The African Swine Fever Isolate ASFV-Kenya-1033-IX is highly virulent and stable after growth in the wild boar cell line WSL

Abstract

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.

Cell Lines for the Development of African Swine Fever Virus Vaccine Candidates: An Update

“…Nevertheless, WSL did not show efficient production of E70 or Armenia/07 [ 18 , 115 ]. More recently, two studies reported that WSL cells supported the growth of the virulent ASFV-Kenya1033-IX strain and of its derived mutant ASFV-KeΔA238L strain (generated by deletion of the gene A238L) [ 118 , 119 ]. ASFV-Kenya1033-IX reached levels in WSL similar to those obtained in PAMs when using an MOI of 1 [ 118 ].…”

“…At the genomic level, Hemmil and colleagues observed only a few single nucleotide differences between ASFV-Kenya1033-IX grown in macrophage or passaged in WSL [ 118 ], and Abkallo and colleagues confirmed the stability of the virus in this cell line; no SNPs, deletions, or rearrangement were observed in ASFV-Kenya1033-IX and its derived mutant ASFV-KeΔA238L replicating in WSL [ 119 ]. In addition, both NH/P68 and ASFV-Kenya1033-IX passaged several times in WSL were still able to infect and replicate in both primary macrophages and pigs; all of these studies are paving the way for the development of an ASFV-LAV in the future [ 18 , 118 ].…”

“…Overall, these studies suggested that WSL cells are suitable for the production of attenuated and highly virulent ASFV virus strains [ 18 , 118 ] and as an alternative method to study ASFV infection biology; nevertheless, they cannot be used for titration because the cytopathic effects are slight or unnoticeable [ 20 , 30 ]. This cell line can be a cell model for the development of antiviral protection using the CRSPR-Cas9 method [ 121 ].…”

“… - PK9, and PK0809 no susceptible to Benin 97/1, Virulent Uganda [ 46 ]. - PK15 no able to support the growth of D/ASF/POT/Vietnam/2019, D/ASF/POB/Vietnam/2019 [ 95 ] - Spencer, Portuguese, Gasson adapted to PK cells → genomic mutation during adaptation with reduction of virulence in pigs but only partial protection against challenge with homologous strain [ 110 ] - Hinde isolates passaged 75 times in PK2a → reduction of virulence in pigs but only partial protection to challenge to parental strain; survived pigs only partial protection to other ASFV isolates [ 65 ] WSL Wild boar (fetus) [ 20 , 30 , 116 ] Lung [ 20 , 30 , 116 ] Spontaneously immortalized cell line [ 130 ] - CC83, Malawi 82, Uganda, Lisbon 57, E70 [ 30 ] -ASFV-Kenya1033-IXL [ 118 ] - NH/P68 [ 18 , 115 ] - ΔEP153R, Hinde attenuated, Uganda attenuated NH/P68, BA71V [ 30 ] - NHV-dTK-EGFP [ 116 ] - ASFV-KeΔA238L strain (generated by deletion of the gene A238 by ASFV-Kenya1033- IXL) [ 119 ] - OUTR88/3 [ 102 ] - ASFV-Kenya1033-IXL and ASFV-KeΔA238L → genomic stability during passages in WSL [ 118 , 119 ] - NH/P68 ASFV strain produced in WSL cells (10 passages) → in vivo in pigs induced a similar infection pattern to that of NH/P68 passaged in PAM [ 18 , 115 ] - ASFV-Kenya-1033-IX produced in WSL cells (more than 20 passages) → retained the virulence in vivo [ 118 ] ...…”