Diagnostics available for African Swine Fever

virus

Dr John Flannery

Regional workshop on swine disease diagnosis

30th October 2019

Beijing, Peoples Republic of China

Overview

• The Pirbright Institute

• Reference laboratories based at Pirbright

• Sample matrices

• Diagnostic tools to combat ASFV

• Antibody detection tests

• Virus isolation

• Antigen detection tests

• Molecular tests

• Field-based or penside tests

• Summary of diagnostic techniques

The Pirbright Institute is a world leading centre ofexcellence in research and surveillance of virus diseasesof farm animals and viruses that spread from animals tohumans.

The Pirbright Institute

• Contributing to global food security and health, improving quality of life for animals and people

• Annual income >£29M (354 million CNY)

• Over 180 scientists working on a range of viruses in specialist areas

• CL2 and CL3 laboratory space

• OIE CL4 containment facility (BSL3)

• High-containment (BSL3) animal facilities

Plowright building, Pirbright

Reference laboratories at Pirbright

Vesicular disease

reference laboratories

Non-vesicular disease

reference laboratories

• Whole blood

• alive/dead pigs

• Lymph nodes

• submandibular,

• Inguinal

• gastrohepatic

• Spleen

• Lung

• Kidney

• Tonsils

• Bone marrow

• Tonsil scrapings/ swabs

ASF sample matrices and tests

Serological analyses

Virological & molecular analyses

• ELISA • Lateral flow device (antibody)• Immunoblotting test• Indirect fluorescent antibody test• Immunoperoxidase test

• Antigen ELISA• Lateral flow device (antigen)• Virus isolation and HAD• Direct fluorescent antibody test• PCR and real-time PCR• Sequencing

• OIE indirect ELISA

• Commercial antibody ELISA

• Immunoblotting test (IBT)

• Indirect fluorescent antibody test (IFAT)

• Indirect immunoperoxidase test (IPT)

ASFV serological tests

Screening assays

Confirmatory assays

ASFV antibodies can be detected from 12 days to 5 years post infection

• The OIE indirect ELISA (Pastor et al., 1990) uses cytoplasm

soluble antigen (ASFV Spanish strain E70 grown in MS cells)

• Antigen coated onto wells and sample serum added

• Samples with ASFV antibodies against ASF will form an

antigen-antibody complex

• Addition of conjugate, washing and addition of substrate will

visualise positive samples

Indirect ELISA- OIE manual

Substrate

Indirect ELISA

Secondary antibody conjugate

ASFV antibodies in sample

Substrate

• OIE indirect ELISA test for ASF has high specificity (95.8%)

and sensitivity (97.3%) (Gallardo et al., 2013)

• to allow confident diagnosis of ASF independent of the

viral genotypes circulating in a particular region.

• However, reagents are not commercially available but can be

requested from OIE reference laboratory, CISA-INIA, Spain

Indirect ELISA- OIE manual

Substrate

Indirect ELISA

Secondary antibody conjugate

ASFV antibodies in sample

Substrate

• Commercial ELISAs are available worldwide

• INGEZIM PPA COMPAC K3 (INGENASA)→ blocking ELISA which uses a monoclonal

antibody (MAb) specific of VP72 ASFV protein

• SVANOVIR® ASFV-Ab → indirect ELISA based on a recombinant antigen - the p30

(screening format)

• ID Screen® ASF → Competition ELISA kit for the detection of antibodies against P32

ASFV protein

• ID Screen® ASF → Indirect Multi-antigen indirect ELISA kit for the detection of

antibodies against P32, P62 and P72 ASFV proteins

All kits have undergone validation and are used in European Union proficiency tests

Commercially-available antibody-detection ELISAs

Antibody complex

Antigen (P72)

HRP substrate

• Described by Pastor et al, 1989 and Escribano et al., 1990

• Immunoblotting test (IBT) is a sensitive assay based on antigen-

antibody recognition (Gallardo et al., 2013)

• ASFV viral proteins (electrophoretically separated using SDS-

PAGE) are transferred to a nitrocellulose membrane.

• The membrane is then blocked using Tween/milk protein and cut

into strips

• Serum is overlaid onto the antigen strip

• Specific antibodies against ASF are visualised by addition of an A-

peroxidase conjugate protein and chloronaphtol as substrate

Immunoblotting test (IBT)

Increasing dilution of serum

Courtesy: CISA-INIA, Spain

• Test takes ~3 hours to complete

• Reagents (strips) are not commercially available and must be

requested from OIE reference laboratory, CISA-INIA, Spain

The IBT is the recommended test by the World Organization for

Animal Health (OIE, 2012) in the Manual of Diagnostic Tests and

Vaccines for Terrestrial Animals for the confirmation of positive

and doubtful samples by ELISA

Immunoblotting test (IBT)

Increasing dilution of serum

Courtesy: CISA-INIA, Spain

• Developed by Pan et al., 1974, Pan et al., 1982

• ASFV-infected Vero or MS cells are fixed and used as antigens

• Antibody-antigen complexes are visualised by the use of fluorescein isothiocyanate or peroxidase conjugated IgG

• IPT considered easier to interpret results and uses simpler equipment

IFAT is used as a confirmatory test (areas free of ASFV but with positive/inconclusive ELISA results)

Indirect fluorescent antibody test (IFAT)Indirect immunoperoxidase (IPT)

ASFV Ab+++ ASFV Ab+ ASFV Ab-

IFAT

IFAT

• ASFV infects and replicates in primary leukocyte cultures from

pig peripheral blood (Malmquist and Hay, 1960)

• Inoculation of sample material on susceptible primary cell

cultures of porcine origin, monocytes and macrophages cells

• ASFV replicates in the cells and the CPE (haemadsorption and

cell lysis) will be produced in the infected cells after 48 hours

• Rosettes form due to haemadsorption (HAD) of porcine

erythrocytes on ASFV infected macrophages

Virus isolation and HAD

Virus isolation and HAD

• Test is highly specific: only ASFV is capable of haemadsorbing

in leukocyte cultures

• Primary cell culture is required

• Not all ASFV strains haemadsorb

• Test duration of 3- 10 days

Virus isolation and identification by HAD are recommended as

a reference test for the confirmation of positive results

Antigen detection by ELISA

Coated monoclonal antibody

HRP substrate

ASFV antigen

HRP-conjugated Ab

• Viral antigens can also be detected using ELISA, but it is only recommended for acute forms of disease

• Monoclonal anti-VP72 antibody coated plates

• ASFV antigen in the sample will bind to the specific anti-VP72

• HRP-conjugated MAb specific for a different epitope of VP72 is added

• Colorimetric reaction after the addition of the substrate

The antigen ELISA has a poor sensitivity and is recommended to use the antigen ELISA only as a ‘herd’ tests and in conjunction with other virological tests

• Microscopic detection of viral antigens on

impression smears or thin cryosections

• Intracellular antigens are detected using

FITC-conjugated specific antibodies.

• Fluorescent inclusion bodies or granules

appear in the cytoplasm of infected cells.

• Useful to detect non-haemadsorbing ASFV

strains antigen in leucocyte cultures in which

no HAD is observed

Direct fluorescent antibody test (FAT)

Courtesy: Dr Pippa Hawes, Pirbright

• Highly sensitive for peracute and acute ASFV

infection

• Less sensitive for subacute and chronic disease due

to the presence of antigen-antibody complexes

• Host antibodies compete with the ASF-

conjugated antibody

Direct fluorescent antibody test (FAT)

Courtesy: Dr Pippa Hawes, Pirbright

ASF PCR

• PCR can be directed to conserved genome regions to detect

all ASFV isolates.

• Genome is stable and can be detected in many sample types

even if infectious virus is inactivated

• PCR enables specific and sensitive detection with 5 hours

(including DNA extraction)

ASF PCR

• One conventional PCR (Agüero et al., 2003) and 2 real-time

PCRs in OIE Manual

• ASF/CSF duplex assay (Haines et al., 2013)

• Commercial assays are now available (IDVet, ThermoFisher)

• Additional assays being developed due to lucrative

market

Real-time PCR assay Target IC Analytical sensitivity

King et al., 2003 VP72 “Mimic” 10-100

Zsak et al., 2005 VP72 None 1.4-8.4

McKillen et al., 2010 9GL None 20

Tignon et al., 2011 VP72 β-Actin 5.7-57

Fernandez Pinero et al., 2012 VP72 β-Actin 18

ASFV genotyping is based on the analysis of three independent regions located at the

conserved central area of the ASFV genome comprising:

ASF Sequencing targets

• Partial sequence of the C-terminal end of the B646L gene encoding

VP72 (Bastos et al., 2003)

• Classification of the ASFV genotypes (Boshoff et al., 2007)

• Full sequence of E183L-gene encoding the p54 protein

• Additional epidemiological information of p72 genotype I viruses

(Gallardo et al., 2009)

• Sequencing of the central variable region within B602L-gene

(CVR) Determining the origin and helps map the spread of closely related

ASFV strains (Nix et al., 2006; Gallardo et al., 2011)

P72 gene sequencingIV RSA_1999_RSA/1/99W_AF449477.1

XIX South_Africa_SPEC/125_DQ250112.1

XX RSA_1975_24823_DQ250110.1

III Botswana_1999_BOT/1/99_AF504886.1

RSA_1998_RSA/1/98_AF302818.1

Botswana_1987_SPEC/154_DQ250113.1VII

RSA_1979_GR44A2_FJ455835.1

RSA_1992_SPEC/245_DQ250117.1XXII

XXI RSA_1983_SPEC/53_DQ250111.1

XXIV Mozambique_2006_MOZ_10/2006_KY353989.1

VI Mozambique_1994_SPEC/265_AF270710.1

Malawi_2002_MAL/2002/1_AY494553.1

Mozambique_1964_Moz64_FJ174376.1V

Tanzania_2016_TAN/16/Uyole_MF437294

Zambia_1991_LUS/93/1_AY351563.1

Madagascar_2003_Ambilo03_KC610528.1

Poland_2014_Pol14/Sz_KJ627217.1

China_2018_Jilin/2018/boar_MK189456.1

G1/19_01_Mongolia

Georgia_2007/1_FR682468.1

II

XVII Zimbabwe_1992_ZIM/92/1_DQ250119.1

Cameroun_1982_CAM/82_AF301544.1

Belgium_1985_BEL85_AF449466.1

Italy_2002_5/Ca/02_FR668402.1

W34/18_13 Cameroon

I

XVIII Namibia_1995_NAM/1/95_DQ250122.1

XIV Zambia_1986_NYA/1/2_AY351555.1

XV Tanzania_2001_TAN/01/1_AY494552.1

XVI Tanzania_2003_TAN/03/1_AY494550.1

XIII Zambia_1983_SUM/14/11_AY351542.1

XII Malawi_1992_MZI/92/1_AY351543.1

XI Zambia_1983_KAB/62_AY351522.1

Mozambique_1998_MOZ/62/98_AY274457.1

Malawi_1991_BAN/91/1_AY351501.1

Zambia_1988_CHG/88/1_AY351552.1VIII

Ethiopia_2011_ETH/3_KT795360.1

Ethiopia_2013_ET13/1504_KU291454.1XXIII

Uganda_2007_UG07.Wak1_GQ477138.1

Kenya_2008_Ken08WH/4_HM745285.1

Uganda_2003_UGA2003/1_AY351564.1IX

Kenya_1961_Gasson_AY351529.1

Uganda_1995_UGA/95/3_AF449476.1

Kenya_1959_KilleanIII_AY351531.1X99

98

93

87

100

98

99

96

89

92

77

91

0.005

IV RSA_1999_RSA/1/99W_AF449477.1

XIX South_Africa_SPEC/125_DQ250112.1

XX RSA_1975_24823_DQ250110.1

III Botswana_1999_BOT/1/99_AF504886.1

RSA_1998_RSA/1/98_AF302818.1

Botswana_1987_SPEC/154_DQ250113.1VII

RSA_1979_GR44A2_FJ455835.1

RSA_1992_SPEC/245_DQ250117.1XXII

XXI RSA_1983_SPEC/53_DQ250111.1

XXIV Mozambique_2006_MOZ_10/2006_KY353989.1

VI Mozambique_1994_SPEC/265_AF270710.1

Malawi_2002_MAL/2002/1_AY494553.1

Mozambique_1964_Moz64_FJ174376.1V

Tanzania_2016_TAN/16/Uyole_MF437294

Zambia_1991_LUS/93/1_AY351563.1

Madagascar_2003_Ambilo03_KC610528.1

Poland_2014_Pol14/Sz_KJ627217.1

China_2018_Jilin/2018/boar_MK189456.1

G1/19_01_Mongolia

Georgia_2007/1_FR682468.1

II

XVII Zimbabwe_1992_ZIM/92/1_DQ250119.1

Cameroun_1982_CAM/82_AF301544.1

Belgium_1985_BEL85_AF449466.1

Italy_2002_5/Ca/02_FR668402.1

W34/18_13 Cameroon

I

XVIII Namibia_1995_NAM/1/95_DQ250122.1

XIV Zambia_1986_NYA/1/2_AY351555.1

XV Tanzania_2001_TAN/01/1_AY494552.1

XVI Tanzania_2003_TAN/03/1_AY494550.1

XIII Zambia_1983_SUM/14/11_AY351542.1

XII Malawi_1992_MZI/92/1_AY351543.1

XI Zambia_1983_KAB/62_AY351522.1

Mozambique_1998_MOZ/62/98_AY274457.1

Malawi_1991_BAN/91/1_AY351501.1

Zambia_1988_CHG/88/1_AY351552.1VIII

Ethiopia_2011_ETH/3_KT795360.1

Ethiopia_2013_ET13/1504_KU291454.1XXIII

Uganda_2007_UG07.Wak1_GQ477138.1

Kenya_2008_Ken08WH/4_HM745285.1

Uganda_2003_UGA2003/1_AY351564.1IX

Kenya_1961_Gasson_AY351529.1

Uganda_1995_UGA/95/3_AF449476.1

Kenya_1959_KilleanIII_AY351531.1X99

98

93

87

100

98

99

96

89

92

77

91

0.005

CVR gene sequencing provides higher resolution

Genotype X

Genotype IX CVR sequencing Kenya

group 2

Kenya group 1

Kenya group 3

Whole genome sequencing

War

tho

gR

SA

1/9

9/W

66

CH

ZT

90/1

28

RSA

96/1

SPE

C/5

360

15

MO

Z20

06/1

8M

OZ20

06/1

0

MO

Z2006

/11

62

7820M

OZ2006/14

MOZ2006/17

78

Tengani

75

SPEC/265

MOZ94/1

MOZ94/893

89

0

Pr96/4Lillie24823

55

8

RSA96/3SPEC/120SPEC/125

3973

2

SPEC/257

Warm

baths

76

11

SPE

C/245R

SA

98/1

SPE

C/1

54

9954

27

MA

D98/1

GE

O2007/1

66

MO

Z2

00

6/1

67

NA

M95/1

ZIM

92/1

SIN

T90/1

Kim

akia

I

BEN

97/1 68

87

70 2

8 23

69

NY

A1/

2

TAN

2003

/2

TAN2001/137

SUM14/11MZI92/1MFUE6/1

87

KAB6/2MOZ98/1

LIL20/1

Dezda

98

51

43

54

5282

99

ET13/1504

ETH/5a

96

ETH/3

92

Ken08BP/HB

Trench

75

Ken09T

k.13/1

97

Ken08W

H/4

Ken08W

H/5

UG

A2003/1

9984

0.0050

X

VIII

III

IV XXIXXIV

V

VI

XX

XIX

III

VIIXVIIIXVII

XIVXVIXV

XIII

XII

XI

XXIII

IX

• Whole genomes only available for selected ASFV genotypes• Mostly Eurasian and East African strains

• Technically challenging due to contamination with host genome – low read numbers for viral DNA

• Repetitive sequences at termini mean Illumina reads could introduce errors, particularly for novel sequences

• Terminal inverted repeats very difficult to assemble.

Moving towards probe capture technologies to purify viral sequences from host

Capture

Amplification Sequencing

Courtesy: Dr Chris Netherton, Pirbright

ASFV field-based diagnostics

INgezim PPA CROM:

• Direct immunochromatography utilising ASFV VP72

• VP72 and a control protein bound to coloured

latex

• The assay is able to detect ASFV antibodies

• 10 to 21 days post infection (Cappai et al., 2017)

• High sensitivity: 99% with OIE ELISA

• High specificity: 99.9% with OIE ELISA

Penside test- Lateral flow device to detect ASFV antibodies

LFDs are not considered suitable for confirmatory diagnosis

INgezim ASF CROM Ag:

• Direct immunochromatography utilising ASFV VP72

• VP72-specific MAb and a control protein MAb

bound to coloured latex

• The assay is able to detect ASFV at high titres (CT<30)

• Similar sensitivity to Ag ELISA (Sastre et al., 2016)

• Overall sensitivity: 76% with qPCR

• High specificity: 99.6% with qPCR

Penside test- Lateral flow device to detect ASFV antigen

LFDs are not considered suitable for confirmatory diagnosis

Summary of main diagnostic tests for ASF

Assay Time Sensitivity Specificity Cost Comments

Antigen ELISA 3 hours + ++ $ Screening test

PCR 5 hours +++ +++ $$ Most commonly used assay

HAD 3-10 days ++ +++ $$$ Used in reference laboratories

Direct FAT 75 min +++ +++ $$$ Confirmatory test

Partial sequence analysis

3 days n.a. n.a. $$$ Genotype determinationCan be applied in most laboratories

Full-genome sequence analysis

Weeks-months

n.a. n.a. $$$$$ Used in reference laboratories and involves complex bioinformatics

Assay Time Sensitivity Specificity Cost Comments

ELISA 3 hours + + $ Screening test

Immunoblotting 3 hours ++ ++ $$$ Confirmatory test

Indirect FAT/ IPT 4 hours +++ + $$$ Confirmatory test

Comparison of available tests

Serological detection

Virus detection/characterisation

Sample AbELISA

Conclusion PCR CT

Conclusion Diagnostic conclusion

1- Negative + 20 Positive

Positive: early infection

2-/+ Inconclusive - Negative

Inconclusive: possible false-positive in ELISA

3 + Positive - Negative Positive: late infection

4 + Positive + 32 Positive Positive: current-late infection

5 - Negative + 38 Positive Positive: early infection

Interpretation of diagnostic test results

• Limited diagnostic capabilities limit successful interpretation of results

• Suitable for regional laboratories

• Multiple tests are needed for complete diagnostic conclusion

Sample AbELISA

IBT IFAT/IPT

AgELISA

PCR 1CT

PCR 2CT

CPE HAD FAT Diagnostic conclusion

1- - - + + 20 + 18 + - +

Positive: early infection- non haemadsorbing ASFV

2 -/+ + + - - - n.d. n.d. n.d. Positive: previous infection

3 + - - - - - n.d. n.d. n.d. Negative: false positive ELISA

4 + + + - + 32 + 35 + + + Positive: chronic infection

5- - - - + 38 - - - -

Inconclusive: possible contamination-request re-sample

Interpretation of diagnostic test results

• Ab ELISA: Antibody ELISA

• Ag ELISA: Antigen ELISA

• IBT:Immunoblotting test

• FAT: Fluorescent antibody test

• CPE: Cytopathic effect in cell culture

• HAD: Haemadsorptiontest

• IFAT/IPT: Indirect fluorescent antibody test/ Immunoperoxidasetest

• PCR: Polymerase chain reaction assay

• n.d. test not performed

Serological tests Virological & molecular tests

• Multiple tests should be performed

• Important to consider results of each test and the test limitations

• PCR can give rise to contamination if not properly controlled

• Ensure seamless communication with veterinary authorities/field vets to obtain additional samples

• Liaise with OIE reference laboratories for confirmation of results

The OIE reference laboratory at Pirbright can help with

• Provision of reagents for ASFV and other viruses

• Interpretation of diagnostic results

• Support for ISO/IEC17025 accreditation

• Diagnostic kit evaluation

• Training in diagnostic tests at Pirbright

OIE reference laboratory support

Please do not hesitate to contact

us concerning any of the above

Non Vesicular Reference laboratories

• Dr Carrie Batten (Head of laboratory)

• Dr Paulina Rajko-Nenow

• Lorraine Frost

• Dr Mark Henstock

• Dr Martin Ashby

• Dr Simon King

• Dr Amanda Corla

• Matthew Tully

• Katie Harris

• Rebecca Moore

• Laura Marsala

• Sarah Belgrave

• Julie Maryan

Pirbright Institute

ASFV research groups at Pirbright

• Dr Linda Dixon (OIE expert ASFV)

• Dr Chris Netherton

• Dr Pip Beard (OIE expert Capripoxviruses)

• Dr Ana Reis

• Lynnette Goatley

• Dr Raquel Portugal