ASF Virus Genomics and Diagnostics 2nd October 2013

Richard Bishop and Cynthia Onzere

African Swine Fever Epidemiology Project

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?

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

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

Genome Sequencing

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

Un-rooted tree derived from whole genomes showing genetic relationship of East African viruses

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)

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.

Rapid field diagnosis of ASFV

ASFV diagnostic assays

Unknown disease causing deaths in pigs -various diseases may be implicated; is ASF the cause?

Identification and validation of diagnostic assays

Specificity & sensitivity

Reproducibility Throughput Cost Test speed

Availability

considerations

portability

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.

Comparison of nucleic acid-based diagnostic platforms

Molecular platforms

Smart cycler (Cepheid)

ABI thermal cycler (Applied biosystems)

Tetracore (Tetracore)

Piko real (thermoscientific)

Comparison of molecular diagnostic assays

Test Types Reagents/test Total cost/test (USD)

Platforms

Conventional PCR:

- nucleotide mix (Roche) - Twelvepaq amplitaq gold

(Applied biosystems) - Primers - 0.5 ml eppendorf tubes

- 3.421

-ABI thermal cycler

Real-time PCR (qPCR):

UPL PCR:

- 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)

- Cepheid Smart cycler - Piko real - T COR

TCOR PCR - pre packed reagents in Cepheid tubes

> 10 using TCOR kit

- Cepheid Smart Cycler

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

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

Evaluation of field detection by qPCR

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.

Haemadsorption: Binding of red blood cells to virus infected macrophages

Kiambu isolate (2012) Athi river isolate (2012)

Karen isolate (2012)

Sigalame isolate (2012)

Nakuru isolate (2012)

Virus Isolation

Virus isolation is an important confirmatory test and is crucial to facilitate genotyping and experimental infections of pigs for phenotypic characterization

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.

Year Outbreaks reported

Associated genotypes

Cases reported

Animals destroyed

Animals slaughtered

Deaths reported

2000 0 - - - - - 2001 3 IX 1537 745 7196 782 2002 0 - - - - - 2003 0 - - - - - 2004 0 - - - - - 2005 0 - - - - - 2006 5 IX 95 7 0 82 2007 5 IX 1011 7 0 630 2008 0 - - - - - 2009 0 - - - - - 2010 4 IX 200 0 60 165 2011 4 IX 203 1 97 167 2012 4 IX 203 1 97 167 2013 0 - - - - -

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

Genotyping of outbreaks-Background

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:

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 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.

Red Blood cell

Infected leucocyte

The EP402R gene encodes the CD2v protein that is responsible for erythrocytes haemadsorption around ASFV infected cells (Borca et al.,1998).

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.

ASFV marker loci used for genotyping

Materials and Methods ASFV diagnosis and verification using conventional PCR, UPL and TCOR PCR and selection of ASFV positive samples.

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.

Monitoring haemadsorption (HAD)

Harvesting and extraction of DNA from HAD positive cultures.

Genotyping and sequencing of the partial and full length VP72, VP54, and CVR markers.

Nucleotide and molecular evolutionary analyses using CLC workbench, MEGA version 5.2, Mobyle and Bioedit

Blood, tissues and serum samples are obtained from the ASF cross sectional survey, longitudinal survey and suspected outbreak areas

Purification

Genotype IX virus similar to that present in Kenya-Uganda border identified at Kenya coast in 2011 and associated with other recent ASF outbreaks

Kenya outbreaks: Project genomic studies

Coast outbreak IX

IX

IX

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 .

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

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

THANK YOU