Phylogenomic Comparison of Seven African Swine Fever Genotype II Outbreak Viruses (1998-2019) Reveals the Likely African Origin of Georgia 2007/1

Abstract

Since the initial report of African swine fever (ASF) in Kenya in 1921, the disease has predominantly been confined to Africa. However, in 2007, an ASF genotype II virus of unknown provenance was introduced to Georgia. This was followed by its rampant spread to 73 countries, and the disease is now a global threat to pig production, with limited effective treatment and vaccine options. Here, we investigate the origin of Georgia 2007/1 through genome sequencing of three viruses from outbreaks that predated the genotype II introduction to the Caucasus, namely Madagascar (MAD/01/1998), Mozambique (MOZ/01/2005), and Mauritius (MAU/01/2007). In addition, genome sequences were generated for viruses from East African countries historically affected by genotype II (Malawi (MAL/04/2011) and Tanzania (TAN/01/2011)) and newly invaded southern African countries (Zimbabwe (ZIM/2015) and South Africa (RSA/08/2019). Phylogenomic analyses revealed that MOZ/01/2005, MAL/04/2011, ZIM/2015 and RSA/08/2019 share a recent common ancestor with Georgia 2007/1 and that none contain the large (~550 bp) deletion in the MGT110 4L ORF observed in the MAD/01/1998, MAU/01/2007 and TAN/01/2011 isolates. Furthermore, MOZ/01/2005 and Georgia 2007/1 only differ by a single synonymous SNP in the EP402R ORF, confirming that the closest link to Georgia 2007/1 is a virus that was circulating in Mozambique in 2005.

Keywords: African swine fever virus; complete genome sequencing; genotype II; phylogenetics; single nucleotide polymorphisms (SNPs).

Figure 1

Maximum likelihood phylogenetic tree using the complete genome sequences of genotype II ASFVs from seven SADC countries, Georgia, Russia and Nigeria. The complete genome sequences generated in this study are indicated with black dots, whilst the remaining sequences were obtained from GenBank. Bootstrap support values from 100 replications, ≥75 are shown next to the relevant nodes, with the five major clades recovered being indicated by Roman numerals (I–V).

Figure 2

Agarose gel indicating the size difference pertaining to the approximately 550 bp deletion in MAD/01/1998, MAU/01/2007 and TAN/01/2011 when compared to MOZ/01/2005, MAL/04/2011, RSA/08/2019 and ZIM/2015. A no-template negative control was included.

Figure 3

Sequence alignment of ORF EP402R from ASFVs isolated in Africa and Eurasia. The genome sequences generated in this study are denoted by a closed circle.

Figure 4

Sequence alignment of ORFs MGF-360-13L (A), A859L (B) and O61R (C). The sequences of MOZ/01/2005 and RSA/08/2019 cluster with Georgia 2007/1 and the selected Eurasian isolates, whilst the remainder of African isolates cluster together. The genome sequences generated in this study are denoted by a closed circle.

Figure 5

Sequence alignment of ORFs MGF-360-9L (A), G1211R (B) and I267L (C). The sequences of MOZ/01/2005, RSA/08/2019 and ZIM/2015 cluster with Georgia 2007/1 (A), whilst the sequences of MOZ/01, RSA/08, ZIM/2015 and MAL/04 cluster with Georgia 2007 and the selected Eurasian isolates (B,C). In contrast, the remainder of African isolates cluster together. The genome sequences generated in this study are denoted by a closed circle.

Figure 6

Sequence alignment of the IGR between ORFs I73R and I329L, with the 10 bp repeat regions indicated with blue arrows. The genome sequences generated in this study are denoted by a closed circleFifteen of the 51 non-synonymous SNPs observed in the sequences under study were non-conservative replacements. The results from this study showed that within the MGF100-3L, MGF110-3L and MGF110-5L-6L, ZIM/2015 had amino acid substitutions as aspartic acid (D) at position 85 replacing glycine (G) (D85G), G91D and leucine (L) at position 35 replacing histidine (H) (L35H), respectively, whereas in MGF360-13L, the substitution of G177D occurred in all the African isolates except for MOZ/01/2005 and RSA/08/2019, which were the same as Georgia 2007/1. Similarly, alanine (A) at position 427 replaced glutamic acid (E) (A427E) within the ORF A859L, occurring in the same manner as mentioned above. Other non-synonymous SNPS, where non-conservative replacement occurred, involved isolate MAL/04/2011, which is where Q115R, R39Q, aspartic acid (D) at position 35 replaced asparagine (N) (D35N) and R42G, occurring in MGF110-13LB, MGF505-9R, MGF360-2L and MGF360-19Rb, respectively. In RSA/08/2019, D151N in the ORF B169L, and E125V in E199L; and in ZIM/2015, L351D in the ORF QP509L and Q104H in E199L, non-conservative replacements occurred. TAN/01/2011 had an amino acid exchange of W109R in MGF 110-1L, the absence of serine in position 164 of MGF 110-5L-6L, threonine (T) at position 81 replaced by methionine (M) [T81M] in MGF110-8L, G177D in MGF 360-13L, T78M in C275L, N1002I in G1211R, M288T in D245L, D130N in E165R, and A83T in MGF505-11L.