# US 2008/0187933 A1 · Hdv nucleic acid molecules, fragments and applications thereof

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- Number: US 2008/0187933 A1
- Document ID: US20080187933A1
- Status: Published application
- Kind: A1
- Application number: 12/024,243
- Filed: 2008-02-01
- Published: 2008-08-07
- Earliest claimed: 2001-09-24
- Assignees: Assistance Publique-Hopitaux de Paris
- Inventors: Paul DENY; Nadjia RADJEF; Patricia HUC-ANAIS
- Web page: https://patentgrep.com/patent/US20080187933A1

## CPC

- C07K 14/005: from viruses
- C12Q 1/706: {for hepatitis}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C12N 2760/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- Y10S 530/806: Antigenic peptides or proteins
- Y10S 530/82: Proteins from microorganisms
- Y10S 530/826: Viruses
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention concerns nucleic acid molecules derived from novel hepatitis D virus strains or isolates constituting genotypes different from known I, II and III genotypes, their fragments, corresponding proteins and their uses as diagnostic reagents. The invention also concerns a method for sensitive diagnosis of the hepatitis D virus (or delta hepatitis virus) and a method for epidemiologic monitoring of HDV-related infections.

## Claims

### Claim at position 1

1-2. (canceled)

### Claim 3

A nucleic acid molecule, comprising at least one fragment of a variant HDV selected from the group consisting of the genome of an HDV, which in molecular terms exhibits, over its entire genome, a genetic divergence or distance ≧20% (less than 80% similarity) with respect to the sequences of an HDV genotype I, of an HDV genotype II or of an HDV genotype III,
the genome of an HDV, which in molecular terms exhibits a genetic divergence or distance≧25% (less then 75% similarity), over a region referred to as R0, delimited by positions 889 to 1289 of the HDV genome, with respect to the corresponding R0 sequences of an HDV genotype I, of an HDV genotype II or of an HDV genotype III,
the complete genomes of the HDV isolates or variants referred to as dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644, which exhibit, respectively, the sequences SEQ ID NOS: 1, 6, 11, 16, 21 and 26, and
the genome of an HDV which exhibits a genetic divergence or distance≦15% with at least one of the sequences SEQ ID NOS: 1, 6, 11, 16, 21 and 26,
wherein the at least one fragment is selected from the group consisting of:
a) the R0 fragments of the following isolated variant HDVs: dFr45, dFr47, dFr48, dFr69, dFr73, dFr644, dFr910, dFr1843, dFr1953, dFr2020 and dFr2066 which exhibit, respectively, the following sequences: SEQ ID NO: 48 to SEQ ID NO: 58,
b) the R1 fragment which extends from position 307 to position 1289 of the HDV genome,
c) the R2 fragment which extends from position 889 to position 328 of the HDV genome,
d) the R3 fragment which extends from position 1486 to position 452 of the HDV genome,
e) the R′1 fragment which extends from position 305 to position 1161 of the HDV genome,
f) the R′2 fragment which extends from position 984 to position 331 of the HDV genome,
g) the R644 fragment which extends from position 889 to position 446 of the HDV genome,
h) the G910 fragment which extends from position 1206 to position 929 of the HDV genome,
i) the p910 fragment which extends from position 553 to position 1550 of the HDV genome,
j) the cDNAs encoding the sHD protein, of sequences SEQ ID NOS: 4, 9, 14, 19, 24 and 29,
k) the cDNAs encoding the LHD protein, of sequences SEQ ID NOS: 2, 7, 12, 17, 22 and 27, and
l) the primers of sequence SEQ ID NO: 33 to SEQ ID NO: 47.

### Claim 4 (depends on 3)

A method for detection of a variant HDV as defined in claim 3, by hybridization and/or amplification, carried out from a biological sample, which method comprises:
(1) extracting the nucleic acid to be detected, belonging to the genome of the virus possibly present in the biological sample,
(2) carrying out at least one gene amplification using a pair of primers selected from the group consisting of the primers capable of amplifying one of the following regions of the HDV genomic RNA: R0, R1, R2, R3, R644, G910, p910, R′1 and R′2, and
(3) analyzing the amplified product by comparison with one of the molecules of sequences SEQ ID NOS: 1, 6, 11, 16, 21 and 26, corresponding respectively to the complete genomes of the isolates or variants referred to as dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644.

### Claim 5 (depends on 4)

The method of detection as claimed in claim 4, wherein the analyzing in (3) is carried out by restriction, sequencing or hybridization.

### Claim 6 (depends on 4)

The method as claimed in claim 4, wherein the specific primers for amplifying the regions R0, R1, R2, R3, R644, G910, p910, R′1 and R′2 used in step (2), are selected from the group consisting of:
the primers 900S (SEQ ID NO:33) and 1280AS (SEQ ID NO:34), for the amplification of R0 (approximately 400 pb),
the primers 320S (SEQ ID NO:39) and 1280AS (SEQ ID NO:34), for the amplification of the R1 fragment (approximately 960 pb),
the primers 900S (SEQ ID NO:33) and 320AS (SEQ ID NO:45), for the amplification of R2 (approximately 1100 pb), which contains the sHD gene corresponding to positions 1598-950,
the primers 1480S (SEQ ID NO:46) and 440AS (SEQ ID NO:47), for the amplification of R3 (approximately 650 pb),
the primers 900S (SEQ ID NO:33) and 420AS (SEQ ID NO:40), for the amplification of the region R644 (approximately 1250 pb) of the isolate dFr644,
the primers 318S (SEQ ID NO:35) and 1150AS (SEQ ID NO:36), for the amplification of R′1 (approximately 850 pb),
the primers 906S (SEQ ID NO:37) and 345AS (SEQ ID NO:38), for the amplification of R′2 (approximately 1050 pb),
the primers R910S (SEQ ID NO:41) and R910AS (SEQ ID NO:42), for the amplification of the region G910 (approximately 1400 pb) of the isolate dFr910,
the primers S1910R (SEQ ID NO:43) and AS1910R (SEQ ID NO:44), for the amplification of the region p910 (approximately 650 pb) of the isolate dFr910.

### Claim 7

A method for detection and for genotyping of HDV from a biological sample, which method comprises:
(a) extracting the nucleic acid belonging to the genome of the HDV virus,
(b) amplifying the region R0 delimited by position 889 to position 1289 of the HDV genome,
(c) a first treatment of the amplified nucleic acid molecules with the SmaI and XhoI restriction enzymes, so as to produce a first set of restriction fragments,
(d) a second treatment of nucleic acid molecules with the SacII restriction enzyme, so as to produce a second set of restriction fragments,
(e) the combined analysis of the two sets of restriction fragments produced by RFLP (Restriction Fragment Length Polymorphism), so as to detect the presence and/or to determine the type of HDV present in said biological sample.

### Claim 8 (depends on 7)

The method as claimed in claim 7, wherein (b) comprises amplifying the nucleic acid molecules of said sample by RT-PCR with the primers 900S (SEQ ID NO:33) and 1280AS (SEQ ID NO:34).

### Claim 9 (depends on 7)

The method as claimed in claim 7 which comprises:
(f) amplification of the nucleic acid molecules of said sample by RT-PCR with the primers 900S (SEQ ID NO:33) and 320AS (SEQ ID NO:45), so as to amplify the R2 region, and
(g) direct sequencing of the amplified R2 region and comparison with one of the RNA molecules of sequences SEQ ID NOS: 1, 6, 11, 16, 21 and 26, corresponding respectively to the complete genomes of the isolates or variants as dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644.

### Claim 10 (depends on 3)

A recombinant vector comprising a nucleic acid molecule as claimed in claim 3.

### Claim 11 (depends on 3)

A cell transformed with a nucleic acid molecule as claimed in claim 3.

### Claim 12

A translation product encoded by one of the RNA molecules of sequences SEQ ID NOS: 1, 6, 11, 16, 21 and 26 corresponding respectively to the complete genomic RNAs of the isolates or variants referred to, respectively, as dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644, or by the sense or antisense sequences complementary thereto.

### Claim 13 (depends on 3)

A protein encoded by one of the nucleic acid molecules as claimed in claim 3.

### Claim 14 (depends on 13)

The protein as claimed in claim 13, selected from the group consisting of:
the LHD protein of dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644 which exhibit, respectively, the sequences SEQ ID NOS: 3, 8, 13, 18, 23 and 28, and
the sHD protein of dFr45, dFr47, dFr73, dFr910, dFr48 and dFr644 which exhibit, respectively, the sequences SEQ ID NOS: 5, 10, 15, 20, 25 and 30.

### Claim 15 (depends on 13)

A peptide which comprises a fragment of a protein as claimed in claim 13 selected from the group consisting of:
peptide A consisting of the 19 amino acids of the carboxy-terminal end of the sequences SEQ ID NOS:3, 8, 13, 18, 23 and 28,
peptide B of sequence RLPLLECTPQ (SEQ ID NO:59) consisting of the 10 amino acids of the carboxy-terminal end of the sequences SEQ ID NOS:3, 8, 13, 18, 23 and 28, and
peptide C consisting of the 9 amino acids preceding the sequence SEQ ID NO:59 (SEQ ID NO:60 to SEQ ID NO:65).

### Claim 16

(canceled)

### Claim 17 (depends on 12)

A kit comprising at least one protein as claimed in claim 12 and at least one reagent.

### Claim 18 (depends on 3)

A kit comprising at least one nucleic acid molecule as claimed in claim 3 and at least one reagent.