Citation: Spinard, E.; Rai, A.; Osei-Bonsu, J.; O’Donnell, V.; Ababio, P.T.; Tawiah-Yingar, D.; Arthur, D.; Baah, D.; Ramirez-Medina, E.; Espinoza, N.; et al. The 2022 Outbreaks of African Swine Fever Virus Demonstrate the First Report of Genotype II in Ghana. Viruses 2023, 15, 1722. https://doi.org/10.3390/ v15081722 Academic Editor: HuaJi Qiu Received: 27 June 2023 Revised: 2 August 2023 Accepted: 5 August 2023 Published: 11 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article The 2022 Outbreaks of African Swine Fever Virus Demonstrate the First Report of Genotype II in Ghana Edward Spinard 1,2,3,† , Ayushi Rai 1,2,†, Jehadi Osei-Bonsu 4,5, Vivian O’Donnell 6 , Patrick T. Ababio 4, Daniel Tawiah-Yingar 4, Daniel Arthur 4, Daniel Baah 4, Elizabeth Ramirez-Medina 1,2, Nallely Espinoza 1,2, Alyssa Valladares 1,2, Bonto Faburay 3,5, Aruna Ambagala 3,5 , Theophlius Odoom 3,6, Manuel V. Borca 1,2,3,* and Douglas P. Gladue 1,2,3,* 1 U.S. Department of Agriculture, Agricultural Research Service, Foreign Animal Disease Research Unit, Plum Island Animal Disease Center, Orient, NY 11957, USA; edward.spinard@usda.gov (E.S.); elizabeth.ramirez@usda.gov (E.R.-M.); nallely.espinoza@usda.gov (N.E.) 2 U.S. Department of Agriculture, Agricultural Research Service, Foreign Animal Disease Research Unit, National Bio and Agro-Defense Facility, Unit Name, Manhattan, KS 66502, USA 3 Center of Excellence for African Swine Fever Genomics, Guilford, CT 06437, USA; aruna.ambagala@inspection.gc.ca (A.A.); theodoom@yahoo.com (T.O.) 4 Accra Veterinary Laboratory of Veterinary Services Directorate, Accra P.O. Box GA184, Ghana; ginolapaatee@gmail.com (P.T.A.); nanayawtee@icloud.com (D.T.-Y.); danielarthur42681@gmail.com (D.A.); dannyneph@gmail.com (D.B.) 5 Animal and Plant Inspection Service, USDA, Greenport, NY 11944, USA 6 Departmenr of Libral Arts & Sciences, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA; vivian.odonnell@usda.gov * Correspondence: manuel.borca@usda.gov (M.V.B.); douglas.gladue@usda.gov (D.P.G.) † These authors contributed equally to this work. Abstract: African swine fever (ASF) is a lethal disease of domestic pigs that has been causing outbreaks for over a century in Africa ever since its first discovery in 1921. Since 1957, there have been sporadic outbreaks outside of Africa; however, no outbreak has been as devastating and as far-reaching as the current pandemic that originated from a 2007 outbreak in the Republic of Georgia. Derivatives with a high degree of similarity to the progenitor strain, ASFV-Georgia/2007, have been sequenced from various countries in Europe and Asia. However, the current strains circulating in Africa are largely unknown, and 24 different genotypes have been implicated in different outbreaks. In this study, ASF isolates were collected from samples from swine suspected of dying from ASF on farms in Ghana in early 2022. While previous studies determined that the circulating strains in Ghana were p72 Genotype I, we demonstrate here that the strains circulating in 2022 were derivatives of the p72 Genotype II pandemic strain. Therefore, this study demonstrates for the first time the emergence of Genotype II ASFV in Ghana. Keywords: African swine fever; ASFV; Ghana; genome 1. Introduction African swine fever (ASF) is a deadly contagious hemorrhagic disease of domestic and wild pigs that was first reported in Kenya in 1921 [1]. ASF has continuously caused outbreaks throughout different parts of Africa since its discovery, with a few sporadic outbreaks outside of Africa that were resolved without spreading worldwide. In 2007, this situation began to change after an outbreak of Genotype II ASF occurred in the Republic of Georgia [2]. This outbreak continued to spread to neighboring countries, and in 2018 an outbreak occurred in China [3], followed by rapid spread of ASF throughout southeast Asia. In addition, the disease has continued to spread to east and central Europe. In 2021 an outbreak occurred in the Dominican Republic, marking the first outbreak in recent history of ASF in the western hemisphere [4]. Viruses 2023, 15, 1722. https://doi.org/10.3390/v15081722 https://www.mdpi.com/journal/viruses https://doi.org/10.3390/v15081722 https://doi.org/10.3390/v15081722 https://creativecommons.org/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://www.mdpi.com/journal/viruses https://www.mdpi.com https://orcid.org/0000-0001-7268-6580 https://orcid.org/0000-0002-7169-3954 https://orcid.org/0000-0003-1783-2105 https://orcid.org/0000-0002-7894-0233 https://doi.org/10.3390/v15081722 https://www.mdpi.com/journal/viruses https://www.mdpi.com/article/10.3390/v15081722?type=check_update&version=2 Viruses 2023, 15, 1722 2 of 12 The causative agent of the disease, ASF virus (ASFV), is a large DNA virus belonging to the family Asfarviridae; it contains a large dsDNA genome ranging from 170–192 kb that encodes 150 to 200 proteins [5–9]. Until recently the ability to perform full-length genome sequencing of the ASFV genome was costly and difficult, which has restricted full-length genome sequencing to only a very few virus isolates. Accordingly, ASFV strains are often broadly characterized by genotype; currently ASFV is classified by 24 genotypes based on the sequencing of a 478 bp fragment of the B646L gene, which encodes for the ASFV capsid protein p72 [10–13]. Further classifications have been implemented by combination of the sequences of genes encoding for p54, p72, and the central variable region (CVR) of pB602L [14]. However, due to the complexity inherent to the ASFV genome (large size, over 150 different proteins, different number of proteins between isolates, deletions and fusions within the multi-gene family (MGF) genes), classifications based on a small subset of genes are insufficient. Sequencing of the entire genome of ASFV can provide a better understanding of how many different ASFV genomes are currently circulating in the world, and perhaps allow for better prediction of cross-protection through sequence homology. Currently, there is only one vaccine available for commercial use for ASF, and its use is limited to Vietnam, with several other experimental vaccines under different stages of development [15]. Therefore, in most of the world control of ASF relies on movement restrictions and culling of infected animals on farms. To more efficiently develop vaccines that can offer protection against different isolates, a more thorough classification of ASFV based on full length sequencing instead of p72 genotyping is needed. Indeed, the molecular basis of cross-protection between or within genotypes is largely unknown, and the signifi- cance of genotypes for predicting cross-protection is uncertain, as historically genotyping of ASFV was performed under convivence for disease tracking, rather than because any scientific reason suggested cross-protection within or between genotypes. However, it has been shown that live-attenuated ASFV with specific genetic deletions offers protection against the parental homologous strain of ASFV; see Gladue et.al., 2022 [15] for a review of ASFV vaccines. Here, we sequenced the full genomes of ASFV circulating in 2022 in Ghana, revealing that they belong to Genotype II and are a derivative of the current pandemic strain ASFV- Georgia/2007. Full-genome sequencing of the 2022 outbreak strains in Ghana revealed a unique deletion in the 5′ end of the genome, completely deleting eight MGF genes. The remaining genes in the Ghana isolates of ASFV are a 99% match to the current pandemic strain. While previous reports determined that Genotype I was the main p72 genotype in Ghana causing outbreaks, this report determines for the first time that Genotype II of ASFV is causing outbreaks in domestic swine in Ghana [16]. The results presented here show the further spread of the pandemic strain of ASFV into Ghana and evidence genetic differences that would not be noted by purely genotyping these isolates by partial gene sequence analysis. This additional knowledge is important for future outbreak mapping to determine both the introduction of these isolates into new areas as well as to detect the introduction of new isolates into areas where outbreaks have previously occurred. This information is critical for both epidemiological control of the disease, and contributes to determining vaccine matching. 2. Materials and Methods 2.1. Sample Collection and Next Generation Sequencing Four isolates of ASFV were obtained from swine from various regions of Ghana that were suspected to have died from ASF during the first few months of 2022. ASFV Ghana2002- 35 was collected on 4 January 2022 from a spleen sample, ASFV isolate Ghana2022-34 was collected on 7 March 2022 from a kidney sample. ASFV isolate Ghana2022-40 was col- lected on 11 March 2022 from a spleen sample, and ASFV isolate Ghana 2022-62 was col- lected on 3 February 2022 from a spleen sample. ASFV isolates Ghana2022-34, Ghana2022-35, Ghana2022-40, and Ghana2022-62 were passed one time in blood-derived primary swine macrophage cultures produced as previously described in [17]. Viral genomes were sequenced Viruses 2023, 15, 1722 3 of 12 using an Illumina Nextseq500 sequencing platform, as previously described [18]. Additionally, for assembly across low-complexity regions of the genome, Ghana2022-35 was sequenced using the Oxford nanopore minion [16] sequencing platform using previously described methods [19]. In brief, viral DNA was extracted from infected macrophage cultures using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). The cytoplasmic fraction was used for sequencing of ASFV DNA. Libraries were created using the Nextera XT kit (Illumnia, San Diego, CA, USA) following the manufacturer’s protocol. Sequence analysis was performed using CLC Genomics Workbench software (CLCBio, Waltham, MA, USA). 2.2. Genome Assembly All steps were performed using the CLC Genomics Workbench (version 21). Illumina reads were trimmed for quality (limit = 0.05), ambiguous base pairs (max = 2), adapters, min- imum size (min = 50), and from the 5′ (20 nucleotides) and 3′ terminal end (5 nucleotides). Ghana2022-35 was first constructed via de novo assembly using the following methodology. To remove reads resulting from the host sequence, 500,497,223 paired-end and 73,874 or- phaned Illumina reads were mapped to ASFV strain ASFV-G (Accession: FR682468.2) and collected, resulting in 249,624 paired-end reads and 105,492 orphaned Illumina reads. Next, 131,918 Minion and the collected Illumina reads were entered into the “De Novo Assembly Long Reads and Polish with Short Reads” pipeline using the default parameters. From the assembly, a contig that matched to ASFV was extracted, and all Minion and trimmed Illumina reads were mapped back to the genome using the default parameters, resulting in an average depth of coverage of 313 reads. The consensus sequence was extracted, resulting in a 184,773 nt genome. To construct the genomes of Ghana2022-34, Ghana2022-40, and Ghana2022-62, Illumina reads originating from each sample were trimmed as previously described and separately mapped to the Ghana2022-35 genome, and the consensus se- quences were extracted. Ambiguous nucleotides were manually resolved based on the mapped reads. 2.3. Annotation of the Genome ORF prediction was performed on Ghana2022-35 using CLC Genomics workbench v21 using the “Find Open Reading Frames” module with the following parameters: both strands, min. size = 50 nt, genetic code = standard, start codon AUG, resulting in 427 ORFs. Protein translations of the ORFs were compared against all 195 protein sequences of the ASFV strain ASFV-G (Accession: FR682468.2) using the default parameters of BLASTP. ORFs with >80% sequence identity and coverage were annotated with the top ASFV-G protein hit. The strand and the start and end nucleotide positions for each gene were extracted from the output file and, when required, were manually edited to include the correct start and stop codon. To cull the remaining unannotated ORFs, the nucleotide sequences of the remaining ORFs were compared to the nucleotide sequence of ASFV-G using the default parameters of TBLASTN. ORFs that matched ASFV-G with over 80% identification and coverage were discarded as incorrectly predicted ORFs. Translated sequences from the remaining unannotated ORFs were compared to proteins translated from our previously described ASFV database [5]. ORFs with no matches or with matches to a hypothetical protein were removed, and the remaining ORFs were annotated with the top ASFV protein hit. The strand, start, and end nucleotide positions for each gene were extracted from the output file and manually edited when required to include the correct start and stop codon. Annotations were entered on the Ghana2022-35 genome in CLC Genomics Workbench, resulting in 167 annotations. Annotations were transferred to Ghana2022-34, Ghana2022-40, and Ghana2022-62 using the Genome Annotation Transfer Utility [20]. Concurrently, proteins with sequences that differed compared to Ghana2022-35 and annotations that failed to transfer were collected and recorded. Viruses 2023, 15, 1722 4 of 12 2.4. Identification of Single-Nucleotide Polymorphisms (SNPs) Ghana2022-35 was mapped to ASFV-G (Accession: FR682468.2) using the Map Long Reads to Reference module in CLC Genomics with long-read splice alignment enabled. The Basic Variant Detection module within CLC Genomics was then used, followed by the Amino Acid Changes module. The same methodology was used with Ghana2022- 34, Ghana2022-40, and Ghana2022-62 separately mapped to Ghana2022-35 in order to determine the SNPs between the samples originating from Ghana. 2.5. Genome Alignment A database of 32 ASFV genomes was created by downloading the top 11 ASFV genomes on NCBI based on percent identity and coverage with respect to Ghana2022-34, Ghana2022- 35, Ghana2022-40, and Ghana2022-62 using the default parameters of BLASTN [21–24]; the genomes of curated historical isolates were downloaded from 4virology.net accessed on, 21 May 2023 and any remaining full length ASFV sequences originating from Africa were downloaded from NCBI. Genomes were aligned against the sequences originating from Ghana using the Create Whole Genome Alignment module in CLC Genomics, and a phylogeny was constructed using four combinations of comparisons (Average Nucleotide Identity (ANI) or Average Alignment Percentage (AP)) and methods (Neighbor Joining (NJ) or Unweighted Pair Group Method with Arithmetic Mean (UPGMA)). 2.6. Protein Alignment The protein translations from all annotated ORFs originating from the genomes iso- lated from Ghana were compared against the NCBI database using the default parameters of BLASTP [21–24]. Proteins containing unique sequences were collected along with their historical isolates’ homologs; these were downloaded from 4virology.net accessed on 21 May 2023 and aligned in CLC Genomics using the following parameters: Gap Open = 10, Gap extension = 1, End Gap Cost = free, Alignment = very accurate. 3. Results 3.1. Characteristics of Collected 2022 Outbreak Samples Four isolates of ASFV were obtained from outbreaks in the first few months 2022 from the Ashanti, Northern, and Eastern regions of Ghana (Figure 1). ASFV Ghana2002-35 was collected on 4 January 2022 from the Ashanti region during an outbreak at a small farm with 20 head of swine, where 11 swine had died and 8 additional swine were showing clinical signs of ASF including red patches on the skin, vomiting blood, swollen head, and were off feed. The ASFV isolate was sequenced from a spleen sample obtained from one of the animals that died. ASFV isolate Ghana2022-34 was obtained from the Eastern region of Ghana on 7 March 2022 from a farm with 416 head of swine, where 186 swine were dead and 45 additional animals were showing clinical signs of ASF, including abortion, loss of coordination, difficulty breathing, and were off feed. The virus was isolated from a kidney sample obtained from an animal that died. ASFV isolate Ghana2022-40 was obtained from the Eastern region of Ghana on 11 March 2022 from a farm containing 50 head of swine, where 4 had died and 11 more were showing clinical symptoms, including loss of appetite, dullness and weakness, reluctance to stand, red pigmentation of the skin, fever, and difficulty breathing. A spleen was collected from one of the animals that died, and the ASFV isolate was sequenced. ASFV isolate Ghana 2022-62 was obtained in the Northern region of Ghana from an outbreak occurring on February 3, 2022 at a farm consisting of 70 head of swine, with 59 swine found dead and 8 more showing clinical symptoms consisting of anorexia, lethargy, and hyperemia of the whole body. The virus was obtained from a spleen sample from one of the animals that died and was sequenced. After the clinical sample was submitted and confirmed positive, no further information was provided by the farmers about the outcome of the outbreak. Nonetheless, these samples from three distinct regions of Ghana (Figure 1) can provide an idea of the circulating strains of ASFV. Viruses 2023, 15, 1722 5 of 12 Viruses 2023, 15, x FOR PEER REVIEW  5  of  12      obtained from a spleen sample from one of the animals that died and was sequenced. After  the clinical sample was submitted and confirmed positive, no  further  information was  provided by the farmers about the outcome of the outbreak. Nonetheless, these samples  from  three distinct  regions of Ghana  (Figure 1)  can provide an  idea of  the  circulating  strains of ASFV.    Figure 1. A map of the different regions in Ghana with the indicated outbreaks identified. Sample  35 from the Ashanti region was collected 4 January 2022; Sample 62 from the Northern region was  collected 3 February 2022; Sample 34 was collected 7 March 2022; Sample 40 was collected 11 March  2022.  3.2. Genotyping of the ASFV Isolates  Genotyping was  performed,  and  all  four  isolates  (Ghana2022-34,  Ghana2022-35,  Ghana2022-40, and Ghana2022-62) were determined to be Genotype II from the coding  sequences of p72 (B646L) and Genotype IIa based on p54 (E183L) (Table 1).  Table 1. Origin and genotyping of Ghana samples.  Sample  GenBank  Accession    Location of  Animal/Farm  Region  Date of  Outbreak  Genotype    p72/VP73  (B646L)  Genotype    p54 (E183L)  35  OP479889  AKUMADAN  Ashanti    1/4/2022  II  IIa  62  OP718535  CENTRAL  GONJA  DISTRICT  Northern    2/3/2022  II  IIa  34  OP718533  BUNSO  Eastern    3/7/2022  II  IIa  Figure 1. A map of the different regions in Ghana with the indicated outbreaks identified. Sample 35 from the Ashanti region was collected 4 January 2022; Sample 62 from the Northern region was collected 3 February 2022; Sample 34 was collected 7 March 2022; Sample 40 was collected 11 March 2022. 3.2. Genotyping of the ASFV Isolates Genotyping was performed, and all four isolates (Ghana2022-34, Ghana2022-35, Ghana2022-40, and Ghana2022-62) were determined to be Genotype II from the coding sequences of p72 (B646L) and Genotype IIa based on p54 (E183L) (Table 1). Table 1. Origin and genotyping of Ghana samples. Sample GenBank Accession Location of Animal/Farm Region Date of Outbreak Genotype p72/VP73 (B646L) Genotype p54 (E183L) 35 OP479889 AKUMADAN Ashanti 1/4/2022 II IIa 62 OP718535 CENTRAL GONJA DISTRICT Northern 2/3/2022 II IIa 34 OP718533 BUNSO Eastern 3/7/2022 II IIa 40 OP718534 SUHYEN Eastern 3/11/2022 II IIa 3.3. ASFV Full-Genome Alignments Using whole-genome alignments for ASFV isolates available on NCBI, phyologenic trees were constructed using four different parameters: ANI + NJ, AP +NJ, UPGMA + ANI, and UPGMA + NJ. All methods grouped the Ghana 2022 genomes (blue font) together as a sister group to RV502 (Accession: OP672342), a 2020 isolate from Nigeria [25]. However further analysis relied only on ANI + NJ, as it correctly grouped the prototypical isolates. Viruses 2023, 15, 1722 6 of 12 The Ghana 2022 genomes shared a sister group with Estonia-2014 (Accession: LS478113.1) as well (Figure 2). RV502 was recently shown to have a ~6500 bp deletion resulting in the loss of MGF_110-8L, MGF_110-XR, ACD 00190, MGF_110-9L, ACD 00210, MGF_110-10L-14 L, G ACD 00240, MGF_110-12L, MGF_110-13La, MGF_110-13Lb, ACD 00270, MGF_360-4L, ACD 00300, and G ACD 00350 [25]. Estonia-2014 has been previously shown to have a ~14,500 bp deletion of the 5′ end, resulting in the loss of the MGF genes MGF_110-1L through MGF_110-14L and MGF_360-1L through MGF_360-3L, along with the partial deletion of MGF_110-13 L [26]. Viruses 2023, 15, x FOR PEER REVIEW  6  of  12      40  OP718534  SUHYEN  Eastern    3/11/2022  II  IIa  3.3. ASFV Full‐Genome Alignments  Using whole-genome alignments for ASFV isolates available on NCBI, phyologenic  trees were constructed using four different parameters: ANI + NJ, AP +NJ, UPGMA + ANI,  and UPGMA + NJ. All methods grouped the Ghana 2022 genomes (blue font) together as  a sister group to RV502 (Accession: OP672342), a 2020 isolate from Nigeria [25]. However  further analysis relied only on ANI + NJ, as it correctly grouped the prototypical isolates.  The Ghana 2022 genomes shared a sister group with Estonia-2014 (Accession: LS478113.1)  as well (Figure 2). RV502 was recently shown to have a ~6500 bp deletion resulting in the  loss of MGF_110-8L, MGF_110-XR, ACD 00190, MGF_110-9L, ACD 00210, MGF_110-10L- 14  L,  G  ACD  00240,  MGF_110-12L,  MGF_110-13La,  MGF_110-13Lb,  ACD  00270,  MGF_360-4L, ACD 00300, and G ACD 00350 [25]. Estonia-2014 has been previously shown  to  have  a  ~14,500  bp  deletion  of  the  5′  end,  resulting  in  the  loss  of  the MGF  genes  MGF_110-1L through MGF_110-14L and MGF_360-1L through MGF_360-3L, along with  the partial deletion of MGF_110-13 L [26].    Figure 2. Whole genome alignment of ASFV isolates using API and NJ. Ghana genomes are high- lighted in blue font.  3.4. Genetic Variation between Ghana 2022 Isolates  To determine genetic differences between the four Ghana  isolates, the genomes of  Ghana2022-34, Ghana2022-40,  and Ghana2022-62 were  compared  to  the first outbreak  strain in Ghana identified in 2022, Ghana2022-35 (Supplementary Table S1). Note that the  following  genes  could  not  be  compared  because  of  gaps  in  the  assembly:  EP402R  (Ghana2022-34,  Ghana2022-40,  and  Ghana2022-62),  DP79L  (Ghana2022-62),  EP153R  (Ghana2022-34 and Ghana2022-40), and MGF_360-6L (Ghana2022-40).  Comparison between  the Ghana2022-35 and Ghana2022-34 genome  resulted  in 34  SNPs. Comparison  of  the  genome  of Ghana2022-35  and Ghana2022-40  resulted  in  26  SNPs, seven of which led to a change in the amino acid (AA) sequence of seven ORFs:  X69R  (P16L),  MGF_505-1R  (R450I),  K145R  (Y166H),  C315R  (Q30H),  E199L  (P85A),  MGF_505-11L (I280V), and a frameshift in MGF_360-13L at A262. Comparison of the ge- nome Ghana2022-35 and Ghana2022-62 contained thirteen SNPs, twelve of which led to a  change  in  AA  sequence  of  six  ORFs:  X69R  (P16L),  C475L  (Q148H),  E199L  (P85A),  MGF_505-11L  (I280V),  a  frameshift  in  MGF_360-13L  at  A262  and  a  frameshift  in  MGF_360-18R at L54.  These results show  that even  in early 2022  there were unique genomic  features at  both the nucleotide and protein level between closely related outbreaks of ASFV in Ghana,  suggesting variation between isolates causing outbreaks even within a restricted area and  time of outbreak in Ghana.  Figure 2. Whole genome alignment of ASFV isolates using API and NJ. Ghana genomes are high- lighted in blue font. 3.4. Genetic Variation between Ghana 2022 Isolates To determine genetic differences between the four Ghana isolates, the genomes of Ghana2022-34, Ghana2022-40, and Ghana2022-62 were compared to the first outbreak strain in Ghana identified in 2022, Ghana2022-35 (Supplementary Table S1). Note that the follow- ing genes could not be compared because of gaps in the assembly: EP402R (Ghana2022-34, Ghana2022-40, and Ghana2022-62), DP79L (Ghana2022-62), EP153R (Ghana2022-34 and Ghana2022-40), and MGF_360-6L (Ghana2022-40). Comparison between the Ghana2022-35 and Ghana2022-34 genome resulted in 34 SNPs. Comparison of the genome of Ghana2022-35 and Ghana2022-40 resulted in 26 SNPs, seven of which led to a change in the amino acid (AA) sequence of seven ORFs: X69R (P16L), MGF_505-1R (R450I), K145R (Y166H), C315R (Q30H), E199L (P85A), MGF_505-11L (I280V), and a frameshift in MGF_360-13L at A262. Comparison of the genome Ghana2022-35 and Ghana2022-62 contained thirteen SNPs, twelve of which led to a change in AA sequence of six ORFs: X69R (P16L), C475L (Q148H), E199L (P85A), MGF_505-11L (I280V), a frameshift in MGF_360-13L at A262 and a frameshift in MGF_360-18R at L54. These results show that even in early 2022 there were unique genomic features at both the nucleotide and protein level between closely related outbreaks of ASFV in Ghana, suggesting variation between isolates causing outbreaks even within a restricted area and time of outbreak in Ghana. 3.5. Analysis of a 6534 nt Deletion and Individual MGFs When Ghana2022-35 was compared to the original outbreak strain ASFV-Georgia/2007, there were a total of 69 SNP, insertions, and deletions, of which 37 resulted in an AA change in 34 ORFs, as detailed in Supplementary Table S2. Compared to ASFV-Georgia/2007, there was a 6534 nt deletion in the Ghana genomes that is not present in other isolates that are decedents of the original Georgia/2007 outbreak. This deletion is between the coding regions of MGF_110-8L and MGF_360-6L, and resulted in the loss of the multi-gene family (MGF) proteins MGF_110-8L, MGF_100-1R, MGF_110-9L, 0 MGF_110-10-14L fusion protein, MGF_110-12L, MGF_110-13La, MGF_110-13Lb, and MGF_360-4L. This deletion additionally consists of the open reading frames (ORFs) ASFV G ACD 00190, ASFV G ACD Viruses 2023, 15, 1722 7 of 12 00300, ASFV G ACD 00210, ASFV G ACD 00240, and ASFV G ACD 00270. This deletion resulted in the replacement of the 35 C-terminal of MGF_360-6L with the sequence “RFT- TNPLSS*” that originated from an off-frame translation within the original MGF_110-8L ORF (Figure 3). Further, a mutation led to the loss of the start methionine codon in the genome that originated from Ghana2022-40, leading to a further shortening of MGF_360-6L. When compared to ASFV-G, an additional 688 nt deletion was observed within the Ghana isolates, which created an MGF_110-3L-4L fusion protein (Figure 3). Further differences that led to truncation or extensions were seen in the coding sequences of other MGF proteins (Supplementary Table S2 and Table 2). KP360L is a fusion of MGF_360-1la and MGF_360-1lb resulting from a single nucleotide insertion in MGF_360-1Lb (Figure 3 and Supplementary Figure S1), though the amino acid sequence is closer to ASFV-G MGF_360- 1La and MGF_360-1Lb than to the sequence of KP360L encoded by historical ASFV isolates. Additional differences in the genes of the MGF families include an extension of MGF_110- 1L (all Ghana 2022 genomes), a truncation of MGF_360-13L (all 2022 Ghana genomes, and samples Ghana2022-40 and Ghana2022-62 are further truncated compared to Ghana2022-34 and 35), a truncation of MGF_360-16R that may possibly lead to the encoding of MGF_360- 16Ra and MGF_360-16Rb (all 2022 Ghana genomes), a truncation of MGF_300-2R that may possibly lead to the encoding of MGF_300-2Ra and MGF_300-2Rb (all Ghana genomes), an extension of MGF_100-3L (all Ghana 2022 genomes), and a truncation of MGF_360-18R that may lead to the encoding of MGF_360-18Ra and MGF_360-18Rb (Ghana2022-62 only). Mutations that led to the encoding of MGF genes with unique AA sequences are further explored in the next section. Viruses 2023, 15, x FOR PEER REVIEW  7  of  12      3.5. Analysis of a 6534 nt Deletion and Individual MGFs  When  Ghana2022-35 was  compared  to  the  original  outbreak  strain ASFV-Geor- gia/2007, there were a total of 69 SNP, insertions, and deletions, of which 37 resulted in an  AA change in 34 ORFs, as detailed in Supplementary Table S2. Compared to ASFV-Geor- gia/2007, there was a 6534 nt deletion in the Ghana genomes that is not present in other  isolates that are decedents of the original Georgia/2007 outbreak. This deletion is between  the coding regions of MGF_110-8L and MGF_360-6L, and resulted in the loss of the multi- gene family (MGF) proteins MGF_110-8L, MGF_100-1R, MGF_110-9L, 0 MGF_110-10-14L  fusion protein, MGF_110-12L, MGF_110-13La, MGF_110-13Lb, and MGF_360-4L. This de- letion additionally consists of the open reading frames (ORFs) ASFV G ACD 00190, ASFV  G ACD 00300, ASFV G ACD 00210, ASFV G ACD 00240, and ASFV G ACD 00270. This  deletion  resulted  in  the  replacement of  the 35 C-terminal of MGF_360-6L with  the  se- quence “RFTTNPLSS*” that originated from an off-frame translation within the original  MGF_110-8L ORF (Figure 3). Further, a mutation led to the loss of the start methionine  codon in the genome that originated from Ghana2022-40, leading to a further shortening  of MGF_360-6L. When compared to ASFV-G, an additional 688 nt deletion was observed  within  the Ghana  isolates, which created an MGF_110-3L-4L  fusion protein  (Figure 3).  Further differences that led to truncation or extensions were seen in the coding sequences  of  other MGF  proteins  (Supplementary  Table  S2  and  Table  2). KP360L  is  a  fusion  of  MGF_360-1la and MGF_360-1lb resulting from a single nucleotide insertion in MGF_360- 1Lb (Figure 3 and Supplementary Figure S1), though the amino acid sequence is closer to  ASFV-G MGF_360-1La and MGF_360-1Lb  than  to  the sequence of KP360L encoded by  historical ASFV isolates. Additional differences in the genes of the MGF families include  an extension of MGF_110-1L (all Ghana 2022 genomes), a truncation of MGF_360-13L (all  2022 Ghana genomes, and samples Ghana2022-40 and Ghana2022-62 are further truncated  compared to Ghana2022-34 and 35), a truncation of MGF_360-16R that may possibly lead  to the encoding of MGF_360-16Ra and MGF_360-16Rb (all 2022 Ghana genomes), a trun- cation  of MGF_300-2R  that may  possibly  lead  to  the  encoding  of MGF_300-2Ra  and  MGF_300-2Rb  (all Ghana genomes),  an  extension of MGF_100-3L  (all Ghana  2022 ge- nomes), and a truncation of MGF_360-18R that may  lead to the encoding of MGF_360- 18Ra and MGF_360-18Rb (Ghana2022-62 only). Mutations that led to the encoding of MGF  genes with unique AA sequences are further explored in the next section.    Figure 3. Graphic illustrating MGF genes that contain truncations, elongations, and fusions encoded  by the 2022 Ghana isolates compared to the original outbreak strain ASFV-Georgia/2007. Note that  the MGF_360-6L fusion was not annotated in Ghana2022-34 because of a single nucleotide gap in  the genome.  Table 2. Proteins with unique amino acid sequences encoded by the indicated Ghana samples.  Gene  Ghana2022-34  Ghana2022-35  Ghana2022-40  Ghana2022-62  B117L  Y98F  Y98F  Y98F  Y98F  B602L  V13I  V13I  V13I  V13I  C315R  no mutation  no mutation  Q30H  no mutation  Figure 3. Graphic illustrating MGF genes that contain truncations, elongations, and fusions encoded by the 2022 Ghana isolates compared to the original outbreak strain ASFV-Georgia/2007. Note that the MGF_360-6L fusion was not annotated in Ghana2022-34 because of a single nucleotide gap in the genome. 3.6. Analysis of Ghana 2022 Proteins All proteins encoded by Ghana2022-34, Ghana2022-35, Ghana2022-40, and Ghana2022- 62 were submitted to BLASTP on NCBI [21–24]. Sequences that did not have 100% identity and 100% coverage matches were considered to be unique protein sequences specific to the 2022 Ghana genomes. As summarized in Table 2, the following proteins encoded from the specified Ghana genome contained unique amino acid sequences that can be used as markers for the 2022 Ghana genomes (Supplementary Figure S1): B117L (Y98F, all genomes), B602L (V13I, all genomes), C315R (Q30H, Ghana2022-40), C475L (Q148H, Ghana2022-62), EP402R (L300V and P313L, Ghana2022-35), EP424R (N155S, all genomes), F334L (S282G, all genomes), H339R (Q319R, all genomes), K145R (Y116R, Ghana2022-40), KP360L (F19L, H41Y, and K47E, all genomes), MGF_110-1L (an additional 54 amino acids to the C-terminus, all genomes), MGF_110-7L (Y112, all genomes), MGF_360-13L (G177D, all genomes, NINQAM-LTSVQYYNIGNIFFCID 262-284 ATSTKLCLLQYNIITSVIYFSV*, (Ghana2022-34 and Ghana2022-35 and NINQAMLTSVQYYN 262-276 QHQPSYAY-FSTIL*, (Ghana2022-40 and Ghana2022-62), MGF_360-14L (P351L, all genomes), MGF_360-16R (split into MGF_360-16Ra and MGF_360-16Rb, all genomes), MGF_360-18R (split into Viruses 2023, 15, 1722 8 of 12 MGF_360-168a and MGF_360-18Rb, Ghana2022-62), MGF_360-6L (LHKKILEPSE 341-350 RFTTNPLSS*, Ghana2022-34, Ghana2022-35, and Ghana2022-62), MGF_505-11L (S231L, all genomes) and (I280V, Ghana2022-40 and Ghana2022-62), MGF_505-1R (I450R, all genomes), MGF_505-2R (T355A, all genomes), MGF_505-5R (G477S, all genomes), NP868R (A9T and A589V, all genomes), and X69R (L16P, Ghana2022-34 and Ghana2022-35). Table 2. Proteins with unique amino acid sequences encoded by the indicated Ghana samples. Gene Ghana2022-34 Ghana2022-35 Ghana2022-40 Ghana2022-62 B117L Y98F Y98F Y98F Y98F B602L V13I V13I V13I V13I C315R no mutation no mutation Q30H no mutation C475L no mutation no mutation no mutation Q148H EP402R na L300V, P313L na na EP424R N155S N155S N155S N155S F334L S282G S282G S282G S282G H339R Q319R Q319R Q319R Q319R K145R no mutation no mutation Y116R no mutation KP360L F19L, H41Y, K47E F19L, H41Y, K47E F19L, H41Y, K47E F19L, H41Y, K47E MGF_110-1L 54 AA extension to the C-terminus 54 AA extension to the C-terminus 54 AA extension to the C-terminus 54 AA extension to the C-terminus MGF_110-7L Y112R Y112R Y112R Y112R MGF_360-13L G177D, NIN- QAMLTSVQYYNIGNIF- FCID 262-284 ATSTKLCLLQYNI- ITSVIYFSV* G177D, NIN- QAMLTSVQYYNIGNIF- FCID 262-284 ATSTKLCLLQYNI- ITSVIYFSV* G177D, NINQAMLTSVQYYN 262-276 QHQPSYAYFSTIL* G177D, NINQAMLTSVQYYN 262-276 QHQPSYAYFSTIL* MGF_360-14L P351L P351L P351L P351L MGF_360-16R D92*, split into MGF_360-16Ra and MGF_360-16Rb D92*, split into MGF_360-16Ra and MGF_360-16Rb D92*, split into MGF_360-16Ra and MGF_360-16Rb D92*, split into MGF_360-16Ra and MGF_360-16Rb MGF_360-18R no mutation no mutation no mutation Split into MGF 360-18Ra and MGF 360-18Rb MGF_360-6L LHKKILEPSE 341-350 RFTTNPLSS* LHKKILEPSE 341-350 RFTTNPLSS* na LHKKILEPSE 341-350 RFTTNPLSS* MGF_505-11L S231L S231L S231L, I280V S231L, I280V MGF_505-1R I450R I450R I450R I450R MGF_505-2R T355A T355A T355A T355A MGF_505-5R G477S G477S G477S G477S NP868R A9T, A589V A9T, A589V A9T, A589V A9T, A589V X69R L16P L16P no mutation no mutation na: Gene was not annotated. 4. Discussion Here, we report four full-length sequences from the 2022 outbreaks in Ghana. This is the first report of Genotype II strains circulating in Ghana that are derivatives of the current Eurasia strain that caused the 2007 outbreak in the Republic of Georgia, as previous outbreaks of ASFV in Ghana were identified as Genotype I [16]. It is interesting that derivatives of ASF can occur with distinct genetic markers even within a few months of outbreaks. However, common markers can be seen when evaluating the differences between the four isolates, suggesting that they originated from the same parental strain or outbreak in recent history. While the exact number of ASFV strains currently circulating in Africa is unknown, the information provided by full-genome sequencing of outbreak strains is imperative to track the continued evolution of ASFV strains. Historically, genotyping was performed to track ASF outbreaks out of convenience; however, partial genome sequencing does not provide an accurate representation of the currently circulating strains. This study and others [25–27] based on ASFV whole-genome sequencing clearly indicate that there are Viruses 2023, 15, 1722 9 of 12 different circulating strains of Genotype II, an observation that would be missed by only sequencing a few selected genes. For example, the isolates from Ghana have a deletion of eight MGF family genes. Recently, a 6534 nt deletion, MGF_110-3L-4L fusion, and MGF_360-1La-ILb fusion were observed in RV502 [25]. Interestingly, unlike RV502, the Ghana isolates do not contain the reverse complement duplication of the 5′ end of the genome on the 3′ end of the genome, and no fusion between MGF_360-21L and MGF_360- 2L was observed [25]. There have been previous reports that deletion of six MGF genes can result in full or partial attenuation of ASFV [28–33]. It is important to note that the deletion observed in the Ghana isolates is not the same or even close to the deletion observed in the genetically engineered strains that were attenuated. Other isolates such as Nigeria-RV502 had deletions of MGF genes; however, these do not show any decreased virulence in domestic swine [25]. Conversely, large deletions that include many MGF genes of the p72 Genotype I or Genotype II strains have been found in animals surviving outbreaks [25,34], although when tested in swine these strains had a level of residual virulence. It should be noted that there are multiple families of MGF genes. Furthermore, even MGF genes within the same family appear to be unique based on structural prediction [9], suggesting that different MGF genes have different specific molecular functions and that different deletions of specific MGF genes would have a differential effect on ASFV [35]. It is possible that these genetic deletions have occurred as a fit for a purposely smaller genome in domestic swine, and that these strains could have reduced replication in ticks (as was observed in other isolates [36]) or perhaps in wild suidae; however, further experiments would have to be performed to evaluate this theory. Although in this study there were minor differences in ASFV proteins when comparing the genomes of the outbreak strains in Ghana to that of the original ASFV-Georgia/2007 backbone, our results would nonetheless suggest that vaccines using the ASFV-Georgia/2010 backbone [28,37–40] would be effective against the outbreak strain occurring in Ghana, as previous reports using the ASFV-G-∆I177L vaccine strain have determined this vaccine to be effective against both the original ASFV-Georgia/2010 strain and recent strains in Vietnam [41]. It is important to note that domestic pigs in Ghana are a crossbreed between European pigs and the local Ashanti Dwarf pig, a breed that is noted for being hardy and less susceptible to local diseases [42]. It would be expected that an ASFV vaccine would show similar results in these pigs, as it did in local Vietnamese breeds [41], though the efficacy of any vaccine would have to be tested under local conditions. When vaccination becomes a method of controlling ASF worldwide, it will become increasingly important to determine the full-length genome of ASFV in order to accurately predict the effectiveness of vaccines rather than relying on one individual protein, as historically used out of convenience for genotyping, particularly as the persistence of Genotype II continues to evolve throughout different parts of the world. The information presented in this manuscript is important, as we have discovered currently circulating strains of ASFV in Africa, providing information on the potential to use either commercial or experimental ASF vaccines in particular areas. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/v15081722/s1, Figure S1: Protein Alignments; Table S1: SNPs present in Ghana2022-34, Ghana2022-40 and Ghana2022-62 compared to Ghana2022-35; Table S2: SNPs present in Ghana2022-45 compared to ASFV-G. Author Contributions: Conceptualization, E.S., J.O.-B., T.O., M.V.B. and D.P.G.; Data curation, A.R., J.O.-B., P.T.A., D.T.-Y., D.A., D.B., E.R.-M., N.E., A.V., B.F., A.A. and T.O.; Formal analysis, E.S., T.O., M.V.B. and D.P.G.; Funding acquisition, D.P.G.; Methodology, A.R. and V.O.; Writing—original draft, E.S., T.O., M.V.B. and D.P.G.; Writing—review and editing, E.S., A.R., J.O.-B., V.O., P.T.A., D.T.-Y., D.A., D.B., E.R.-M., N.E., A.V., B.F., A.A., T.O., M.V.B. and D.P.G. All authors have read and agreed to the published version of the manuscript. https://www.mdpi.com/article/10.3390/v15081722/s1 https://www.mdpi.com/article/10.3390/v15081722/s1 Viruses 2023, 15, 1722 10 of 12 Funding: This work was supported by USDA internal funding (CRIS 301-3022-505-63) and NBAF partnership funding, and in part by an appointment to the Plum Island Animal Disease Center (PIADC) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Genome sequences have been deposited in GenBank under the ac- cession nos. OP718533 (Ghana2022-34), OP479889 (Ghana2022-35), OP718534 (Ghana2022-40), and OP718535 (Ghana2022-62). 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MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. https://doi.org/10.1111/tbed.14329 https://www.ncbi.nlm.nih.gov/pubmed/34582622 https://doi.org/10.1186/s12864-017-3536-6 https://www.ncbi.nlm.nih.gov/pubmed/28219344 Introduction Materials and Methods Sample Collection and Next Generation Sequencing Genome Assembly Annotation of the Genome Identification of Single-Nucleotide Polymorphisms (SNPs) Genome Alignment Protein Alignment Results Characteristics of Collected 2022 Outbreak Samples Genotyping of the ASFV Isolates ASFV Full-Genome Alignments Genetic Variation between Ghana 2022 Isolates Analysis of a 6534 nt Deletion and Individual MGFs Analysis of Ghana 2022 Proteins Discussion References