ASFV genome sequencing

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

Text of ASFV genome sequencing

  • 1.ASFV genome sequencing Etienne de Villiers International Livestock Research InstituteASFV 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 thedifferences in virulence, and potential diagnosticantigens, we are comparing the genome sequence ofnon-pathogenic and pathogenic ASF virus isolates. 3. ASFV genomes The first complete ASFV genome was generated from theavirulent VERO cell culture adapted isolate BA71V. In 2008 a virulent isolate from Benin and an avirulent tickisolate from Portugal were sequenced and analysed byChapman et al. 2008. Seven additional complete genomes of southern and easternAfrican origin are available in GenBank. Preliminary annotation is available on a publicly accessiblewebsite(http://athena.bioc.uvic.ca/database.php?db=asfarviridae). In 2010 we sequenced and annotated the complete genomesequence of E75, a second virulent isolate classified withinp72 genotype I, originating from Spain using Roche 454technology. 4. Evolutionary relationships of ASFV isolatesbased 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 concatenatedmulti gene approach can resolve ambiguities inphylogenetic reconstructions based on single genes. The amino acid sequences of the core set oforthologous genes from each of the 11 ASFV isolateswere concatenated into a single pseudo-sequence. A neighbor-joining phylogenetic tree wasconstructed from a multiple amino acid sequencealignment of the concatenated sequences. 8. Phylogenetics based on core set of genes Phylogenetic analysis from 123 concatenated genesseparated the viruses into two major clusters thatcorrelate with their geographical distribution. West Africa - Iberian Peninsula (p72 genotype group I) Southern African ASFV isolates 9. Positive selection Investigated positive selection at the individualamino 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 positiveselection and might be a good phylogenetic marker. Trees based on this sequence reproduced the geneticrelationships indicated by analysis of theconcatenated 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 inBusia district Genotype IX 12. Ken.05 assembly Performed mapped assembly of Roche 454 data toASFV-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 toASFV-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 CISAINIA Carmina Gallardo Marisa Arias Raquel Martin University of Victoria Melissa da Silva Chris Upton Inqaba Biotec