Analysis of the Unique Historical Isolate of African Swine Fever Virus Isolate Spencer from Outbreaks in 1951.

African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.

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.

A Standardized Pipeline for Assembly and Annotation of African Swine Fever Virus Genome.

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

African Swine Fever Virus Protein-Protein Interaction Prediction.

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein-protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein-protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein-protein interactions, further validating their relevance. Two protein-protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

African swine fever virus P72 genotyping tool.

Historically, genotyping of African swine fever virus was based on partial sequencing of B646L (p72). Until recently, the number of differences that defined genotypes was ambiguous. This tool allows a sequence to be uploaded and will report its closest matches along with its likely p72 genotype.

A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups.

The African swine fever virus (ASFV) is currently causing a world-wide pandemic of a highly lethal disease in domestic swine and wild boar. Currently, recombinant ASF live-attenuated vaccines based on a genotype II virus strain are commercially available in Vietnam. With 25 reported ASFV genotypes in the literature, it is important to understand the molecular basis and usefulness of ASFV genotyping, as well as the true significance of genotypes in the epidemiology, transmission, evolution, control, and prevention of ASFV. Historically, genotyping of ASFV was used for the epidemiological tracking of the disease and was based on the analysis of small fragments that represent less than 1% of the viral genome. The predominant method for genotyping ASFV relies on the sequencing of a fragment within the gene encoding the structural p72 protein. Genotype assignment has been accomplished through automated phylogenetic trees or by comparing the target sequence to the most closely related genotyped p72 gene. To evaluate its appropriateness for the classification of genotypes by p72, we reanalyzed all available genomic data for ASFV. We conclude that the majority of p72-based genotypes, when initially created, were neither identified under any specific methodological criteria nor correctly compared with the already existing ASFV genotypes. Based on our analysis of the p72 protein sequences, we propose that the current twenty-five genotypes, created exclusively based on the p72 sequence, should be reduced to only six genotypes. To help differentiate between the new and old genotype classification systems, we propose that Arabic numerals (1, 2, 8, 9, 15, and 23) be used instead of the previously used Roman numerals. Furthermore, we discuss the usefulness of genotyping ASFV isolates based only on the p72 gene sequence.

Reclassification of ASFV into 7 Biotypes Using Unsupervised Machine Learning.

In 2007, an outbreak of African swine fever (ASF), a deadly disease of domestic swine and wild boar caused by the African swine fever virus (ASFV), occurred in Georgia and has since spread globally. Historically, ASFV was classified into 25 different genotypes. However, a newly proposed system recategorized all ASFV isolates into 6 genotypes exclusively using the predicted protein sequences of p72. However, ASFV has a large genome that encodes between 150-200 genes, and classifications using a single gene are insufficient and misleading, as strains encoding an identical p72 often have significant mutations in other areas of the genome. We present here a new classification of ASFV based on comparisons performed considering the entire encoded proteome. A curated database consisting of the protein sequences predicted to be encoded by 220 reannotated ASFV genomes was analyzed for similarity between homologous protein sequences. Weights were applied to the protein identity matrices and averaged to generate a genome-genome identity matrix that was then analyzed by an unsupervised machine learning algorithm, DBSCAN, to separate the genomes into distinct clusters. We conclude that all available ASFV genomes can be classified into 7 distinct biotypes.