# US 12,522,879 B2 · Fish Totivirus

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- Number: US 12,522,879 B2
- Document ID: US12522879B2
- Status: Granted
- Kind: B2
- Application number: 17/433,416
- Filed: 2020-02-04
- Published: 2025-08-28
- Granted: 2026-01-13
- Earliest claimed: 2019-02-05
- Assignees: Pharmaq AS
- Applicants: Pharmaq AS
- Inventors: Stian Nylund; Liv Sandlund; Arnfinn L. Okland
- Web page: https://patentgrep.com/patent/US12522879B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- A61K 39/12: Viral antigens
- C07K 14/005: from viruses
- C07K 16/10: RNA viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/1131: {against viruses}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2720/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2720/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2720/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to a novel fish virus, indicated to be a Totivirus, which causes mortality in fish, and to methods of detecting said virus in fish, and related uses.

## Claims

### Claim 1

A nucleic acid, wherein the sequence of said nucleic acid comprises at least one open reading frame (ORF) sequence selected from the group consisting of ORF-1, ORF-2, ORF-X,
ORF-Y and ORF-Z; wherein
ORF-1 is at least 80% identical to the nucleic acid sequence of SEQ ID NO:1,
ORF-2 is at least 80% identical to the nucleic acid sequence of SEQ ID NO:2,
ORF-X is at least 80% identical to the nucleic acid sequence of SEQ ID NO:3,
ORF-Y is at least 80% identical to the nucleic acid sequence of SEQ ID NO:4, and
ORF-Z is at least 80% identical to the nucleic acid sequence of SEQ ID NO:5.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein
(a) the sequence of said nucleic acid comprises at least ORF-1 and ORF-2, as defined in claim 1; and/or
(b) the sequence of said nucleic acid is at least 80% identical to the virus genome according to SEQ ID NO:6.

### Claim 3

A nucleic acid, wherein
(a) the sequence of said nucleic acid is complementary to SEQ ID NO: 1 and/or to SEQ ID NO: 2; and/or
(b) the sequence of said nucleic acid is complementary to SEQ ID NO: 6.

### Claim 4 (depends on 1, 2, 3)

A virus that infects and is capable of killing lumpsucker fish (Cyclopterus lumpus), wherein the virus genome comprises the sequence of a nucleic acid of any one of claims 1 to 3, wherein said nucleic acid sequence contains the base uracil (U) instead of the base thymine (T).

### Claim 5 (depends on 1, 4)

The virus of claim 4, wherein said virus comprises ORF-1, ORF-2, ORF-X, ORF-Y and ORF-Z as defined in claim 1, and wherein said ORF-1, ORF-2, ORF-X, ORF-Y and ORF-Z encode viral polypeptides that are at least 80% identical to SEQ ID NOs 7-11, respectively.

### Claim 6 (depends on 5)

The virus of claim 5, wherein said ORF-1, ORF-2, ORF-X, ORF-Y and ORF-Z encode viral polypeptides that are at least 95% identical to SEQ ID NOs 7-11, respectively.

### Claim 7 (depends on 5, 6)

The virus of claim 5 or claim 6, wherein said ORF-1, ORF-2, ORF-X, ORF-Y and ORF-Z encode viral polypeptides that are conservatively substituted variants of SEQ ID NOs 7-11, respectively, or viral polypeptides comprising the amino acid sequences of SEQ ID NOs 7-11, respectively.

### Claim 8 (depends on 4, 5, 6, 7)

The virus of any one of claims 4-7, wherein the infection of lumpsucker fish by the virus causes the following symptoms in the fish:
(i) tissue damage in the intestine, and/or
(ii) diarrhoea; or
(iii) cardiomyopathy.

### Claim 9 (depends on 4, 5, 6, 7, 8)

The virus of any one of claims 4-8, wherein
(a) the virus is a non-enveloped virus; and/or
(b) the virus is a Totivirus.

### Claim 10

An oligonucleotide primer which
(a) comprises at least 18 consecutive nucleotides of a sequence which is, or which is complementary to, a portion of a reference nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO:6; or
(b) comprises at least 18 consecutive nucleotides of a sequence which is at least 95% identical to a sequence which is, or which is complementary to, a sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO:40;
with the proviso that said oligonucleotide primer does not comprise a sequence selected from the group consisting of SEQ ID NO:41 to SEQ ID NO:49.

### Claim 11 (depends on 4, 5, 6, 7, 8, 9)

Use of at least one oligonucleotide primer in a method of detecting the virus of any one of claims 4 to 9, wherein the at least one primer
(a) comprises at least 9 consecutive nucleotides of a sequence which is, or which is complementary to, a portion of a reference nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO:6; or
(b) comprises at least 9 consecutive nucleotides of a sequence which is at least 80% identical to a sequence which is, or which is complementary to, a sequence selected from the group consisting of SEQ ID NO:12 to SEQ ID NO:40;
with the proviso that said oligonucleotide primer does not comprise a sequence selected from the group consisting of SEQ ID NO:41 to SEQ ID NO:49.

### Claim 12 (depends on 4, 5, 6, 7, 8, 9)

A method for detecting a virus that infects and is capable of killing fish, comprising the steps of:
(a) contacting a nucleic acid extracted from a biological sample of a fish with at least one oligonucleotide primer to form a mixture, wherein the at least one oligonucleotide primer is complementary to a nucleic acid sequence which is comprised within the genome of the virus of any one of claims 4 to 9, and
(b) determining whether upon subjecting the mixture of a) to amplification an amplification product is present, wherein the presence of amplification product indicates the presence of RNA associated with the virus, and hence the presence of the virus in the biological sample, wherein the oligonucleotide primer
(i) comprises at least 9 consecutive nucleotides of a sequence which is, or which is complementary to, a portion of a reference nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO:6; or
(ii) comprises at least 9 consecutive nucleotides of a sequence which is at least 80% identical to a sequence which is, or which is complementary to, a sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO:40;
with the proviso that said oligonucleotide primer does not comprise a sequence selected from the group consisting of SEQ ID NO:41 to SEQ ID NO:49.

### Claim 13

A method for detecting a virus that infects and is capable of killing fish, comprising the steps of:
(a) sequencing a nucleic acid extracted from a biological sample of a fish, and
(b) comparing the resulting nucleic acid sequence with a nucleic acid sequence which is, or which is complementary to, a reference sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, wherein an at least 80% sequence identity between the two sequences indicates the presence of the virus in the biological sample.

### Claim 14 (depends on 4, 5, 6, 7, 8, 9)

An antibody that binds a polypeptide, wherein the polypeptide is encoded by a nucleic acid sequence which is comprised within the genome of the virus of any one of claims 4 to 9,
wherein the polypeptide is selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence which is at least 80% identical to SEQ ID NO:7;
(ii) a polypeptide comprising an amino acid sequence which is at least 80% identical to SEQ ID NO:8;
(iii) a polypeptide comprising an amino acid sequence which is at least 80% identical to SEQ ID NO:9;
(iv) a polypeptide comprising an amino acid sequence which is at least 80% identical to SEQ ID NO:10; and
(v) a polypeptide comprising an amino acid sequence which is at least 80% identical to SEQ ID NO:11.

### Claim 15 (depends on 10, 14)

A kit for detecting a virus in a biological sample from fish, wherein the kit comprises an oligonucleotide primer of claim 10 and/or an antibody of claim 14,
optionally wherein the kit is a real-time RT-PCR assay.

### Claim 16 (depends on 4, 5, 6, 7, 8, 9, 14)

The antibody of claim 14 for use in treating fish infected with a virus of any one of claims 4 to 9.

### Claim 17 (depends on 4, 5, 6, 7, 8, 9)

Use of the virus of any one of claims 4 to 9 for producing a vaccine.

### Claim 18

A viral polypeptide comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS 7-11.

### Claim 19 (depends on 18)

The viral polypeptide according to claim 18, comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS 7-11.

### Claim 20 (depends on 18)

The viral polypeptide according to claim 18, comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs 7-11.

### Claim 21 (depends on 18, 19, 20)

The viral polypeptide according to any one of claims 18-20, comprising an amino acid sequence comprising any one of SEQ ID NO: 7-11 or a conservatively substituted variant thereof.

### Claim 22 (depends on 18, 19, 20)

The viral polypeptide according to any one of claims 18-20, comprising an amino acid sequence comprising any one of SEQ ID NOS 7-11.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 18, 19, 20, 21, 22)

A vaccine comprising:
(i) a nucleic acid sequence as claimed in any one of claims 1 to 3;
(ii) a viral polypeptide according to any one of claims 18-22; or
(iii) a virus of any one of claims 4 to 9.

### Claim 24 (depends on 1, 2, 3, 23)

The vaccine of claim 23, wherein the sequence of the nucleic acid is the sequence of the nucleic acid claimed in any one of claims 1 to 3, wherein said nucleic acid sequence contains the base uracil (U) instead of the base thymine (T).

### Claim 25

An interfering RNA (iRNA) molecule for use in treating fish infected with a virus, wherein the IRNA molecule comprises at least 12 consecutive nucleotides of, or complimentary to, a nucleic acid sequence which is at least 80% identical to the sequence of the virus genome according to SEQ ID NO:6.