ASFV genome sequencing Etienne de Villiers International Livestock Research InstituteASFV CSIRO-AusAID workshop, 20th-21 July 2011, Nairobi
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.
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.
Evolutionary relationships of ASFV isolates based on C-terminal sequence of p72 Eastern and Southern African isolates
Comparative genome analysis of Southern African (A) and West African-European (B) genomes
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.
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
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.
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.
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
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
Neighbour-joining phylogenetic tree constructed from Helicase genes
Ken.06 assembly• Performed mapped assembly of Roche 454 data to ASFV-Kenya genome – numMappedReads = 1452, 2.00% – numberOfContigs = 156 – numberOfBases = 154118
Genome sequence of additional Kenyan ASFV isolates• Gap closure of both Ken.05 and Ken.06 are ongoing.
Acknowledgments• ILRI – Richard Bishop• CISA–INIA – Carmina Gallardo – Marisa Arias – Raquel Martin• University of Victoria – Melissa da Silva – Chris Upton• Inqaba Biotec