ASF Virus Genomics and Diagnostics
2nd October 2013
Richard Bishop and Cynthia Onzere

African Swine Fever Epidemiology Pr...
Virus Prevalence and Diversity:
Selected Questions
• How diverse are ASFV isolates associated with
Kenyan disease outbreak...
Significance
• Rapid diagnosis of ASFV is critical for
implementation of control measures by veterinary
authorities
• Viru...
Summary of portfolio of Activities
• Whole genome sequencing using Illumina
platform
• Genotyping using PCR-sequencing fro...
Genome Sequencing

Un-rooted tree derived from whole
genomes showing genetic

Analysis of complete
genome sequences has
sh...
Summary Genome sequencing
• The complete genomes of genotype IX (from a
clinically reacting pig) and genotype X (from a ti...
Rapid field diagnosis of ASFV
Kenya and Uganda veterinarians
at Project workshop in Kisumu,
July 2011:
• Testing labs are ...
ASFV diagnostic assays
Unknown disease causing deaths in pigs
-various diseases may be implicated; is ASF the cause?

Iden...
DNA extraction in the field

 Magnetic beads (Roche Magna kit) and magnetic strips used for DNA extraction; the
Roche pro...
Comparison of nucleic acid-based
diagnostic platforms
Molecular platforms
ABI thermal cycler
(Applied biosystems)

Smart c...
Comparison of molecular diagnostic
assays
Test

Types

Conventional
PCR:

Reagents/test

-

Real-time PCR
(qPCR):

UPL PCR...
Serology
ELISA for Detection of anti-p72 antibodies
Blocking ELISA

 Ingenasa kit (Spain) used for detection
 Can curren...
Evaluation of field detection by qPCR
Here is
the Lab
Field laboratory test run from a basic set-up (i.e. table) or back o...
Real Time-PCR (qPCR) Diagnostics Data



UPL Real time PCR assay was the best assay due to:
Thermostability of reagents
...
Virus Isolation
Haemadsorption: Binding of red blood cells to virus infected macrophages

Kiambu isolate (2012)
Athi river...
Diagnosis: Conclusions
 PCR Diagnosis is recommended relative to serology for use in
confirming outbreaks in Kenya-Uganda...
Genotyping of outbreaks-Background
 Twenty-two ASFV genotypes (I-XII) have been identified on the basis of nucleotide seq...
ASFV marker loci used for genotyping
The study explores other genetic markers in addition to the B646L gene to identify ge...
Materials and Methods
Blood, tissues and serum
samples are obtained from
the ASF cross sectional
survey, longitudinal surv...
Kenya outbreaks: Project genomic studies
Genotype IX virus
similar to that
present in KenyaUganda border
identified at Ken...
Summary results genotyping
 Sequences are highly conserved within p72 and p54 from isolates from 2010 to
date in Kenya an...
Key Conclusions- Prevalence and
Genotyping
 Longitudinal survey in the Kenya-Uganda study area indicated that
6 animals w...
Implications for ASF control
Field detection of ASFV is possible but cheap user
friendly platform linked to rapid feedbac...
THANK YOU
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African Swine Fever (ASF) virus genomics and diagnostics

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Presented by Richard Bishop and Cynthia Onzere at the Closing workshop of the BecA‐ILRI‐CSIRO‐AusAID project on Understanding ASF epidemiology as a basis for control, Nairobi, Kenya, 2‐3 October 2013

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African Swine Fever (ASF) virus genomics and diagnostics

  1. 1. ASF Virus Genomics and Diagnostics 2nd October 2013 Richard Bishop and Cynthia Onzere African Swine Fever Epidemiology Project
  2. 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. 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. 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. 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. 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. 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. 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. 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. 10. Comparison of nucleic acid-based diagnostic platforms Molecular platforms ABI thermal cycler (Applied biosystems) Smart cycler (Cepheid) Tetracore (Tetracore) Piko real (thermoscientific)
  11. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 24. THANK YOU
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