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Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system.

ABSTRACT: A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus V?MGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus V?MGF?GUS. The EP402R and EP153R genes were subsequently deleted from the genome of V?MGF?GUS, using the same GUS marker gene, to construct virus V?MGF?CD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.

SUBMITTER: Abrams CC

PROVIDER: S-EPMC3526793 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system.

Abrams Charles C CC Dixon Linda K LK

Virology 20120815 1

A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus VΔMGF-GUS. Subsequently the GUS gene was removed ...[more]

PMID: 22902236

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Project description:African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.

Dataset Information

Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system.

Publications

Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system.

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