Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes

Abstract

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach.

Genotyping of African swine fever virus (ASFV) isolates associated with disease outbreaks in Uganda in 2007

“…For molecular epidemiological analysis, DNA was extracted from cell cultures and three separate polymerase chain reactions (PCRs) were set up; for p72 genotyping classification amplifying the Cterminal region of p72 protein using the primers p72-U/D as previously described (Bastos et al, 2003) for p54 genotyping amplifying the complete gene encoding the p54 protein using the primers PPA722/PPA89 (Gallardo et al, 2009); for CVR sub-typing using the primer pairs ORF9L-F/9L-R to amplify the CVR located in the B602L gene (Nix et al, 2006). Amplicons of the expected size were excised, purified by Quiaex gel extraction (QIAGEN) and cloned into a pGEMT-Easy vector (Promega) according the manufacturer's instructions.…”

“…Recent studies have demonstrated the value of p54-gene sequencing as an additional, intermediate-reso-lution, molecular epidemiological tool for typing of ASFV viruses (Gallardo et al, 2009). Amplification of the fragment containing the complete p54-gene from all the Ugandan isolates produced PCR products of 550 bp, which were identical in sequence.…”

“…The genotyping involved sequencing the 3' end of the gene encoding the p72 protein (Bastos et al, 2003;Boshoff et al, 2007;Lubisi et al, 2005) and the full length p54-gene (Gallardo et al, 2009) to place isolates into major subgroups, followed by higher resolution sub-typing through analysis of tandem repeat sequences (TRS) in the central variable region (CVR) of the ASFV B602L gene (Irusta et al, 1996;Lubisi et al, 2007;Nix et al, 2006;Phologane et al, 2005). The very close genetic similarity of isolates associated with recent disease outbreaks in both Uganda and Kenya emphasizes the value of molecular epidemiology for tracing the source and dynamics of ASF infections.…”

Risk factors for farm-level African swine fever infection in major pig-producing areas in Nigeria, 1997–2011

“…Intensive efforts have been made in the use of genetic epidemiology to analyse the ASF viruses circulating in different parts of Africa in order to gain an significant understanding of the relation between and geographic spread of each circulating genotype (Bastos et al, 2003;Bastos et al, 2004;Lubisi et al, 2005;Boshoff et al, 2007;Lubisi et al, 2007;Gallardo et al, 2009;Owolodun et al, 2010;Gallardo et al, 2011). However, the causes/factors that support the continued circulation of ASF viruses in pig herds in various parts of Africa in general and in Nigerian pig populations in particular remain poorly understood or at best hypothetical.…”

Molecular characterisation of African swine fever viruses from Nigeria (2003–2006) recovers multiple virus variants and reaffirms CVR epidemiological utility

“…Whilst alternative genes with greater intra-genotypic resolution capabilities than p72 have been identified [11,19], these alternative genome targets are of limited use for geographically and temporally constrained viruses such as those causing outbreaks in Kenya from 2006-7 [19]. Indeed, for the three genome regions compared in the Kenyan study, only the CVR recovered more than one virus variant [19], and it therefore remains the genome target of choice when attempting to determine the origin and map the spread of closely related virus. It was for this reason that, following genotype confirmation by p72 gene characterisation, that the CVR was used to investigate ASF outbreaks occurring between 2003 and 2006 in the main pig producing areas of Nigeria ( Fig.…”