
partially degraded RNA, it is interesting that in this
template the AUG triplet correspcnding to the fifth in-
frame start codon of isolate 35 is replaced by an ACG
codon, that has been found to act as initiation codon in
other mRNAs (Boeck, R., and D. Kolakofsky. l994. EMBO J.
13:3608-3617), including a transcript derived from a GBV-
C mutated sequence (Simons et al. 1996 supra).
Reactions programmed with the deletion mutants
derived from pCR3064/AC1S construct provided no
detectable bands (data not shown). Since in this
CA 02251745 1998-10-14
WO 97I39129 PCT/1T96/00248
22
constructs the only available initiation codon is the AUG
corrisponding to the fourth start codon of the isolate
35, these results confirmed the data obtained with the
mutants of isolate 35 and with the pCR3064/AC1S and
pCRMG/AC1S plasmids, suggesting that this particular
codon, as already reported (Simons et al. 1996 supra), is
silent, probably due to the composition of the flanking
sequences.
Finally, evidence for a regulatory role of the
partial 5'UTR were obtained with the deleted 2095
plasmids (Fig. 9C). In this isolate the only available
site of transcription initiation corresponds to the fifth
AUG of isolate 35, that is however silent in construct
pCR2095/AC1S. The expected 7 kDa product was surprisingly
detected as a faint band with pCR2095/01, pCR2095/02 and
pCR2095/~3 and the intensity of this band increased
progressively by programming the reactions with
pCR2095/04 and pCR2095/05. These data clearly mean that,
in this particular template, the translation of the
polypeptide is strongly influenced by the partial 5'UTR
present in the undeleted construct that, in this case,
seems to inhibit the translation process.
Finally, we must stress that we have verified that
none of the bands obtained with the deletion mutants can
be accounted for by unrelated products of translation
encoded by the other two possible reading frames.
o . ~ CA 02251745 1999-04-27
- 2 3 -~ ;
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: WABCO BV
(ii) TITLE OF INVENTION: Non-A non-E Hepatitis virus
having a
translatable core region, reagents and methods for their
use
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dennison Associates
(B) STREET: 133 Richmond Street West, Suite 301
(C) CITY: Toronto
(D) PROVINCE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE: M5H 2L7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30(EPO)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,251,745
(B) FILING DATE: 09-DEC-1996
(C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Dennison Associates
(B) REFERENCE/DOCKET NUMBER: JJ-10288CA
(viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416-368-8313
(B) TELEFAX: 416-368-1645
(2)
INFORMATION
FOR
SEQ
ID
NO:
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
Arg Val Arg Asp Val Ala Arg Gly Cys Gly Val Gln Leu Leu Tyr
Val
1 5 10 15
Ala Thr Ala Thr Pro Pro Gly Ser Pro Met Thr Gln His Ser Ile
Pro
20 25 30
Ile Glu Thr Lys Leu Asp Val Gly Glu I1e Pro Phe Tyr His Gly
Gly
CA 02251745 1998-10-14
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24
35 40 45
Ile Pro Leu Glu Arg Met Arg Thr Gly Arg His Leu Val Phe Cys His
50 55 60
Ser Lys Ala Glu Cys Glu Arg Leu Ala Gly Gln Phe Ser Ala Arg Gly
65 70 75 80
Val Asn Ala Ile Ala Tyr Tyr Arg Gly Lys Asp Ser Ser Ile Ile Lys
85 90 95
Asp Gly Asp Leu Val AlaThrAsp AlaLeu SerThrGly Tyr
Val Cys
100 105 110
Thr Gly Asn Phe Ser ThrAspCys GlyLeu ValValGlu Glu
Asp Val
115 120 125
Val Val Glu Val Leu ProThrIle ThrIle SerLeuArg Thr
Thr Asp
130 135 140
Val Pro Ala Ser Glu SerMetGln ArgArg GlyArgThr Gly
Ala Leu
145 150 155 160
Arg Gly Arg Ser Arg TyrTyrAla GlyVal GlyLysAla Pro
Gly Tyr
2 165 170 175
5
A1a Gly Val Val Ser ProValTrp SerAla ValGluAla Gly
Arg Gly
180 185 190
Val Thr Trp Tyr Met ProAspLeu ThrAla Asn
Gly Glu
195 200 205
(2) INFO RMATION FOR ID NO: :
SEQ 2
3 (i) SEQUENCE CHARACTERISTICS:
5
(A) LENGTH: 7 aminoacids
10
(B) TYPE: o acid
amin
(C) STRANDEDNESS: le
sing
(D) TOPOLOGY:linear
(ii) MOLECULE TYPE:peptide
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25
(xi)SEQUENCE DESCRIPTION: 2:
SEQ ID NO:
Met Ser Leu Asn Arg Ala TyrPro ProGlyLeu ThrThr ProThr
Arg
1 5 10 15
Tyr Gly Pro Arg Arg Pro MetSer LeuLeuThr AsnArg PheAsn
Ser
20 25 30
Arg Arg Val Asp Lys Asp TrpGly ProGlyVal MetGly LysAsp
Gln
35 40 45
Pro Lys Pro Cys Pro Ser ArgThr GlyLysCys MetGly ProPro
Arg
50 55 60
Ser Ser Ala Ala Ala Cys ArgGly SerProArg IleLeu ArgVal
Ser
65 70 75 80
Arg Ala Gly Gly Ile Ser ProTyr ThrIleMet GluAla LeuLeu
Leu
85 90 95
Phe Leu Leu Gly Val Glu GlyAla IleLeu
Ala
100 105
(2) INFORMATION
FOR SEQ
ID N0:
3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino
acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(xi)SEQUENCE DESCRIPTION: 3:
SEQ ID NO:
Met Ser Leu Leu Thr Asn PheAsn ArgArgVaI AspLys AspGln
Arg
1 5 10 15
Trp Gly Pro Gly Val Met LysAsp ProLysPro CysPro SerArg
Gly
20 25 30
Arg Thr Gly Lys Cys Met ProPro SerSerA1a AlaAla CysSer
Gly
CA 02251745 1998-10-14
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PCT/IT96/00248
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35 40 45
Arg Gly Ser Pro Arg Ile Leu Arg Val Arg Ala Gly Gly Ile Ser Leu
50 55 60
Pro Tyr Thr Ile Met Glu Ala Leu Leu Phe Leu Leu Gly Val Glu Ala
65 70 75 80
Gly A1a Ile Leu
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
1 5 (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Met Ser Leu Asn Arg Ala Arg Tyr Pro Pro Gly Leu Thr Thr Pro Thr
1 5 10 15
Tyr Gly Pro Arg Arg Pro Ser
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AGGGTTSGWW GGTSGTAAAT CC 22
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27
(2) INFORMATION FOR SEQ ID NO:
6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE; nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleicacid
(xi) SEQUENCE DESCRIPTION: SEQ NO: 6:
ID
CGCCTGRTAN ARNGGCCARC A 21
1 (2) INFORMATION FOR SEQ ID NO:
5 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B} TYPE: nucleic acid
2 (C) STRANDEDNESS; single
0
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleicacid
2 (xi) SEQUENCE DESCRIPTION: SEQ N0: 7:
5 ID
ACGTAAGCTT AGGTGTWGGY CCTACCG 27
(2) INFORMATION FOR SEQ ID NO:
8:
30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3 (D) TOPOLOGY: linear
5
(ii) MOLECULE TYPE: other nucleicacid
(xi) SEQUENCE DESCRIPTION: SEQ NO: 8:
ID
40
ACGTAAGCTT GTACGGYCCA CGTCGCC 27
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(2) INFORMATION FOR SEQ ID NO:
9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ N0: 9:
ID
ACGTAAGCTT GCGAGTTGRC AAGGACCAG 29
1 (2) INFORMATION FOR SEQ ID N0:
5 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
2 (C) STRANDEDNESS: single
0
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
2 (xi) SEQUENCE DESCRIPTION: SEQ NO: 10:
5 ID
ACGTAAGCTT GRRVCGGGAA ATGCATG 27
(2) INFORMATION FOR SEQ ID NO:
11:
30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
3 (D) TOPOLOGY: linear
5
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ NO: 11:
ID
40
ACGTAACCTT TGAGGGCGGG TGGCATT 27
