The C962R ORF of African Swine Fever Strain Georgia Is Non-Essential and Not Required for Virulence in Swine

Abstract

African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.

Evaluation in Swine of a Recombinant Georgia 2010 African Swine Fever Virus Lacking the I8L Gene

“…There is no information regarding the possible biological function(s) of the I8L gene, including virus replication. Mounting evidence suggests an increasing number of virus genes are not essential for ASFV replication [ 4 , 5 , 7 , 8 , 9 , 10 , 11 , 17 , 27 ]. It is possible that I8L gene function is duplicated by a different ASFV gene that is yet to be elucidated.…”

“…Therefore, identifying viral proteins that are important for in vitro and in vivo virus replication, and importantly in virus virulence in swine, is essential for the development of novel countermeasures to control the disease. Just a small number of genes have been successfully deleted from the ASFV genome, producing novel recombinant viruses (e.g., 9GL, UK, TK, MGF, NL, CD2, Lectin, DP148R, L83L, I177L, C962R, X69R) [ 3 , 4 , 5 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ] with a subset of deleted genes determined to be essential for virus replication (e.g. : EP152R, p30, p54, and p72) [ 17 , 18 , 19 , 20 ].…”

X69R Is a Non-Essential Gene That, When Deleted from African Swine Fever, Does Not Affect Virulence in Swine

“…Discovery of ASFV gene function via genetic manipulation has enabled the production of experimental live-attenuated ASFV vaccine candidates by different research groups [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 25 ]. Interestingly, just a small number of virus genes have been successfully deleted from the ASFV genome, producing a novel recombinant virus (e.g., 9GL, UK, TK, MGF, NL, CD2, Lectin, DP148R, I177L, and C962R) [ 4 , 5 , 6 , 7 , 8 , 18 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]), and another small number of genes were determined to be essential for virus replication (e.g., EP152R, p30, p54, and p72) [ 27 , 33 , 34 , 35 ]. The absence of experimental information restricts the knowledge for most ASFV proteins to ORF analysis by functional genomics, predicting the functions of these ORFs.…”

In-yeast reconstruction of the African swine fever virus genome isolated from clinical samples

“…This section describes the necessary steps for the engineering of the ASFV genome using the previously isolated sub-genomic fragments. Here, we substituted one non-essential gene in Fragment 4 (namely the C962R gene; Figure 5 , in dark gray) ( Ramirez-Medina et al., 2020 ) and replaced it by chemically-synthetized DNA cassettes carrying reporter genes as a proof of concept. Design of the chemically-synthetized DNA cassettes carrying the reporter genes Sma I restrictions sites (indicated with asterisks in Figure 5 ): Two Sma I restrictions sites were added at the 5′ and 3′ ends of the DNA cassettes to ensure linearization and purification from the pUC57 backbone plasmid.…”