Presented by Etienne de Villiers at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011

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