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Multi-locus sequence typing of African swine fever viruses from endemic regions of Kenya and Eastern Uganda (2011-2013) reveals rapid B602L central variable region evolution.

ABSTRACT: The central variable region (CVR) within the B602L gene of the African swine fever virus (ASFV) is highly polymorphic within the 23 ASFV genotypes defined by sequencing of the C-terminal end of the p72 locus. Sequencing the p54 gene further discriminates ASFV genotypes that are conserved at the p72 locus. Variation in the thymidine kinase locus is a novel additional tool for ASFV genotyping whose application for this purpose is described for the first time herein. We evaluated genetic variation at these four polymorphic loci in 39 ASFV isolates obtained from outbreaks in Kenya and a region of Eastern Uganda between 2011 and 2013. Analysis of the p72 and p54 loci revealed high genetic conservation among these isolates; all clustered within p72 genotype IX and were similar to isolates associated with earlier outbreaks in East Africa. The thymidine kinase gene of the Kenyan isolates in this study were distinct relative to Southern African isolates and synonymous substitutions were observed among viruses from central Kenya. Analysis of the CVR within the B602L gene revealed two previously unknown polymorphisms that were restricted to Western Kenya and Eastern Uganda. A novel variant was revealed within CVR subgroup XXIV and a novel CVR subgroup XXIVa that contains tetrameric repeat F which has previously only been associated with p72 genotype I, was also identified for the first time in East Africa. Phylogeographic analysis of isolates based on CVR polymorphisms revealed rapid evolution and dissemination of variants present within ASFV genotype IX in East Africa.

SUBMITTER: Onzere CK

PROVIDER: S-EPMC5847163 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Multi-locus sequence typing of African swine fever viruses from endemic regions of Kenya and Eastern Uganda (2011-2013) reveals rapid B602L central variable region evolution.

Onzere C K CK Bastos A D AD Okoth E A EA Lichoti J K JK Bochere E N EN Owido M G MG Ndambuki G G Bronsvoort M M Bishop R P RP

Virus genes 20171115 1

The central variable region (CVR) within the B602L gene of the African swine fever virus (ASFV) is highly polymorphic within the 23 ASFV genotypes defined by sequencing of the C-terminal end of the p72 locus. Sequencing the p54 gene further discriminates ASFV genotypes that are conserved at the p72 locus. Variation in the thymidine kinase locus is a novel additional tool for ASFV genotyping whose application for this purpose is described for the first time herein. We evaluated genetic variation ...[more]

PMID: 29143187

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Project description:BackgroundAfrican swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in Eastern Africa particularly in Uganda where outbreaks regularly occur. Sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of African swine fever virus (ASFV) isolates. By combining p72, P54 and pB602L (CVR), a high level resolution approach is achieved for viral discrimination. The major aim of this study therefore, was to investigate the genetic relatedness of ASF outbreaks that occurred between 2010 and 2013 in Uganda to contribute to the clarification of the epidemiological situation over a four year period.MethodsTissue samples from infected domestic pigs associated with an ASF outbreak from 15 districts in Uganda were confirmed as being infected with ASFV using a p72 gene-based polymerase chain reaction amplification (PCR) assay recommended by OIE. The analysis was conducted by genotyping based on sequence data from three single copy ASFV genes. The E183L gene encoding the structural protein P54 and part of the gene encoding the p72 protein was used to delineate genotypes. Intra-genotypic resolution of viral relationships was achieved by analysis of tetramer amino acid repeats within the hypervariable CVR of the B602L gene.ResultsTwenty one (21) ASF outbreaks were confirmed by the p72 ASF diagnostic PCR, however; only 17 isolates were successfully aligned after sequencing. Our entire isolates cluster with previous ASF viruses in genotype IX isolated in Uganda and Kenya using p72 and P54 genes. Analysis of the CVR gene generated three sub-groups one with 23 tetrameric amino acid repeats (TRS) with an additional CAST sequence, the second with 22 TRS while one isolate Ug13. Kampala1 had 13 TRS.ConclusionWe identified two new CVR subgroups different from previous studies. This study constitutes the first detailed assessment of the molecular epidemiology of ASFV in domestic pigs in the different regions of Uganda.

Project description:Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

Dataset Information

Multi-locus sequence typing of African swine fever viruses from endemic regions of Kenya and Eastern Uganda (2011-2013) reveals rapid B602L central variable region evolution.

Publications

Multi-locus sequence typing of African swine fever viruses from endemic regions of Kenya and Eastern Uganda (2011-2013) reveals rapid B602L central variable region evolution.

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