African swine fever (ASF) is a disease that affects domestic pigs and wild boars, resulting in up to 100% case fatality rate, and there is currently no effective treatment or vaccine. To date, there are 67 ASFV complete genome sequences available, but most of the sequences represent only genotypes I-V and VII-X of the 24 genotypes identified based on p72 sequencing, limiting inter and intra-genotype comparative studies. ASFVs encode several multigene families (MGFs) involved in virulence and host range which are found at the genomic termini and the majority of genomic differences between isolates are due to the composition of these MGFs. The comparison of the MGFs across ASFV isolates is of utmost importance in understanding genome variability and their contribution to virulence. The p72 gene has historically been used in phylogenetic analysis of ASFV. However, it lacks the capacity for higher resolution between isolates belonging to the same genotype. This study aimed to analyse and characterise six novel ASFV isolates of African origin from a domestic pig, warthog, wild boar and ticks in terms of genomic makeup, MGF composition and phylogenetic relationships, including identification of additional phylogenetic markers, specifically for use in discrimination between closely related isolates. Genomes of six novel isolates were sequenced and annotated by identifying open reading frames (ORFs) with a methionine START codon and performing BLASTx searches of each ORF against the NCBI data base. Differences between the genomes were analysed by generating dotplots and using Base-By-Base which showed them to be mostly collinear, but regions of difference were observed at the termini and the CCR. MGF analysis using sorting and clustering in Morpheus software, based on genotype, serogroup, country, host, virulence, and year, showed that genotype and serogroup play a role in the MGF arrangement patterns. Loci corresponding to regions of difference in the CCR were used for phylogenetic comparison to the previously identified marker p72. The tree topology of all of the alternative phylogenies differed from the current p72 classification. B117L and B169L provided slightly better resolution of genotypes I and II, respectively, and viruses from East Africa that are classified as belonging to genotype IX based on p72 were separated when using EP364R. This data adds to the pool of diverse ASFV isolates available for comparative genomics studies, and to the knowledge of ASFV in Africa. The sequencing of more diverse ASFV isolates of each genotype will help characterise the MGFs arrangement patterns among isolates. The novel alternative phylogenetic markers should further be investigated using more ASFV isolates representing the 24 genotypes described to date.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/33876 |
| Date | 14 September 2021 |
| Creators | Ndlovu, Sandy Sibusiso |
| Contributors | Williamson, Anna-Lise, Carulei, Olivia, Heath, Livio |
| Publisher | Faculty of Health Sciences, Department of Clinical Laboratory Sciences |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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