African swine fever virus (ASFV) is the etiological agent of a contagious and fatal disease of domestic pigs that has significant economic consequences for the global swine industry. Due to the lack of effective treatment and vaccines against African swine fever, there is an urgent need to leverage cutting-edge technologies and cost-effective approaches for generating and purifying recombinant virus to fast-track the development of live-attenuated ASFV vaccines. Here, we describe the use of the CRISPR/Cas9 gene editing and a cost-effective cloning system to produce recombinant ASFVs. Combining these approaches, we developed a recombinant virus lacking the non-essential gene A238L (5EL) in the highly virulent genotype IX ASFV (ASFV-Kenya-IX-1033) genome in less than 2 months as opposed to the standard homologous recombination with conventional purification techniques which takes up to 6 months on average. Our approach could therefore be a method of choice for less resourced laboratories in developing nations.

Infection of pigs with the African swine fever virus (ASFV) leads to a devastating hemorrhagic disease with a high mortality of up to 100%. In this study, a CD2v gene deletion was introduced to a genotype IX virus from East Africa, ASFV-Kenya-IX-1033 (ASFV-Kenya-IX-1033-∆CD2v), to investigate whether this deletion led to reduced virulence in domestic pigs and to see if inoculation with this LA-ASFV could induce protective immunity against parental virus challenge. All pigs inoculated with ASFV-Kenya-IX-1033-ΔCD2v survived inoculation but presented with fever, reduced appetite and lethargy. ASFV genomic copies were detected in only one animal at one time point. Seven out of eight animals survived subsequent challenge with the pathogenic parental strain (87.5%) but had mild to moderate clinical symptoms and had a gross pathology compatible with chronic ASFV infection. All mock-immunised animals developed acute ASF upon challenge with ASFV-Kenya-IX-1033 and were euthanised upon meeting the humane endpoint criteria. ASFV genome copy numbers after challenge were similar in the two groups. ASFV-Kenya-IX-1033-∆CD2v is therefore a useful tool to investigate the development of immunity to ASFV genotype IX, but safety concerns preclude its use as a candidate vaccine without further attenuation.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), resulting in up to 100% mortality in pigs. Although endemic in most sub-Saharan African countries, where all known ASFV genotypes have been reported, the disease has caused pandemics of significant economic impact in Eurasia, and no vaccines or therapeutics are available to date. In endeavors to develop live-attenuated vaccines against ASF, deletions of several of the ~170 ASFV genes have shown contrasting results depending on the genotype of the investigated ASFV. Here, we report the in vivo outcome of a single deletion of the A238L (5EL) gene and double deletions of A238L (5EL) and EP402R (CD2v) genes from the genome of a highly virulent genotype IX ASFV isolate. Domestic pigs were intramuscularly inoculated with (i) ASFV-Ke-ΔA238L to assess the safety of A238L deletion and (ii) ASFV-Ke-ΔEP402RΔA238L to investigate protection against challenge with the virulent wildtype ASFV-Ke virus. While A238L (5EL) gene deletion did not yield complete attenuation, co-deletion of A238L (5EL) and EP402R (CD2v) improved the safety profile of the single deletions, eliciting both humoral and cellular immune responses and conferred partial protection against challenge with the virulent wildtype ASFV-Ke virus.

We describe the characterization of an African swine fever genotype IX virus (ASFV-Kenya-IX-1033), which was isolated from a domestic pig in western Kenya during a reported outbreak. This includes the efficiency of virus replication and in vivo virulence, together with genome stability and virulence, following passage in blood macrophages and in a wild boar lung cell line (WSL). The ASFV-Kenya-IX-1033 stock retained its ability to replicate in primary macrophages and retained virulence in vivo, following more than 20 passages in a WSL. At the whole genome level, a few single-nucleotide differences were observed between the macrophage and WSL-propagated viruses. Thus, we propose that the WSL is suitable for the production of live-attenuated ASFV vaccine candidates based on the modification of this wild-type isolate. The genome sequences for ASFV-Kenya-IX-1033 propagated in macrophages and in WSL cells were submitted to GenBank, and a challenge model based on the isolate was developed. This will aid the development of vaccines against the genotype IX ASFV circulating in eastern and central Africa.

In this study, we describe an African swine fever genotype IX virus (ASFV-Kenya-1033-IX), which was isolated from a domestic pig in Western Kenya during a reported outbreak, including efficiency of virus replication, in vivo virulence, and genome stability in pulmonary alveolar macrophages (PAM) and in a wild boar cell line (WSL). The ASFV-Kenya-1033-IX stock, which underwent multiple passages in WSL (more than 20), retained its ability to replicate in primary macrophages and it also retained the virulence in vivo. At the genomic level, only a few single nucleotide differences were observed between the macrophage and WSL-grown virus. Thus, we propose that the WSL cell line is suitable to produce live attenuated ASFV vaccine candidates based on this isolate and probably of similar viruses. The genome sequences for ASFV-Kenya-1033-IX grown in macrophages and in WSL cells was submitted to GenBank and a challenge model based on this isolate was set up, which will aid the development of vaccines against genotype IX ASFV circulating in Eastern and Central Africa.