# US 9,474,797 B1 · African swine fever virus georgia strain adapted to efficiently grow in the vero cell line

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- Number: US 9,474,797 B1
- Document ID: US9474797B1
- Status: Granted
- Kind: B1
- Application number: 14/309,548
- Filed: 2014-06-19
- Granted: 2016-10-25
- Earliest claimed: 2014-06-19
- Assignees: The United States of America as represented by the Secretary of Agriculture; University of Connecticut
- Applicants: The United States of America, as Represented by the Secretary of Agriculture; The University of Connecticut
- Inventors: Manuel V. Borca; Luis L. Rodriguez; Peter W. Krug; Vivian K. O'Donnell
- Web page: https://patentgrep.com/patent/US9474797B1

## CPC

- A61K 39/12: Viral antigens
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/525: {Virus}
- C12N 2710/12021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/12034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)

## Abstract

We have developed an ASFV Georgia strain adapted to grow in Vero cell line. The resulting virus, ASF-GVAV, efficiently grows in Vero cells although it still is able to significantly replicate in primary cell cultures of swine macrophages. ASF-GVAV virus was successfully used as parental virus to develop several recombinant ASF viruses. The development of an ASFV adapted to grow in an established cell line is a significant advance for research and development of vaccine candidate strains using genetic manipulation based in the process of homologous recombination. The GVAVS can be utilized as a basis for large scale production of ASF vaccines.

## Claims

### Claim 1

An African Swine Fever-Georgia Virus (ASF-GV) encoded by a mutant DNA molecule having the nucleotide sequence of SEQ ID NO: 2 except for a deletion between nucleotide positions 178,643 and 182,578 producing the deletion of the following ORFs: MGF 505-11L, MGF 100-1L, I7L, I8L, ASFV G ACD 01870, I9R, I10L, and I11L and one or more of the following changes: (i) nucleotide insertions (T and A) in the non-coding region at position 434 and 441, respectively; (ii) a double nucleotide (TT) deletion and a nucleotide insertion (T) at positions 1602 and 1620, respectively; (iii) an amino acid substitution (Cys131Tyr in ORF EP424R) in nucleotide position 71002; (iv) a synonymous (A to G) substitution at nucleotide position 97391 inside ORF B438L; (v) an amino acid substitution (Gly257Ser in ORF CP530R) in position 126174; (vi) an amino acid substitution (Gly127Glu in ORF E199L) in position 166065; and a nucleotide deletion (T), a nucleotide substitution (T to C) and a nucleotide insertion (T) in a non coding region at nucleotide positions 182582, 182 591 and 183303, respectively.

### Claim 2

An African Swine Fever-Georgia Vero Adapted Virus (ASF-GVAV) encoded by a mutant cDNA molecule having the nucleotide sequence of SEQ ID NO:1.

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

A method of developing recombinant ASF viruses for possible ASF vaccine candidate strains comprising:
a) producing a genetically manipulated African Swine Fever recombinant virus by combining, in a cell culture, African Swine Fever-Georgia Virus (ASF-GV) of claim 1 or 2 and plasmids having ASFV DNA incorporating a genetically manipulated targeted change, wherein co-culture of said ASF-GV template virus and said plasmids results in homologous recombination events;
b) introducing a reporter gene as part of said recombination events;
c) purifying said genetically manipulated African Swine Fever recombinant virus on the basis of said reporter gene;
d) expanding said genetically manipulated African Swine Fever recombinant virus; and
e) evaluating said genetically manipulated African Swine Fever recombinant virus as a vaccine candidate strain.

### Claim 4

An isolated or recombinant mutant DNA molecule encoding a African Swine Fever virus, said molecule having the nucleotide sequence of SEQ ID NO: 2 except for a deletion between nucleotide positions 178,643 and 182,578 producing the deletion of the following ORFs: MGF 505-11L, MGF 100-1L, I7L, I8L, ASFV G ACD 01870, I9R, I10L, and I11L and one or more of the following changes: (i) nucleotide insertions (T and A) in the non-coding region at position 434 and 441, respectively; (ii) a double nucleotide (TT) deletion and a nucleotide insertion (T) at positions 1602 and 1620, respectively; (iii) an amino acid substitution (Cys131Tyr in ORF EP424R) in nucleotide position 71002; (iv) a synonymous (A to G) substitution at nucleotide position 97391 inside ORF B438L; (v) an amino acid substitution (Gly257Ser in ORF CP530R) in position 126174; (vi) an amino acid substitution (Gly127Glu in ORF E199L) in position 166065; and a nucleotide deletion (T), a nucleotide substitution (T to C) and a nucleotide insertion (T) in a non coding region at nucleotide positions 182582, 182 591 and 183303, respectively.

### Claim 5

The isolated or recombinant mutant DNA molecule encoding an African Swine Fever virus adapted to growth in Vero cells, said molecule having the nucleic acid sequence is of SEQ ID NO: 1.

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

An expression vector comprising the DNA molecule of any one of claims 4 and 5.

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

A host cell containing the cDNA molecule of any one of claims 4 and 5.