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African Journal of Pig Farming ISSN 2375-0731 Vol. 2 (10), pp. 001-010, October, 2014. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 
 

Full Length Research Paper 

 

Genotyping of African swine fever virus (ASFV) 

isolates associated with disease outbreaks in 

Uganda in 2007 
 

Carmina Gallardo1*, Anna R. Ademun2, Raquel Nieto1, Noelina Nantima 2, Marisa 
Arias1, Elena Martín1, Virginia Pelayo1 and Richard P. Bishop3

  
1
Centro de Investigación en Sanidad Animal, INIA. Valdeolmos, Madrid 28130, Spain. 

2
Ministry of Agriculture, Animal Industry and Fisheries. Entebbe, Uganda. 

3
International Livestock Research Institute (ILRI), P. O. Box 30709, Nairobi, Kenya. 

 
Accepted 11 January, 2014 

 
Samples from infected domestic pigs associated with an outbreak of African swine fever (ASF) in three districts of 
central Uganda in 2007 were confirmed as being infected with African swine fever virus (ASFV) using a P72 gene- 
based polymerase chain reaction amplification (PCR) assay combined with restriction analysis. None of the sera 
collected from pigs with clinical symptoms were positive using the OIE serological prescribed tests. However, seven 
haemadsorbing viruses were isolated in macrophage culture and genotyped by partial p72 and full length p54-gene 
sequencing. Four of these viruses were isolated directly from serum samples. All the viruses were classified within 
the domestic-pig cycle- associated p72 and p54 genotype IX which also includes viruses responsible for ASF 
outbreaks in Kenya in 2006 and 2007 and Uganda in 2003. To define virus relationships at higher resolution, typing 
was performed by analysis of tetrameric amino acid repeat regions within the central variable region (CVR) of the 
B602L gene. Ugandan isolates sequences exhibited 100% identity to viruses isolated from outbreaks in Kenya in 2007. 
The identity was greater than the viruses obtained from an earlier outbreak in Kenya in 2006. This provides further 
evidence that genetically similar ASFV virus within p72 Genotype IX may be circulating between Kenya and Uganda. 
  

Key words: African swine fever virus (ASFV), restriction analysis, serological detection, genotyping, p72, p54, central variable 

region (CVR). 

 
INTRODUCTION 

 
African swine fever (ASF) is caused by a virus, ance-strally 
associated with argasid ticks and wild suids that can infect 
domestic pigs, frequently resulting in a rapidly lethal disease. 
However, depending on the combination of virus genotype 
and pig breed a range of outcomes varying from acute to 
chronic disease and apparently asymptomatic animals that 
are carriers of the virus can be observed. Virulent genotypes 
induce acute haemor-rhagic disease, with symptoms 
including high fever, heamorrhages in the skin and internal 
organs and death within three to ten days.  

ASFV is classified within the Asfarviridae family, genus 

Asfivirus (Dixon et al., 2000, 2005). It is highly contagious 

and can spread very rapidly in pig populations by direct or 

indirect contact. The disease has been reported in  
 
 
 
*Corresponding author. E-mail: gallardo@inia.es 

 
 
 
 
more than twenty African countries, where the virus is 
maintained either through a sylvatic cycle involving warthogs 
( Phacochoerus aethiopicus) and soft ticks in the genus 
Ornithodoros or in a domestic cycle that involves pigs of 
local breeds, with or without tick involvement (Haresnape 
and Wilkinson, 1989; Plowright and Pierce, 1969; Sanchez-
Botija, 1963; Thomson, 1985). ASF is endemic in most of 
sub-Saharan Africa, including the island of Madagascar; the 

highest incidence of disease being recorded from the 
equator to the northern Transvaal in southern Africa. 
Disease outbreaks have also occurred in Europe, South 
America and the Caribbean. In 2007, it was introduced 
into Georgia, most probably through infected pig-meat 
that was unloaded from a ship, recycled and fed to local 
pigs and has since spread throughout the Caucasus and 
into southern Russia (Rowlands et al., 2008).  

Uganda, situated in the eastern-central part of Africa, is 

divided into 80 districts and shares borders with Kenya, 



 
 
 

 

the Sudan, DRC, Rwanda and Tanzania. Currently, 
African swine fever is officially described as endemic in 
the whole country and outbreaks are reported spo-
radically, but without quantitative data on prevalence. 
Although, no new ASF outbreaks have been officially 
notified since 2004 (World Animal Health Organization-
World Organisation for Animal Health (WHO-OIE), occur-
rence of hemorrhagic disease has been reported regu-
larly to the Ugandan veterinary authorities by pig farmers. 
A more recent ASF outbreak occurred in Uganda-Kenyan 
border (Busia district) in 2007, placing a significant 
proportion of the Ugandan pig population at risk. In 
October 2007, an outbreak of virulent hemorrhagic 
disease was notified in three districts of Central Uganda, 
Mukono, Wakiso and Nakasongola. Following these outbreaks, 
blood, serum and tissue samples from pigs with clinical 
signs of hemorrhagic disease were collected for African 
swine fever diagnosis and molecular charac-terization. 

 

We report here the diagnosis and genetic charac-
terization of ASF viruses collected during outbreaks in 
Uganda in 2007. 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 re-
solution 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 em-
phasizes the value of molecular epidemiology for tracing 
the source and dynamics of ASF infections. 

 

MATERIALS AND METHODS 
 
Sampling 
 
During October 2007, the presence of virulent hemorrhagic disease 
suspected as being caused by ASFV was described by Uganda 
authorities in Central Uganda districts of Mukono, Wakiso and 
Nakasongola. Eighty four samples were collected by Ministry of 
Agriculture, Animal Industry and Fisheries (MAAIF) staff from pigs 
with clinical symptoms and submitted to the Animal Health Re-
search Centre (CISA-INIA), Valdeolmos, Spain (EU ASF Reference 
Laboratory). Samples collected comprised sera from 39 pigs 
(Wakiso n = 13; Nakasangola n = 11; Mukono n = 15), EDTA blood 
samples from 39 pigs (Wakiso n = 11; Nakasangola n = 9; Mukono 
n = 19) and six tissue samples from one pig (Wakiso n = 6). They 

were received at CISA-INIA on 23
rd

 October 2007. 

 
ASF diagnosis 
 
ASF antibody detection 
 
39 field sera were tested using OIE (2008) prescribed tests (con-
ventional ELISA and Immunoblotting assays) using as antigen a 
lysate of monkey stable (MS) cells infected with ASF Spanish 
isolate E70MS48 and protein-A/HRPO as the reporter (OIE 2008). 

  
  

 
 

 
Nucleic acid extraction and genomic DNA amplification 
 
DNA was extracted directly from 35 sera, 39 blood samples and 
10% suspensions of 6 ground tissues (from one animal) using a 
nucleic acid extraction kit (Nucleospin/ Machery-Nagel–Cultek) 
following the manufacturer’s procedures. A polymerase chain 
reaction amplification (PCR) assay using the ASF diagnostic 
primers PPA1/PPA2, which generates an amplicon of 257 bp within 
the p72 protein (Aguero et al., 2003), was used to confirm the 
presence of ASFV DNA. The PCR products were analyzed by 
electrophoresis through 2% agarose gels visualized under UV light. 

 
Restriction enzyme analysis 
 
The NdeI restriction endonuclease (New England Biolabs) was 
used to analyze the DNA amplified by PCR. Briefly, 10 µl of the 
PCR sample was digested for 1 h at 37°C with 8 to 20 U of 
restriction enzyme in a total reaction volume of 20 µl in the appro-
priate restriction enzyme buffer. Aliquots of 10 µl of each reaction 
mixture were size fractionated on a 3% agarose gel and the 
restriction patterns were visualized under UV light. 

 
Virus isolation 
 
Primary leukocyte cultures were used for the isolation of samples 
recovered from naïve domestic pigs as previously described 
(Malmquist and Hay, 1960). Briefly, cells were seeded into 96-well 
tissue culture grade microtitre plates (200 µl; 300,000 cells per well) 
in homologous swine serum and incubated in a humidified atmo-
sphere containing 5% CO2 at 37°C. Three day cultures were 
infected at 1:10 dilution with serum or 10% suspensions of ground 
tissues supplemented with 5 µg/ml gentamycine sulphate 
(BioWhittaker) and incubated for 24 h at 37°C. After inoculation, a 
preparation of 1% homologous red blood cells (RBC) in buffered 
saline was added to each well. The plates were examined for 
haemadsorption over a 6 day period. The samples were blind 
passaged three times. 

 

ASF molecular characterization 
 
Genomic amplification and nucleotide sequencing 
 
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 C-
terminal 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. The nucleotide sequence of the purified 
products was determined using SP6/T7 primers specific for the 
pGEMT vector using an automated 3730 genetic analyzer (Applied 
biosystems). 

 
Sequence analysis 
 
Analysis of sequence data was performed with Chromas 
(www.technelysium.com.au), BioEdit (www.mbio.ncsu.edu/ BioEdit/ 
BioEdit.html) and ClustalX version 1.83 (www.clustal.org) . For the 
TRS, analyses including that of the CVR sequences and deduced 
amino acid sequences were manually aligned with gaps being 



 
 
 

 
Table 1. ASFV Uganda isolates obtained from 2007 outbreak characterized in this study.  

 
Isolate name Id. sample Kind of sample Origin 

 

  sequenced  
 

UG07.Wak1 44a Blood  
 

UG07.Wak2 Wakiso 46 Sera 
SSEBUGWAWO mixed farm, Wakiso district Kinoni  

UG07.Wak3 Wakiso 0656 Sera  

 
 

UG07.Wak4 1128 lymph node Tissue  
 

UG07.Mukono 20 Blood Katumba wamala farm, Mukono district 
 

UG07.F7 63/Fattener 7 F7 Sera 
Kinoni produce farm, Nakasongola district  

UG07.F8 63/Fattener 8 F8 Sera  

 
 

 

 
inserted to optimize the alignment. Two datasets were generated 
for phylogenetic analyses conducted using MEGA version 4.0 
(Kumar et al., 2001) (i) A p72-gene dataset comprising 73 taxa (404 
characters) in which p72 nucleotide sequences generated in this 
study from the Ugandan pig viruses were analyzed together with 
homologous sequences from at least two viruses representative of 
each of the 22 p72 genotypes identified in a previous study (Boshoff 
et al., 2007), (ii) A p54-gene dataset comprising 155 taxa in which 
p54 sequences generated in this study were compared with 
homologous sequences available in GenBank. Neighbor joining 
(NJ) and minimum evolution (ME) p72 and p54 trees were 
constructed employing the p-distance nucleotide substitution model 
as implemented in the MEGA v4.0 program. To determine the 
degree of statistical support for each node in the resulting p72 and 
p54 trees, data were re-sampled 1000 times using the bootstrap 
method. 
 

 

RESULTS 

 

ASF diagnosis 

 

Among the 39 porcine serum samples received at CISA 
from the Central Ugandan Districts, ASFV antibody 
detection performed using the OIE-prescribed ELISA and 
immunoblotting (IB) assays revealed that, none of them 
were antibody-positive for ASF.  

For ASFV detection, PCR was performed on 35 serum 
samples, 39 blood samples and also 6 individual tissues 
from one of the sampled animals. Following PCR ampli-
fication, a single major amplicon of approximately 260 bp 
was generated from 9 of the 35 sera, 17 of the 39 blood 
samples and all tissue samples examined. Virus isolation 
was performed using homogenenised pooled tissues and 
also sera and blood from each PCR positive animal. 
Seven ASFV strains with a haemadsorption pattern 
typical of virulent ASF viruses were isolated from the 
Ugandan samples after three passages in leukocytes 
(Table 1). In four cases these virus isolates were made 
directly from serum samples that were negative accor-
ding to the OIE ELISA and IB (Table 1). DNA was 
extracted from the cell cultures and the presence of the 
ASF virus was confirmed using diagnostic PCR. The 
sequencing and compared with sequences available in 

 

 

the GenBank and with additional sequences obtained in a 
separate study from ASF isolates recovered during ASF 
outbreaks that occurred in 2003 and 2006 to 2007, in 
Uganda and Kenya, respectively (previously unpublished 
data held at CISA-INIA). Nucleotide sequence analysis of 
the genome region delimited by the PPA- 1/2 primers 
revealed an NdeI restriction endonuclease site, which 
which could generate two fragments of 204 and 53 bp in 
length. NdeI digestion of the amplicons from the Ugandan 
ASFV isolates resulted in generation of the two expected 
specific restriction fragments (Figure 1). This site is 
conserved only in ASFV isolates obtained from eastern 
African countries, while in sequences from European, 
Caribbean, West and South African countries a mutation 
is present at bp 54 within the NdeI restriction site (Table 
2). 
 

 

ASF molecular characterization 

 

Analysis of the p72-gene partial sequences from each of 
the seven Uganda ASFV isolates showed that, they were 
identical at the nucleotide level (results not shown). 
Comparison of these sequences to the 22 (I-XXII) 
currently known major p72 genotypes (Boshoff et al., 
2007) identified the Uganda 2007 sequences as falling 
within p72 genotype IX (Figure 2).  
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 sequences of the 7 Uganda 
isolates were compared with 155 p54 ASFV sequences 
available in GenBank. As was the case with the p72-
genotyping, the Uganda isolates were clustered together 
with Ugandan and Kenyan viruses obtained from 
domestic pigs (Figure 3). 

In an attempt to discriminate genotypes at higher reso-

amplified products were characterized by nucleotide 

lution, the CVR sequences of viruses from Uganda were 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. NdeI restriction endonuclease analysis of PPA1/2 amplicons derived from different 
representative ASFV isolates. Lanes 1 and 1´, UG07.Wak.1; 2 and 2´ UG07.Wak4; 3 and 3´, 
UG07.Mukono; 4 and 4´, UG07.F7; 5 and 5´, Ken06.Bus; 6 and 6´, Ken07.Eld1; 7 and 7´, Ug03H1; 
8 and 8´, E70; 9 and 9´, Cam; 10 and 10´, Za. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 show 
amplification products. Lanes 1´, 2´, 3´, 4´, 5´, 6´, 7´, 8´, 9´ and 10´ show amplification products after 
digestion with NdeI. Reaction conditions are described in the text. M, molecular weight marker VI. 

 

 
Table 2. Partial nucleotide sequences showing residues 1 to 60 within the p72- gene PCR amplicon used for ASFV diagnosis. The NdeI 

restriction site conserved in Kenya and Uganda ASFV isolates is highlighted in grey.  
 
 Isolate P72 Country of    3´   5´ nucleotide sequences from p72- gene P72-based diagnosis Reference 

 

  genotype origin PCR diagnosis product Genbank  
 

          accession no.  
 

 E70 I Spain AGTTATGGGAAACCCGACCCCGAACCCACTT AY578692 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT        
 

 E75 I Spain AGTTATGGGAAACCCGACCCCGAACCCACTT AY578693 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT        
 

 Ba71V I Spain AGTTATGGGAAACCCGACCCCGAACCCACTT U18466 Complete genome 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT        
 

 Haiti I Haiti AGTTATGGGAAACCCGACCCCGAACCCACTT AY578695 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT        
 

 Tengani62 V Malawi AAGTACGGGAAACCCGACCCCGAACCCACTT AY578704 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTG        
 

 M1 III South Africa AGTTATGGGAAACCCGACCCCGAACCCACTT AY578699 Zsak et al. (2005) 
 

   

DRC
*
 

TGAGTCAAATCGAAGAAACGCATTTGGTG      
 

 Za I AGTTATGGGAAACCCGACCCCGAACCCACTT AY578708 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT       
 

 Mk I South Africa AGTTATGGGAAACCCGATCCCGAACCCACTTT AY578700 Zsak et al. (2005) 
 

    GAGTCAAATCGAAGAAACACATTTGGTG     
 

 Wart IV Namibia AGTTATGGGAAACCCGACCCCGAACCCACTT AY578706 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTG      
 

 Cam I Cameroon AGTTATGGGAAACCCGACCCCGAACCCACTT AY578689 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTT      
 

 Vic I Zimbabwe AGTTATGGGAAACCCGATCCCGAACCCACTTT AY578705 Zsak et al. (2005) 
 

    GAGTCAAATCGAAGAAACACATTTGGTG     
 

 Wb III South Africa AGTTATGGGAAACCCGACCCCGAACCCACTT AY578707 Zsak et al. (2005) 
 

    TGAGTCAAATCGAAGAAACACATTTGGTG      
 

 Cro1.2 IV South Africa AGTTATGGGAAACCTGACCCCGAACCCACTTT AY578690 Zsak et al. (2005) 
 

    GAGTCAAATCGAAGAAACACATTTGGTG     
 

 Cro 3.5 IV South Africa AGTTATGGGAAACCTGACCCCGAACCCACTTT AY578691 Zsak et al. (2005) 
 

    GAGTCAAATCGAAGAAACACATTTGGTG     
 



 
      

  Table 2. Continued.        
             

   F6     III South Africa AGTTATGGGAAACCCGACCCCGAACCCACTT AY578694 Zsak et al. (2005) 
          TGAGTCAAATCGAAGAAACACATTTGGTG     

   K1     III South Africa AGTTATGGGAAACCCGACCCCGAACCCACTT AY578696 Zsak et al. (2005) 
          TGAGTCAAATCGAAGAAACACATTTGGTG     

   O1     III South Africa AGTTATGGGAAACCCGACCCCGAACCCACTT AY578701 Zsak et al. (2005) 
          TGAGTCAAATCGAAGAAACACATTTGGTT     

   Pr4     XX South Africa AGTTATGGGAAACCTGACCCCGAACCCACTT AY578702 Zsak et al. (2005) 
          TGAGTCAAATCGAAGAAACACATTTGGTG     

   Pr5     XX South Africa AGTTATGGGAAACCTGACCCCGAACCCACTT AY578703 Zsak et al. (2005) 
          TGAGTCAAATCGAAGAAACACATTTGGTG     

   Ug03H.1 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916933 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ug03P.4     IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916934 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.Wak1 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916926 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.Wak2 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916927 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.Wak3 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916928 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.Wak4 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916929 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.Mukono IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916930 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.F7 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916931 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   UG07.F8 IX Uganda AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916932 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Kenya1950 X Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT AY261360 Complete genome 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.B1 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916935 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.B2 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916936 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.B3 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916937 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.B4 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916938 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.B5 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916939 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.Bus IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916940 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken06.Kis IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916941 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken07.Eld1 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916942 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken07.Eld2 IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916943 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken07.Kia IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916944 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

   Ken07.Nak IX Kenya AGTTATGGGAAACCCGACCCCGAACCCACTT GQ916945 This study 
          TGAGTCAAATCGAAGAAACACATATGGTG   

 

 

determined. Amplification of the CVR fragment generated 

PCR products of approximately 350 b from all Ugandan 

 

 

isolates. As in the case of the other genes, the Uganda 

isolates characterized in this study clustered with the 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Minimum evolution (ME) tree representing the 22 p72 genotypes that distinguish African swine 
fever viruses (labelled I-XXII) illustrating the phylogenetic position of the Ugandan isolates from 2007. 
The optimal tree with the sum of branch lengths = 0.23816201 is shown. The percentage of replicate 
trees in which the associated taxa clustered together according to bootstrap analysis (1000 replicates) is 
marked adjacent to the nodes. The ME tree was searched using the Close-Neighbor - Interchange (CNI) 
algorithm at a search level of 1. A neighbor-joining algorithm was used to generate the initial tree. The 
seven Ugandan viruses characterized in this study are highlighted in grey ( ) within genotype IX (marked 
in grey). 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Minimum Evolution phylogenetic tree based on full length p54 gene sequences illustrating the position of Uganda 
2007 isolates relative to other ASFV viruses. An unrooted minimum evolution tree was derived from alignments of complete 
p54-gene nucleotide sequences. The ME tree was searched using the Close- Neighbor-Interchange (CNI) algorithm at a 
search level of 1. A Neighbor-joining algorithm was used to generate the initial tree. The seven Ugandan viruses 
characterized in this study are highlighted in black ( ). 

 

 

Uganda isolates (UGA 95/1; Ug03H1-3, Ug03P4-6) and 
Kenya isolates (Ken06B1-5, Ken06.Bus, Ken06.Kis, 
Ken07Eld1-2, Ken07.Nak, Ken07.Kia) within CVR sub-
group XXIV. Differences were identified mainly in the 
number of tetrameric amino acid repeats. When the tetra-
meric repeats within the B602L gene from the Uganda 

isolates were compared with viruses included within the 

 
 

 

CVR sub-group XXIV (Nix et al., 2006), the isolates from 
Uganda obtained in October 2007, contained a minor 
change due to the absence of a single internally located 
tetrameric repeat (CADT) present in the viruses 
recovered from earlier Uganda outbreaks that occurred in 
2003 and in the Kenyan ASF Visolates obtained in May 
2006 (Ken06.B1-5). However, they were identical to 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. Amino acid sequence alignment of tetrameric tandem repeats within the central variable region (CVR) of gene B602L from Ugandan and Kenyan p72 group IX isolates 

associated with recent outbreaks. The isolates from the Ugandan 2007 outbreaks are underlined. The Ugandan 2007 isolate sequences determined in this study were aligned with 
additional Kenyan and Ugandan CVR sequences available in GenBank (UGA95/1, Accession No. CAJ90783; Ug03H1-3 and Ug03P4-6 Accession Nos FJ174339-44; Ken06.B1-B5, 

Ken06.Bus, Ken06.Kis, Ken07Eld1-2, Ken07.Nak, Ken07.Kia, Accession Nos FJ174329-39) belonging to B602L sub-group XXIV. 
 

 

those obtained from the second outbreak that 
occurred in western Kenya in 2006 (Ken06.Bus) 
and to all isolates from subsequent outbreaks in 
the country (Figure 4).  
DISCUSSION 

 

Outbreaks of African swine fever have been 

reported in Uganda sporadically, but regularly in 

 
 

 

specific areas since 1996 and the pig industry has 
been very significantly affected. The last major 
outbreak, according to the OIE reports was in 
2004 when an estimated 7,500 pigs were 
destroyed or slaughtered in twelve districts across 
the country. Since then, outbreaks of ASF have 
occurred on a regular basis in Uganda, causing 
serious losses to both smallholder pig keepers 

 
 

 

and commercial pig farms. At present, ASF is 
officially described as "present in the whole 
country but without quantitative data on the pre-
valence of the virus". In October 2007, a 
suspected African swine fever outbreak was 
reported by farmers in three central Uganda 
districts. Sampling in these areas in collaboration 
with the Uganda MAAIF resulted in confirmation of 



 
 
 

 

gene based PCR and virus isolation at the CISA-INIA EU 
reference laboratory. Subsequent nucleotide sequence 
determi-nation of the PCR product allowed identification 
of a new NdeI restriction site, specific for a particular 
group of viruses belonging to east African countries. The 
res-triction fragment pattern enables the specificity of the 
amplicons to be validated within hours. However, the 
method does not permit unambiguous classification of 
virus isolates because of the high variability observed in 
east African ASVF isolates. Although, nucleotide sequen-
cing of the p72 amplicon would be more definitive, the 
technique is expensive, time consuming and less 
universally available in African laboratories. Restriction 
digestion with Nde1 therefore, represents a useful rapid 
method for confirmation of the presence of ASFV 
following p72 PCR.  

Interestingly, no positive antibody response was 
detected using OIE-prescribed serological methods in 
any of the serum samples collected from these out-
breaks. This data reconfirms previous studies that des-
cribe a low incidence of detectable serological response 
to ASFV infection in pigs from east African countries 
using both recombinant antigen and OIE-prescribed 
African swine fever serological methods (Perez-Filgueira 
et al., 2006). Since the OIE-prescribed methods are 
based on the use of total ASF virus as antigen that 
includes many viral proteins, a possible explanation for 
the unexpectedly low seropositive response with east 
African sera may reside in the immunogenetics of the 
indigenous pig populations in the region and is unlikely to 
be due to polymorphisms in immunodominant viral anti-
gens. The area of host-virus interaction, including 
possible differences in both innate and acquired immune 
responses therefore, represents an important area for 
future research on African swine fever (ASF).  
To characterize the viruses responsible for the out-break 
of ASF in Uganda in 2007 and identify genetic 
relationships with recent ASFV isolates from neighboring 
countries, the sequence of three regions of the ASFV 
genome was determined. Sequence analysis of the C-
terminal end of p72-gene and complete p54-gene placed 
the Uganda isolates within genotype IX together with 
isolates from Kenya and Uganda. This is a domestic pig-  
associated genotype that contains 8 isolates from Western 

and Eastern Uganda (UGA 1/95; Ug03H1-3, Ug03P4-6, 

UGA2003/1), isolated from outbreaks in 1995 and 2003 

(Bastos et al., 2003; Gallardo et al., 2009) and 11 isolates 

from Western ands Central Kenya (Ken06B1-5, Ken06.Bus, 

Ken06.Kis, Ken07Eld1-2, Ken07.Nak, Ken07.Kia) obtained 

from outbreaks in 2006 to 2007 (Gallardo et al., 2009). One 

additional genetic type of ASFV was also identified in this 
study as having occurred in Uganda; this is classified within 

p72 genotype X that comprises viruses recovered from both 

domestic and wild pigs and the arthropod tick vector. 
 

The data generated using three genes indicated that, 

African swine fever outbreaks occurring in the three 

viruses whilst the 1995 outbreak was caused by two diffe- 

 
 
 
 

 

rent viruses belonging to p72 genotypes IX and X 
(UGA/1/95 and UGA/3/95) (Boshoff et al., 2007; Lubisi et 
al., 2005).  

Sequence analysis of the central variable region (CVR) 
from the Ugandan isolates identified 22 different 
tetrameric amino acid units. The sequence of the CVR 
from the Ugandan isolates was identical to the sequence 
of 6 isolates responsible for the second wave of infec-
tions that occurred in Western and Central Kenya from 
October 2006 to January 2007, but was distinguishable 
from Kenyan isolates recovered from an earlier outbreak 
in May 2006 and from Ugandan viruses recovered from 
the outbreaks that occurred in Western and Eastern 
Uganda in 2003. A deletion of one tetrameric repeat that 
was observed in viruses recovered from the 2007 Ugan-
dan outbreak was also present in viruses responsible for 
an outbreak that occurred in the Uganda-Kenya border 
district, subsequently spreading to central Kenya, in 2006 
to 2007. This is consistent with a single origin for the 
Kenyan 2006 to 2007 and Ugandan 2007 outbreaks, if 
there is rapid cross-border movement of the virus. The 
data from the CVR analysis are consistent with the 
hypothesis that, ASFV virus exchange between the two 
countries has occurred on more than one occasion, 
although, an alternative explanation would be that identi-
cal mutations have occurred independently in the central 
variable region of two closely related virus lineages. Such 
information should be proved useful to veterinary 
authorities for better disease control and highlight the 
requirement for improved regulation and monitoring of 
domestic pig movement and the pork product trade. 
 

 

ACKNOWLEDGEMENTS 
 
The present work was partly supported under the grant 
ILRI2003-001 funded by the Spanish Ministerio de 
Innovacion y Ciencia and by the European Union through 
the network of excellence "EPIZONE” and also the 
ASFRISK project (Grant Agreement) 211691. We greatly 
appreciate the intellectual and practical contributions of 
our colleagues at Centro de Investigación en Sanidad 
Animal (CISA-INIA) and International Livestock Research 
Institute (ILRI). This ILRI publication number IL-201003. 

 
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