The document discusses a workshop focused on the genome sequencing of the African Swine Fever Virus (ASFV), detailing its genetic characteristics, pathogenicity, and comparative analyses of various ASFV isolates. It outlines significant findings on the evolutionary relationships and phylogenetic clustering of these isolates, with an emphasis on genes under positive selection and ongoing genome sequencing efforts for additional Kenyan isolates. Acknowledgments are made for contributions from various research institutions and contributors involved in the study.

1. ASFV genome sequencing
Etienne de Villiers
International Livestock Research Institute
ASFV CSIRO-AusAID workshop, 20th-21 July 2011, Nairobi

2. African Swine Fever Virus
• African swine fever (ASF) virus
– an acute, highly contagious and often fatal disease of
domestic pigs
– a large cytoplasmic virus of the family Asfaviridae
– linear double-stranded DNA genome (170-190 kbp)
• To identify the putative genetic basis for the
differences in virulence, and potential diagnostic
antigens, we are comparing the genome sequence of
non-pathogenic and pathogenic ASF virus isolates.

3. ASFV genomes
• The first complete ASFV genome was generated from the
avirulent VERO cell culture adapted isolate BA71V.
• In 2008 a virulent isolate from Benin and an avirulent tick
isolate from Portugal were sequenced and analysed by
Chapman et al. 2008.
• Seven additional complete genomes of southern and eastern
African origin are available in GenBank.
• Preliminary annotation is available on a publicly accessible
website
(http://athena.bioc.uvic.ca/database.php?db=asfarviridae).
• In 2010 we sequenced and annotated the complete genome
sequence of E75, a second virulent isolate classified within
p72 genotype I, originating from Spain using Roche 454
technology.

4. Evolutionary relationships of ASFV isolates
based on C-terminal sequence of p72
Eastern and Southern
African isolates

5. Phylogenetic analyses of ASFV isolates

6. Comparative genome analysis of Southern African
(A) and West African-European (B) genomes

7. Identification of core set of orthologous genes
• Several studies have shown that a concatenated
multi gene approach can resolve ambiguities in
phylogenetic reconstructions based on single genes.
• The amino acid sequences of the core set of
orthologous genes from each of the 11 ASFV isolates
were concatenated into a single pseudo-sequence.
• A neighbor-joining phylogenetic tree was
constructed from a multiple amino acid sequence
alignment of the concatenated sequences.

8. Phylogenetics based on core set of genes
• Phylogenetic analysis from 123 concatenated genes
separated the viruses into two major clusters that
correlate with their geographical distribution.
– West Africa - Iberian Peninsula (p72 genotype group I)
– Southern African ASFV isolates

9. Positive selection
• Investigated positive selection at the individual
amino acid sites.
• The most stringent model for positive selection, M8,
identified eighteen genes under positive selection.

10. Helicase gene
• The helicase gene, BA71VA859L is under positive
selection and might be a good phylogenetic marker.
• Trees based on this sequence reproduced the genetic
relationships indicated by analysis of the
concatenated set of orthologous proteins.

11. Genome sequence of additional
Kenyan ASFV isolates
• Ken.05 - tick isolate from a warthog burrow at Kapiti.
– p72 genotype X
• Ken.06 - virulent isolate from a 2006 outbreak in
Busia district
– Genotype IX

12. Ken.05 assembly
• Performed mapped assembly of Roche 454 data to
ASFV-Kenya genome
– numMappedReads = 4959, 7.31%
– numberOfContigs = 45
– numberOfBases = 185832
• Gap closure is ongoing

13. Neighbour-joining phylogenetic tree
constructed from Helicase genes

14. Ken.06 assembly
• Performed mapped assembly of Roche 454 data to
ASFV-Kenya genome
– numMappedReads = 1452, 2.00%
– numberOfContigs = 156
– numberOfBases = 154118

15. Genome sequence of additional
Kenyan ASFV isolates
• Gap closure of both Ken.05 and Ken.06 are ongoing.

16. Acknowledgments
• ILRI
– Richard Bishop
• CISA–INIA
– Carmina Gallardo
– Marisa Arias
– Raquel Martin
• University of Victoria
– Melissa da Silva
– Chris Upton
• Inqaba Biotec