A Retrospective Analysis Reveals That the 2021 Outbreaks of African Swine Fever Virus in Ghana Were Caused by Two Distinct Genotypes

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly infectious and lethal disease of domesticated swine. Outbreaks of ASF have been mostly restricted to the continent of Africa. The outbreaks that have occurred outside of Africa were controlled by extensive depopulation of the domesticated pig population. However, in 2007, an outbreak occurred in the country of Georgia, where ASFV infected wild pigs and quickly spread across eastern Europe. Since the reintroduction of ASF into Europe, variants of the current pandemic strain, ASFV Georgia 2007/01 (ASFV-G), which is classified as Genotype 2 based on p72 sequencing, have been reported in countries within western Europe, Asia, and the island of Hispaniola. Additionally, isolates collected in 2020 confirmed the presence of variants of ASFV-G in Nigeria. Recently, we reported similar variants of ASFV-G collected from domestic pigs suspected of dying of ASF in Ghana in 2022. Here, we retroactively report, based on full-length sequencing, that similar variants were present in Ghana in 2021. The SNP analysis revealed derivatives of ASFV with distinct genetic markers. Furthermore, we identified three full-length ASFV genomes as Genotype 1, indicating that there were two genotypes circulating in proximity during the 2021 ASF outbreaks in Ghana.


Introduction
African swine fever (ASF) is a highly contagious and fatal viral disease of domestic and wild pigs.Its first reported occurrence was in Kenya in 1921, and since then, it has Viruses 2024, 16 caused numerous outbreaks across different regions of Africa [1].Although sporadic outbreaks have been reported outside of Africa, they have been contained and controlled via mass culling and by restricting the transport of susceptible animals and animal products.However, in 2007, an outbreak in the Republic of Georgia quickly spread to neighboring countries and after an introduction into China in 2018, which resulted in the rapid spread of ASF across all of southeastern Asia [2].Concurrently, the disease continued to spread in both eastern and central Europe and has been causing continued outbreaks as far west as Germany, where recently, ASF outbreaks occurred not only in wild boar but also in pig farms.In 2021, the first outbreak in the western hemisphere occurred in the Dominican Republic, and outbreaks have continued since on the island of Hispaniola; this was the first time the island of Hispaniola had ASF outbreaks since the late 1970s which was only resolved by the culling of all swine on the island [1,3,4].
The etiological agent of the disease, African swine fever virus (ASFV), is an icosahedral, enveloped virus belonging to the Asfivirus genus of the Asfarviridae family.The virus's genome is composed of double-stranded DNA that varies in size from 170 to 192 kilobases and encodes between 150 and 200 open reading frames (ORFs).The functions of the majority of genes have not been experimentally verified; rather, some of the gene functions have been predicated based on sequence and structure homology [5,6].The diversity observed in ASFV is largely attributed to the gain, loss, truncation, elongation, and fusion of proteins classified within one of the five multigene families (MGF): MGF-100, MGF-110, MGF-300, MGF-360, and MGF-505 [7][8][9][10].Moreover, structural prediction suggests that MGF genes within a family can have unique functions [6,11].It is noteworthy that virulent and attenuated [12] isolates harboring deletions of MGF genes have been sequenced, emphasizing the complex involvement of these genes in ASFV virulence [13][14][15].
Historically, due to sequencing limitations, ASFV was classified into 25 genotypes based on the partial sequencing of B646L, which encodes for the major capsid protein p72 [16,17].Yet, the number of differences that defined a genotype was never established and led to establishment of genotypes at will.Accordingly, ASFV was recently reclassified into five distinct genotypes based on a maximum allowance of two amino acid differences [18], and a tool for easy analysis was developed [19].Other ASFV classification systems have attempted to include p54 and the central variable region (CVR) of B602L with p72 [20].Still, the complexity of the ASFV genome renders classifications based on a limited number of genes inadequate, and we developed a method for Biotyping [21] based on all the open reading frames.Further, recombination events, such as the one observed in China that resulted in the generation of isolates composed of sequences originating from NHV (Genotype 1) and ASFV-G (Genotype 2), would have remained unreported [22].Accordingly, complete genome sequencing can enhance comprehension of the number of circulating ASFV genomes and is of vital importance to understand and track the continued evolution of ASFV strains.
Between 1996 and 1997, the western African countries of Cote D'Ivoire, Togo, Benin, and Nigeria reported outbreaks of ASF [12].Shortly thereafter, the first reported outbreak of ASF in Ghana occurred in the fall of 1999 [12].Previous studies have reported that Genotype 1 was the predominant p72 genotype responsible for outbreaks of ASF in Ghana [23].However, we recently demonstrated that Genotype 2 was responsible for the 2022 outbreaks of domestic swine in Ghana [24].Here, we examined the genomes of 13 isolates collected from nine different regions experiencing outbreaks in 2021.Ten of the genomes were highly similar to the Genotype 2 isolates sequenced from Ghana in 2022, indicating that derivatives of the current pandemic strain emerged in Ghana earlier than reported.Still, the SNP analysis revealed synonymous and nonsynonymous mutations, including large deletions leading to fusions within proteins of the MGF 360 family, indicating the continued evolution of ASFV and the existence of unique Genotype 2 variants.Furthermore, three of the isolates were demonstrated to be Genotype 1 and were more similar to the Genotype 1 isolate sequenced from Benin in 1997 (Benin 97/1 and AM712239) than to the Genotype 1 genomes sequenced from Europe in the 1960s-1970s (E75, Ourt 88/3, NHV, and L60).The Viruses 2024, 16, 1265 3 of 13 data indicate that Genotype 1 was still causing outbreaks in Ghana during this sampling period.Comparatively, the Genotype 1 isolates sequenced were more diverse than the Genotype 2 isolates, suggesting potential extended evolution of Genotype 1 isolates in Ghana, as expected from previous reports of Genotype 1 in Ghana, where Genotype 2 was likely recently introduced and thus less diverse during the sampling period.These findings are significant, as they expand our understanding of the epidemiology of ASF in Ghana and highlight the need for the continued surveillance and monitoring of ASFV genotypes in domestic swine populations, especially since outbreaks of Genotypes 1 and 2 are occurring in proximity, which could lead to a potential recombination event, as was recently observed in Asia.

Next-Generation Sequencing
ASFV isolates were passed once in blood derived primary swine macrophage cultures produced as previously described [25].Viral genomes were sequenced as previously described by using an Illumina Nextseq500 sequencing platform [26].To summarize, viral DNA was extracted from the cytoplasmic fraction of infected macrophage cultures by using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA).For the creation of libraries, the Nextera XT kit (Illumnia, San Diego, CA, USA) was utilized in accordance with the manufacturer's instructions.Furthermore, to bridge across low-complexity regions of the genome, Ghana2021-95 was sequenced by using the Oxford nanopore GridION sequencing platform with previously described methods [24].

Genome Assembly and SNP Detection
All steps were performed by using QIAGEN CLC Genomics Workbench 23.0 (QIA-GEN, Aarhus, Denmark).Illumina reads were trimmed for quality (limit = 0.05), ambiguous base pairs (max = 2), adapters, and minimum size (min = 50) and from the 5 ′ (20 nucleotides) and 3 ′ terminal end (5 nucleotides).GridION reads were not trimmed for quality.To construct the genome of Ghana2021-95, 2,152,588 GridION reads and 270,361,688 (paired) and 83,058 (orphan) trimmed Illumina reads were assembled by using the default parameters of the "De Novo Assembly Long Reads and Polish with Short Reads" pipeline.Following assembly, the contig corresponding to ASFV was extracted, and all Illumina reads were mapped back to the extracted contig, resulting in an average depth of coverage of 1601 reads.The consensus sequence was extracted, resulting in a 184,940 nt genome.To construct the genomes of Ghana2021-01 and Ghana2022-75, Illumina reads originating from each sample were trimmed as previously described and separately mapped to the Ghana2021-95 genome, and the consensus sequences were extracted.To construct the genomes of Ghana2021-13, Ghana2021-27, Ghana2021-37, Ghana2021-41, Ghana2021-49, Ghana2021-57, Ghana2021-81, Ghana2021-87, Ghana2021-91, and Ghana2021-105, Illumina reads originating from each sample were trimmed as previously described and separately mapped to the Ghana2022-35 genome (OP479889) [24], and the consensus sequences were extracted.To identify Single-Nucleotide Polymorphisms (SNPs), the 2021 Ghana genomes were mapped to their indicated reference by using the Map Long Reads to Reference module with long-read splice alignment disabled, followed by the Basic Variant Detection and Amino Acid Changes modules.

Genome Alignment
A previously described database of genomes consisting of 32 ASFV genomes [24] was aligned against the 2021 and 2022 sequences originating from Ghana by using the Create Whole-Genome Alignment module by CLC Genomics, and a phylogeny was constructed by using four combinations of the comparisons Average Nucleotide Identity (ANI) or Average Alignment Percentage (AP) and the methods Neighbor Joining (NJ) or Unweighted Pair Group Method with Arithmetic Mean (UPGMA).

Protein Alignment
The protein translations from all annotated ORFs originating from the genomes isolated from Ghana were compared against the NCBI database by using the default parameters of BLASTP.Proteins containing unique sequences were collected along with homologs encoded by the Ghana 2022 isolates and a previously described list of historical isolates [24] and were aligned by using MUSCLE [28], using the default parameters Gap Open = 10 and Gap extension = 1.

Characteristics of Collected 2021 Outbreak Samples
Thirteen isolates of ASFV were obtained from outbreaks during 2021, from the Savannah, Greater Accra, Bono East, Eastern, Central East, Volta, and Oti regions of Ghana (Figure 1 and Table 1) on the indicated dates, from farms with suspect ASF outbreaks.
Genotype 1 isolates (Ghana2021-01, Ghana2021-75, and Ghana2021-95) formed a clade that was a sister group to the clade composed of Benin 97, NHV, and OURT-88/3.However, unlike NHV and OURT-88-3, further examination of the genomes between nucleotides positions 10,000 and 35,000 demonstrates that Benin 97, Ghana2021-01, Ghana2021-75, and Ghana2021-95 do not contain deletions of MGF_360-6L, MGF_360-10 to 14L, and MGF_505-1R to -3R (Figure 3).The deletion of these MGF genes is believed to contribute to the attenuation of virulence observed in NHV and OURT-88-3 [5,[29][30][31], and this deletion was not observed in the Ghana Genotype 1 isolates.A super clade composed of all Genotype 1 isolates (the previously mentioned isolates, E75, L60, BA71V, and Mkuzi1979) is a sister group to the Genotype 2 super clade.Taken together, based on whole-genome alignment analysis, the Ghana2021 genomes are the most similar to genomes of the same genotype that were collected from the same region.
However, unlike NHV and OURT-88-3, further examination of the genomes between nucleotides positions 10,000 and 35,000 demonstrates that Benin 97, Ghana2021-01, Ghana2021-75, and Ghana2021-95 do not contain deletions of MGF_360-6L, MGF_360-10 to 14L, and MGF_505-1R to -3R (Figure 3).The deletion of these MGF genes is believed to contribute to the attenuation of virulence observed in NHV and OURT-88-3 [5,[29][30][31], and this deletion was not observed in the Ghana Genotype 1 isolates.A super clade composed of all Genotype 1 isolates (the previously mentioned isolates, E75, L60, BA71V, and Mkuzi1979) is a sister group to the Genotype 2 super clade.Taken together, based on whole-genome alignment analysis, the Ghana2021 genomes are the most similar to genomes of the same genotype that were collected from the same region.
type 2 genomes had on average 14.8 (±6.26)SNPs, which resulted, on average, in a change in the coding sequence of 6.1 (±1.97)ORFs per genome.The data indicate the continued evolution of ASFV Genotype 2 genomes in Africa.Table 3. Unique protein sequences encoded by Ghana2021 Genotype 2 genomes.

Discussion
Here, we report thirteen full-length sequences obtained from the ASF outbreaks that occurred in Ghana in 2021.The data indicate that variants of the 2007 Genotype 2 pandemic strain were in Ghana a year earlier than previously believed.Furthermore, our findings identified the complete genomes of three Genotype 1 isolates that exhibited sequence homology to Benin 97/1, suggesting that some outbreaks can still be attributed to Genotype 1 strains, which historically have been the cause of ASF outbreaks in Ghana [23].The cladistics analysis revealed that the 2021 Genotype 1 strains share a common ancestor and that the Genotype 2 strains share a common ancestor.Further, the examination of the ORFs revealed several distinct mutations that are common among the Ghana 2021 Genotype 1 and Genotype 2 isolates.Still, the SNP analysis identified unique differences among the genomes, indicating the continued evolution of ASFV in Ghana; although this study had limited sampling and it is possible that there were other circulating strains in Ghana, it provides the first body of evidence that in Ghana, there were two distinct circulating strains causing outbreaks in the same time period.Both the Genotype 1 and 2 strains came from swine from suspected cases of ASFV.
As reviewed by Njau et al., based on the full or partial sequencing of p72, 12 different genotypes based on the historic classification (I, II, III, IX, X, XI, XII, XIII, XIV, XV, XVIII, and XXII) or the recent reclassification (1, 2, 8, and 9) have been responsible for ASF outbreaks in

13 Figure 1 .
Figure 1.A map of the different regions in Ghana with the indicated outbreaks identified.The different p72 genotypes are differentiated by red (Genotype 1) or blue (Genotype 2) circles.Sample 1 from the Savannah region was collected on 5-1-2021; Sample 13 from the Greater Accra region was collected on 17-3-2021; Sample 27 from the Bono East region was collected on 3-3-2021; Sample 37 from the Bono East region was collected on 20-9-2021; Sample 41 from the Eastern region was collected on 9-10-2021; Sample 49 from the Greater Accra region was collected on 17-11-2021; Sample 57 from the Eastern region was collected on 17-11-2021; Sample 75 from the Greater Accra region

Figure 1 .
Figure 1.A map of the different regions in Ghana with the indicated outbreaks identified.The different p72 genotypes are differentiated by red (Genotype 1) or blue (Genotype 2) circles.Sample 1

Table 1 .
Origin and genotyping of Ghana samples.

Table 2 .
Summary of the identified SNPs in the Ghana 2021 genomes.

Table 2 .
Summary of the identified SNPs in the Ghana 2021 genomes.