This document summarizes activities related to analyzing the genetics of the African Swine Fever virus (ASFV) in Kenya. It discusses sequencing the whole ASFV genome to analyze diversity and origins of outbreaks. Genotyping using three genetic markers finds that recent Kenyan outbreaks involve genotype IX, the same genotype present in Uganda. While whole genome sequencing and genetic analysis can inform vaccine development and tracing outbreaks, developing low-cost, rapid field diagnostics remains a priority for controlling ASF. Surveillance of pigs in coastal Kenya may also be needed to prevent the spread of genotype IX globally.

1. ASF Virus Genomics and Diagnostics
2nd October 2013
Richard Bishop and Cynthia Onzere
African Swine Fever Epidemiology Project

2. Virus Prevalence and Diversity:
Selected Questions
• How diverse are ASFV isolates associated with
Kenyan disease outbreaks?
• What is most effective platform for monitoring
prevalence?
• How is the virus maintained in endemic areas?
• Is there a role of the sylvatic cycle involving wild
pigs and Argasid ticks in recent outbreaks?

3. Significance
• Rapid diagnosis of ASFV is critical for
implementation of control measures by veterinary
authorities
• Virus genotyping can contribute to identification
of origins and monitoring spread of outbreaks
• Information from whole genome sequencing will
underpin rational strategies for vaccine
development

4. Summary of portfolio of Activities
• Whole genome sequencing using Illumina
platform
• Genotyping using PCR-sequencing from three
polymorphic loci
• Diagnosis using DNA extracted from field pig blood
samples by conventional and real time PCR (in
laboratory and field)
• ELISA for detection of antibodies
• Virus isolation from pig tissue samples associated
with outbreaks

5. Genome Sequencing
Un-rooted tree derived from whole
genomes showing genetic
Analysis of complete
genome sequences has
shown that p72 genotype IX
viruses in East Africa from
2005-2013 Kenyan ASF
outbreaks in pigs cluster
with genotype X from pigs
and ticks.
These Kenyan and Ugandan
genotypes are distinct fro
other sequenced viruses

6. Summary Genome sequencing
• The complete genomes of genotype IX (from a
clinically reacting pig) and genotype X (from a tick
from a warthog burrow) cluster together
• Both genotypes are infective to domestic pigs but
genotype IX is more virulent
• Genotypes IX and X are sympatric at a single
Kenyan locality, in adult warthogs and ticks
respectively (Gallardo et al. 2011)

7. Rapid field diagnosis of ASFV
Kenya and Uganda veterinarians
at Project workshop in Kisumu,
July 2011:
• Testing labs are distant and
hard to access.
• It takes many weeks to get a
confirmed ASF diagnosis.
• The time lag hampers action
to contain ASF outbreaks.

8. ASFV diagnostic assays
Unknown disease causing deaths in pigs
-various diseases may be implicated; is ASF the cause?
Identification and validation of diagnostic assays
considerations
Specificity &
sensitivity
Reproducibility
Throughput
Cost
Test
speed
Availability
portability

9. DNA extraction in the field
 Magnetic beads (Roche Magna kit) and magnetic strips used for DNA extraction; the
Roche protocol modified to accommodate field parameters.
 Method of choice due to thermo stability of reagents and speed.

10. Comparison of nucleic acid-based
diagnostic platforms
Molecular platforms
ABI thermal cycler
(Applied biosystems)
Smart cycler
(Cepheid)
Tetracore
(Tetracore)
Piko real
(thermoscientific)

11. Comparison of molecular diagnostic
assays
Test
Types
Conventional
PCR:
Reagents/test
-
Real-time PCR
(qPCR):
UPL PCR:
-
TCOR PCR
-
Total cost/test
(USD)
Platforms
nucleotide mix (Roche)
Twelvepaq amplitaq gold
(Applied biosystems)
Primers
0.5 ml eppendorf tubes
-
3.421
-ABI thermal cycler
Cepheid tubes/piko real
plates
UPL # 162 probe (Roche)
Taqman master mix (Applied
biosystems)
primers
-
3.386 (smart
cycler)
2.32 (24 well
piko real)
2.25 (96 well
piko real)
-
pre packed reagents in
Cepheid tubes
> 10 using TCOR
kit
-
-
-
Cepheid Smart
cycler
Piko real
T COR
Cepheid Smart
Cycler

12. Serology
ELISA for Detection of anti-p72 antibodies
Blocking ELISA
 Ingenasa kit (Spain) used for detection
 Can currently only be performed in a laboratory
setup although lateral flow assay is under
development

13. Evaluation of field detection by qPCR
Here is
the Lab
Field laboratory test run from a basic set-up (i.e. table) or back of a vehicle
BSL-2 lab
BSL-3 lab

14. Real Time-PCR (qPCR) Diagnostics Data


UPL Real time PCR assay was the best assay due to:
Thermostability of reagents
Sensitivity and specificity of the test
Multiple platform compatibility
Cost
Laboratory confirmation of field qPCR results using conventional PCR is useful.

15. Virus Isolation
Haemadsorption: Binding of red blood cells to virus infected macrophages
Kiambu isolate (2012)
Athi river isolate (2012)
Karen isolate (2012)
Nakuru isolate (2012)
Sigalame isolate (2012)
 Virus isolation is an important confirmatory test and is crucial to facilitate
genotyping and experimental infections of pigs for phenotypic characterization

16. Diagnosis: Conclusions
 PCR Diagnosis is recommended relative to serology for use in
confirming outbreaks in Kenya-Uganda. No positives identified
using serology ( Only 1 out of 1,141 samples tested positive by
ELISA)
 Further research should be done to validate cheaper molecular
diagnostic assays with simple readouts e.g. ASFV LAMP PCR.
 New technologies directly linked to mobile phone readouts should
be developed in order to facilitate direct feedback for
implementation of control.

17. Genotyping of outbreaks-Background
 Twenty-two ASFV genotypes (I-XII) have been identified on the basis of nucleotide sequencing of the
variable 3′-end of the B646L gene encoding the major capsid protein p72 (Bastos, 2003).
 In Kenya, P72 genotypes IX has been associated with recent outbreaks of disease and these
genotypes have been reported to be genetically similar to the genotypes isolated in Uganda
(Gallardo, C., Okoth, E., Bishop, R. et al., 2009).
Table 1: ASF outbreaks reported to the OIE between 2000 and
2013
Year
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
Outbreaks
reported
0
3
0
0
0
0
5
5
0
0
4
4
4
0
Associated
genotypes
IX
IX
IX
IX
IX
IX
-
Cases
reported
1537
95
1011
200
203
203
-
Animals
destroyed
745
7
7
0
1
1
-
Animals
slaughtered
7196
0
0
60
97
97
-
Deaths
reported
782
82
630
165
167
167
-

18. ASFV marker loci used for genotyping
The study explores other genetic markers in addition to the B646L gene to identify genotypes and
determine variations within and between genotypes. The study also intends to eventually evaluate the
effects of the variations on the pathogenicity of the virus. These markers include:
Red Blood cell
The EP402R gene encodes
the CD2v protein that is
responsible for erythrocytes
haemadsorption around
ASFV infected cells (Borca et
al.,1998).
The complete E183L gene that encodes the p54 ASFV protein
essential in the recruitment of envelope precursors to the
assembly site (Rodriguez et al., 2004).
The variable 3′-end of the B646L gene that encodes the
major capsid protein p72
Inner membrane
Matrix
The CP204L gene that encodes
the p30 protein which modifies
the subcellular distribution of
heterogeneous nuclear
ribonucleoprotein K (HNRNPK)
and may modulate functions
related to processing and
export of mRNAs during ASFV
infection.
The B602L gene that encodes the central variable region
(CVR) where repeated amino acid tetramers that vary in
number and type among ASFV isolates are located. This
variation is important in identifying and grouping the ASFV
isolates.
Infected leucocyte

19. Materials and Methods
Blood, tissues and serum
samples are obtained from
the ASF cross sectional
survey, longitudinal survey
and suspected outbreak
areas
Nucleotide and molecular
evolutionary analyses using
CLC workbench, MEGA
version 5.2, Mobyle and
Bioedit
ASFV diagnosis and
verification
using
conventional
PCR,
UPL and TCOR PCR
and selection of ASFV
positive samples.
Purification
Genotyping and sequencing of the
partial and full length VP72, VP54, and
CVR markers.
Harvesting and extraction
of DNA from HAD positive
cultures.
Monitoring haemadsorption (HAD)
Collection of ASFV naïve
blood for PBMC isolation.
Culture of the leucocytes
using RPMI medium and
autologous serum and
infection of the resultant
macrophages with the
ASFV isolates.

20. Kenya outbreaks: Project genomic studies
Genotype IX virus
similar to that
present in KenyaUganda border
identified at Kenya
coast in 2011 and
associated with
other recent ASF
outbreaks
IX
IX
IX
Coast outbreak

21. Summary results genotyping
 Sequences are highly conserved within p72 and p54 from isolates from 2010 to
date in Kenya and Uganda.
 The CVR is highly variable especially the Ugandan 2010 to 2012 isolates that are
very similar to the Ugandan 1995 isolate. For example in containing an insert at
positions 103 to 114 of the alignment.
 There is co-existence of CVR variations in viral isolates between 2006 and 2013 in
both Kenya and Uganda at positions 365, 366 and 381 .

22. Key Conclusions- Prevalence and
Genotyping
 Longitudinal survey in the Kenya-Uganda study area indicated that
6 animals were ASFV positive by PCR in the blood; 2 were positive
in both blood and tissues and 1 positive in tissues but negative in
blood. An indication of possible virus sequestration in tissues.
 Higher prevalence in blood from slaughter slab samples-consistent
with rapid sale of sick animals.
 All outbreaks during 2010-2013 appear to be the p72 genotype IX
associated with domestic pigs.
 No evidence for Warthog-Tick sylvatic cycle contributing to recent
disease outbreaks
 CVR data indicates more than one genotype circulating in East
Africa-interpretation not yet clear

23. Implications for ASF control
Field detection of ASFV is possible but cheap user
friendly platform linked to rapid feedback to
Veterinary authorities still needed for the region
 A regional vaccine for East Africa created by
rational attenuation of the virus may be effective
since genetic diversity in Kenyan and Ugandan
viruses appears limited
Surveillance of pigs will be required at the Kenya
coast in future to prevent possible export of
genotype IX-Threatening global food security

24. THANK YOU