# US 10,067,130 B2 · Infectious genomic DNA clone and serological profile of torque teno sus virus 1 and 2

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- Number: US 10,067,130 B2
- Document ID: US10067130B2
- Status: Granted
- Kind: B2
- Application number: 14/978,413
- Filed: 2015-12-22
- Published: 2016-09-15
- Granted: 2018-09-04
- Earliest claimed: 2009-08-21
- Assignees: Virginia Tech Intellectual Properties, Inc.
- Applicants: Virginia Tech Intellectual Properties, Inc.
- Inventors: Xiang-Jin Meng; Yaowei Huang
- Web page: https://patentgrep.com/patent/US10067130B2

## CPC

- G01N 33/56983: {Viruses}
- A61K 39/12: Viral antigens
- C07K 14/005: from 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 15/86: Viral vectors
- C12Q 1/701: {Specific hybridization probes}
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/552: {Veterinary vaccine}
- C12N 2750/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2750/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2750/00043: viral genome or elements thereof as genetic vector
- G01N 2333/01: DNA viruses
- G01N 2469/20: Detection of antibodies in sample from host which are directed against antigens from microorganisms
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- 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

## Abstract

The present invention also provides infectious DNA clones, biologically functional plasmid or viral vector containing the infectious nucleic acid genome molecule of Torque teno sus virus (TTsuV). The present invention also provides methods for diagnosing TTsuV infection via immunological methods, e.g., enzyme-linked immunoabsorbent assay (ELISA) and Western blot using PTTV specific antigens for detecting serum PTTV specific antibodies which indicate infections TTsuV1, TTsuV2, and individual TTsuV1 genotypes.

## Claims

### Claim 1

A method for diagnosing a Torque Teno Sus Virus (TTsuV) infection in a pig, or determining whether a pig has been exposed to one or more TTSuV genotypes or subtypes, comprising:
immobilizing an immunogenic fragment or a complete protein of a recombinantly expressed and purified polypeptide sequence of an ORF1 protein of one or more TTsuV genotypes or subtypes wherein the immunogenic fragment or complete polypeptide sequence comprises one or more of: amino acids 363 to 375 of a TTSuV2 ORF1 represented by SEQ ID NO: 16, and amino acids 388 to 423 of a TTSuV2 ORF1 represented by SEQ ID NO: 16;
contacting a serum sample from a pig suspected of TTsuV infection with the immobilized immunogenic fragment or complete protein; and
detecting captured antibody specific to the immunogenic fragment.

### Claim 2 (depends on 1)

The method of claim 1, wherein the TTsuV genotypes or subtypes include one or more of TTsuV1a and TTsuV1b in addition to the-TTsuV2 ORF1 immunogenic fragment or complete polypeptide sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polypeptide comprises N-terminal truncated ORF1 proteins of one or more of TTsuV genotypes or subtypes TTsuV1a and TTsuV1b in addition to the TTsuV2 ORF1 immunogenic fragment or complete polypeptide sequence.

### Claim 4 (depends on 2)

The method of claim 2, wherein the polypeptide sequence comprises amino acids 317-635 of the ORF1 protein of TTsuV1a as referenced by SEQ ID NO: 12.

### Claim 5 (depends on 2)

The method of claim 2, wherein the polypeptide sequence comprises amino acids 322-639 of the ORF1 protein of TTsuV1b as referenced by SEQ ID NO: 13.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polypeptide sequence comprises amino acids 310-625 of the ORF1 protein of TTsuV2 as referenced by SEQ ID NO: 16.

### Claim 7 (depends on 1)

The method of claim 1, wherein the detecting captured antibody is by Western blot.

### Claim 8 (depends on 1)

The method of claim 1, wherein the detecting captured antibody is by enzyme-linked immunosorbent assay (ELISA).

### Claim 9

A diagnostic reagent for use in the detection of Torque Teno Sus Virus (TTSuV) infection in a mammal, comprising ORF1 proteins or immunogenic fragments thereof of one or more TTsuV genotypes or subtypes wherein the immunogenic fragments or complete polypeptide sequences are recombinantly expressed and purified and comprise one or more of: amino acids 363 to 375 of a TTSuV2 ORF1 represented by SEQ ID NO: 16, and amino acids 388 to 423 of a TTSuV2 ORF1 represented by SEQ ID NO: 16.

### Claim 10 (depends on 9)

The diagnostic reagent of claim 9, wherein the one or more TTsuV genotypes or subtypes include one or more of TTsuV1a and TTsuV1b in addition to the TTSuV2 immunogenic fragments or complete polypeptide sequences.

### Claim 11

A method for simultaneously detecting TTSuV1 and TTSuV2 infection comprising:
extracting DNA from a sample suspected of TTSuV infection;
performing real-time polymerase chain reaction (RT-PCR) using a TTSuV1-specific primer pair according to SEQ ID NO:29 and SEQ ID NO:30 and a TTSuV2-specific primer pair according to SEQ ID NO:31 and SEQ ID NO:32 in the same real-time PCR reaction; and
determining a presence of TTSuV1 and/or TTSuV2 DNA based on amplified primer specific sequences.

### Claim 12

A method for simultaneously detecting TSSuV1 and TTSuV2 infection by duplex nested polymerase chain reaction (PCR) comprising:
isolating viral DNA from samples of pigs suspected of TTSuV infection;
performing a first round of PCR using one pair of primers Plab-mF according to SEQ ID NO:33 and Plab-mR according to SEQ ID NO:34;
performing a second round of PCR using a mixture of two pairs of primers, Pla-nF according to SEQ ID NO:35 and Pla-nR according to SEQ ID NO:36 for detection of TTSuV1a, and Plb-nF according to SEQ ID NO:37 and Plb-nR according to SEQ ID NO:38 for detection of TTSuV1b, and visualizing the PCR products.