Abstract
African swine fever virus (ASFV) is the causative agent of African swine fever (ASF) which reaches up to 100% case fatality in domestic pigs and wild boar and causes significant economic losses in the swine industry. Lack of knowledge of the function of ASFV genes is a serious impediment to the development of the safe and effective vaccine. Herein, I267L was identified as a relative conserved gene and an early expressed gene. A recombinant virus (SY18ΔI267L) with I267L gene deletion was produced by replacing I267L of the virulent ASFV SY18 with enhanced green fluorescent protein (EGFP) cassette. The replication kinetics of SY18ΔI267L is similar to that of the parental isolate in vitro. Moreover, the doses of 102.0 TCID50 (n = 5) and 105.0 TCID50 (n = 5) SY18ΔI267L caused virulent phenotype, severe clinical signs, viremia, high viral load, and mortality in domestic pigs inoculated intramuscularly as the virulent parental virus strain. Therefore, the deletion of I267L does not affect the replication or the virulence of ASFV. Utilizing the fluorescent-tagged virulence deletant can be easy to gain a visual result in related research such as the inactivation effect of some drugs, disinfectants, extracts, etc. on ASFV.
Highlights
Introduction published maps and institutional affilAfrican swine fever (ASF) is a highly contagious and severe viral disease that infects domestic and wild pigs, which has caused devastating economic losses to the swine industry worldwide
We studied the function of I267L gene by constructing a recombinant African swine fever virus (ASFV) (SY18∆I267L) deleting I267L gene and evaluated the virulence of SY18∆I267L on domestic pigs inoculated intramuscularly with 102.0 Tissue Culture Infectious Dose 50 (TCID50) and 105.0 TCID50
After 72 hpi, the Bone Marrow-Derived Macrophages (BMDMs) infected with SY18∆I267L and ASFV SY18 were stained by FITC-labeled p30 monoclonal antibody prepared by our lab
Summary
The virulent ASFV SY18 was isolated from the spleen of a domestic pig infected with ASFV in July 2018 in China. Bone Marrow-Derived Macrophages (BMDMs) were prepared from 2 to 3-month-old Landrace piglets. The ribs and leg bones were used to separate BMDMs and the red blood cells were lysed with Red Blood Cell Lysis Buffer (BOSTER, Pleasanton, CA, USA). The. BMDMs were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA) and 10 ng·mL−1 granulocyte-macrophage colonystimulating factor (GM-CSF) (E. coli-derived porcine GM-CSF protein prepared by our lab) for 7–10 days to stimulate the differentiation of BMDMs and make them infectious
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