EURL-African swine fever (ASF) activities

Transcription

1 EURL-African swine fever (ASF) activities Dr. Carmina Gallardo Hannover, Germany- 29 th May 2018 Workshop on Laboratory Diagnosis and Control of CSF and ASF NOTE; the use of the content presented here is subjected to the author s permission

2 CONTENT Workshop on Laboratory Diagnosis of African and Classical Swine Fever (ASF and CSF). Hannover, Germany, 29 th May UPDATED ASF SITUATION EU 2. EURL ACTIVITIES FOR Active assistance to EU member states in the diagnosis of ASF outbreaks, and other European countries if requested/needed. Confirmatory diagnosis, characterization and epidemiological studies. Provision of emergency ASF diagnostic kits and reference material Biological characterization of viral isolates collected in the ASF outbreaks worldwide. Prepare and distribute SOPs, reference standards and other biological reagents for ASF serological and virological diagnosis previous request. Technology transfer Evaluation and validation of ASF diagnostic kits/ Interpretation of the results.

3 ASF SITUATION 2018 EAST EUROPE (NON EU COUNTRIES): 7 COUNTRIES AFFECTED (Georgia, Azerbaijan, Armenia, Russia Federation, Ukraine, Belarus, Moldova) EAST EU 7 COUNTRIES AFFECTED

4 ASF SITUATION 2018 EUROPE NO-EU Source: OIE DP 76%; WB 24% HOST Total Domestic pig Armenia Azerbaijan 1 1 Belarus 5 5 Georgia Moldova Russia Ukraine wild boar Armenia 3 3 Moldova 3 3 Russia Ukraine Total general

5 ASF SITUATION 2018 EU countries Source: ADNS DP:2%; WB 98% COUNTRY ADNS EU INFORMATION Date of last outbreak Nº of WB CASES ADNS TOTAL ESTONIA 17/05/ LATVIA 18/05/ LITHUANIA 19/05/ POLAND 20/05/ CZECH REPUBLIC 19/04/ ROMANIA HUNGARY 18/05/ TOTAL WILD BOAR CASES COUNTRY Date of last outbreak ESTONIA 19/09/ LATVIA 10/10/ LITHUANIA 21/09/ POLAND 16/05/ CZECH REPUBLIC ROMANIA 02/05/ TOTAL DOMESTIC PIG OUTBREAKS Nº ofdp OUTBREAKS ADNS TOTAL

6 10,652 wild boar cases 261 domestic pig outbreaks (ADNS data) LITHUANIA ASF (JAN14-MAY18) 2,945 WB CASES 68 DP OUTBREAKS POLAND ASF (JAN14-MAY18) 2,166 WB CASES 109 DP OUTBREAKS LATVIA ASF (JUNE14-MAY18) 2,945 WB CASES 53 DP OUTBREAKS ESTONIA ASF (SEP14-MAY18) 2,609 WB CASES 27 DP OUTBREAKS Czech RP ASF (JULY17-APRIL18) 230 WB CASES ROMANIA ASF (AUG17-MAY18) 5 DP OUTBREAKS HUNGARY ASF (APRIL18-MAY18) 11 WB CASES UPDATED 27 MAY 2018

7 1. Active assistance to EU member states in the diagnosis of ASF outbreaks, and other European countries if requested/needed Confirmatory diagnosis, characterization and epidemiological studies TOTAL = 8,059 (ADNS SOURCE)

8 Confirmatory diagnosis, characterization and epidemiological studies. Since 2014 up to Jan ,724 SAMPLES FROM 1,482 WILD BOAR (18,3% of the total cases declared)

9 Confirmatory diagnosis, characterization and epidemiological studies ,647 wild boar samples from 823 wild boar COUNTRY HOST Animals received Nº SAMPLES 423 WB (2016) 400 WB (2017) LITHUANIA WILD BOAR LATVIA WILD BOAR POLAND WILD BOAR ESTONIA WILD BOAR CZECH REPUBLIC WILD BOAR Total wild boar ,1% BM 20,6% SP

10 Confirmatory diagnosis, characterization and epidemiological studies. MOLDOVA WILD BOAR CONFIRMATION FIRST CASE DECEMBER 2017

11 MAIN FINDINGS Wild boar Ab detection results. COUNTRY TOTAL WB RECEIVED AB DETECTION BY IPT WB TESTED POSITIVE NEGATIVE Nº % Nº % Nº % CZECK Rep % 27 90% 3 10% ESTONIA % 34 60% 23 40% LATVIA % 21 57% 16 43% LITHUANIA % % % POLAND % % 95 29% TOTAL % % % The presence of Abs was confirmed in 528 /823 wild boar which corresponds to the 65% of the animals received Increased percentage of seropositive animals

12 MAIN FINDINGS Wild boar virus detection results. COUNTRY TOTAL WB RECEIVED VIRUS DETECTION BY PCR WB TESTED POSITIVE NEGATIVE Nº % Nº % Nº % CZECK Rep % % 0 0% ESTONIA % 57 98% 1 2% LATVIA % 27 73% 10 27% LITHUANIA % % 92 26% POLAND % % 29 9% TOTAL % % % The presence of the ASFV genome was confirmed in 691 wild boar which corresponds to the 84% of the animals received VIRUS ISOLATION (672 PCR positive samples) The virus was only successfully isolated from 117 samples of 89 wild boar ( 17%) FIRST NON HAD ASFV GENOTYPE II ISOLATED WITHIN THE EU (at the EURL)

13 NON- HAD GENOTYPE II ASFV ISOLATED WITHIN THE EU. Sequence analysis of the EP402R gene responsible of the HAD. Nucleotide frameshift mutation that disrupt the open reading frame. TRUNCATED PROTEIN

14 Confirmatory diagnosis, characterization and epidemiological studies. Since 2014 up to Jan SAMPLES FROM 344 DOMESTIC PIGS (46% of the total OUTBREAKS declared)

15 Confirmatory diagnosis, characterization and epidemiological studies. 441 domestic pig samples 183 DP COUNTRY HOST Animals Nº received SAMPLES LITHUANIA DOMESTIC PIG LATVIA DOMESTIC PIG POLAND DOMESTIC PIG ESTONIA DOMESTIC PIG 0 0 ROMANIA DOMESTIC PIG 4 4 Total domestic pig % SP 25,4% KID

16 Confirmatory diagnosis, characterization and epidemiological studies. Domestic pig virus detection results. The presence of the ASFV genome was confirmed in 181 domestic pigs which corresponds to the 99% of the animals received. Virus was successfully isolated from 40 domestic pig outbreaks (60%) Domestic pig antibody detection results. ASF antibody detection: The presence of antibodies was confirmed by IPT in 65 out of the 179 animals tested resulted in 64% seroconverted domestic pig

17 Confirmatory diagnosis, characterization and epidemiological studies (EU) 601 NEW ASFV CHARACTERIZED COUNTRY ASFV analysed Host Domestic Wild boar pig CZECK Rep Estonia Latvia Lithuania Poland Romania MOLDOVA TOTAL ,115 genotype II EU ASFVs characterized since 2014 from wild and domestic pigs in the EU countries All of them clustered within the p72 genotype II

18 IDENTIFICATION OF DIFERENT GENOTYPE II GENETIC VARIANTS CIRCULATING IN THE EU COUNTRIES. SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene Analysis of the intergenic region located between the I73R and I329L genes (IGR I73R-I329L) Analysis of the intergenic region between the multigene family (MGF) 505 9R and the 10R genes (IGR MGF505)

19 IDENTIFICATION OF DIFERENT GENOTYPE II GENETIC VARIANTS CIRCULATING IN THE EU COUNTRIES. SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene Analysis of the intergenic region located between the I73R and I329L genes (IGR I73R-I329L) Analysis of the intergenic region between the multigene family (MGF) 505 9R and the 10R genes (IGR MGF505)

20 SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene COUNTRY YEAR CVR SUBTYPING Georgia 2007 CVR1 Armenia 2007 CVR1 Azerbaijan 2008 CVR1 Russia Federation CVR CVR1 Ukraine 2012, 2015 CVR1 Belarus 2013 CVR CVR1 Estonia CVR1+ CVR CVR1 + CVR1SNP1* Latvia CVR1 Lithuania CVR CVR1SNP3* CVR1 Poland 2016 CVR CVR1 + CVR1SNP2* Moldova CVR1 Czech Republic 2017 CVR1 Romania CVR1 ESTONIA TARTU

21 SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene COUNTRY YEAR CVR SUBTYPING Georgia 2007 CVR1 Armenia 2007 CVR1 Azerbaijan 2008 CVR1 Russia Federation CVR CVR1 Ukraine 2012, 2015 CVR1 Belarus 2013 CVR CVR CVR1+ CVR 2 Estonia CVR1 + CVR1SNP1* Latvia CVR CVR1 Lithuania Poland 2017 CVR1 + CVR1SNP3* CVR CVR CVR1 + CVR1SNP2* Moldova CVR1 Czech Republic 2017 CVR1 Romania CVR1 SNPs Single nucleotide polymorphisms

22 SNP1 Estonia; A instead of G (A) Nucleotide alignment Est17/WB-Parnu11 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/DP-Parnu1 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/DP-Parnu2 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/DP-Parnu3 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/DP-Parnu4 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/WB-Parnu12 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/WB-Parnu13 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/WB-LANE12 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC Est17/WB-Parnu14 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTATGCAAGCATGTGTGCAGATACC CVR-1 variant AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTGTGCAAGCATGTGTGCAGATACC ************************************* ********************** (B) Aminoacid alignment Est17/WB-Parnu11 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/DP-Parnu1 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/DP-Parnu2 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/DP-Parnu3 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/DP-Parnu4 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/WB-Parnu12 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/WB-Parnu13 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/WB-LANE12 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY Est17/WB-Parnu14 SAYTCADTNVDTYASMCADTNVDTCASMCADTNVDTCASTCTSTEY CVR-1 variant SAYTCADTNVDTCASMCADTNVDTCASMCADTNVDTCASTCTSTEY ************ *********************************

23 SNP2 POLAND; T instead of A (A) Nucleotide alignment Pol17/WB-CASE 316 AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTGTGCAAGCTTGTGTGCAGATACC CVR-1 variant AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTGTGCAAGCATGTGTGCAGATACC ********************************************* ************** Pol17/WB-CASE 316 AATGTAGACACCTGTGCAAGCATGTGTGCAGATACCAATGTAGATACCTGTGCAAGCACT CVR-1 variant AATGTAGACACCTGTGCAAGCATGTGTGCAGATACCAATGTAGATACCTGTGCAAGCACT ************************************************************ Pol17/WB-CASE 316 CVR-1 variant TGTACAAGCACAGAATAC TGTACAAGCACAGAATAC ****************** (B) Aminoacid alignment Pol17/WB-CASE 316 SAYTCADTNVDTCASLCADTNVDTCASMCADTNVDTCASTCTSTEY CVR-1 variant SAYTCADTNVDTCASMCADTNVDTCASMCADTNVDTCASTCTSTEY *************** ****************************** CASE /05/ KM BELARUS BORDER

24 (A) Nucleotide alignment Lt17/WB-Kupiskis-18 CVR-1 variant Lt17/WB-Kupiskis-18 CVR-1 variant Lt17/WB-Kupiskis-18 CVR-1 variant AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTGTGCAAGCATGTGTGCAGATACC AGTGCGTATACTTGTGCAGATACCAATGTAGACACTTGTGCAAGCATGTGTGCAGATACC ************************************************************ AATGTAGACACCTGTGCAAGCATGTGTGCAGATACCAATGTAGATACCTGTGCAAGCACT AATGTAGACACCCGTGCAAGCATGTGTGCAGATACCAATGTAGATACCTGTGCAAGCACT ************ *********************************************** TGTACAAGCACAGAATAC TGTACAAGCACAGAATAC ****************** SNP3 LITHUANIA; C instead of T (B) Aminoacid alignment Lt17/WB-Kupiskis-18 SAYTCADTNVDTCASLCADTNVDTCASMCADTNVDTCASTCTSTEY CVR-1 variant SAYTCADTNVDTCASMCADTNVDTRASMCADTNVDTCASTCTSTEY Lt17/WB-Kupiskis-18 ************************ ********************* 31/08/2017

25 IDENTIFICATION OF DIFERENT GENOTYPE II GENETIC VARIANTS CIRCULATING IN THE EU COUNTRIES. SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene Analysis of the intergenic region located between the I73R and I329L genes (IGR I73R-I329L) Analysis of the intergenic region between the multigene family (MGF) 505 9R and the 10R genes (IGR MGF505)

26 SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the intergenic region located between the I73R and I329L genes (IGR I73R-I329L) COUNTRY YEAR IGR I73R-I329L SUBTYPING Georgia 2007 IGR-1 Armenia 2007 IGR-1 Azerbaijan 2008 IGR IGR IGR-1 + IGR-2 Ukraine 2012, 2015 IGR-2 Russia Federation Belarus 2013 IGR IGR-2 Estonia IGR IGR-2 Latvia IGR-2 Lithuania IGR IGR IGR IGR IGR-1 + IGR-2 Moldova IGR-2 Czech Republic 2017 IGR-2 Romania IGR-2 Poland 14 KM BELARUS BORDER

27 IDENTIFICATION OF DIFERENT GENOTYPE II GENETIC VARIANTS CIRCULATING IN THE EU COUNTRIES. SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the central variable region (CVR) within the B602L-gene Analysis of the intergenic region located between the I73R and I329L genes (IGR I73R-I329L) Analysis of the intergenic region between the multigene family (MGF) 505 9R and the 10R genes (IGR MGF505)

28 SUBTYPING OF ASFV GENOTYPE II ISOLATES Analysis of the intergenic region between the MGF 505 9R and the 10R genes (IGR MGF505) COUNTRY YEAR IGR MGFSUBTYPING Georgia 2007 MGF -1 Armenia 2007 MGF -1 Azerbaijan 2008 MGF MGF -1 Russia Federation MGF -1 + MGF -2 + MGF -3 Ukraine 2012, 2015 MGF -1 Belarus 2013 MGF MGF -1 Estonia MGF MGF -1 Latvia MGF -1 Lithuania MGF -1 MGF MGF -4 + MGF MGF MGF -1 + MGF MGF -1 + MGF -2 Moldova MGF -1 Czech Republic 2017 MGF -1 Romania MGF -1 Poland IGR MGF 5 VARIANTS (4 WITHIN EU COUNTRIES)

29 MGF 2 identified in POLAND ( ) 100% of homology with certain ASFV isolates viruses circulating in the Russia Federation since at least 2012

30 MGF 2 identified in POLAND ( )

31 MGF 4 and MGF 5 identified in LITHUANIA 2017 Not found in other genotype II viruses sequenced at the EURL: maintenance within the WB population?

32 Summary of the molecular characterization results from Eastern European ASFV isolates COUNTRY YEAR P72 CVR IGR I73R-I329L IGR MGF GENOTYPE SUBTYPING SUBTYPING SUBTYPING Georgia 2007 II CVR1 IGR-1 MGF -1 Armenia 2007 II CVR1 IGR-1 MGF -1 Azerbaijan 2008 II CVR1 IGR-1 MGF II CVR1 IGR-1 MGF -1 Russia Federation MGF -1 + MGF II CVR1 IGR-1 + IGR-2 MGF -3 Ukraine 2012, 2015 II CVR1 IGR-2 MGF -1 Belarus 2013 II CVR1 IGR -2 MGF II CVR1 IGR-2 MGF II CVR1+ CVR 2 IGR-2 MGF -1 Estonia CVR II IGR-2 MGF -1 CVR1SNP1* Latvia II CVR1 IGR-2 MGF II CVR1 IGR-2 MGF -1 Lithuania MGF -1+ MGF II CVR1SNP3* IGR-2 MGF II CVR1 IGR-2 MGF II CVR1 IGR-2 MGF -1 + MGF -2 Poland CVR II IGR-1 + IGR-2 MGF -1 + MGF -2 CVR1SNP2* Moldova II CVR1 IGR-2 MGF -1 Czech Republic 2017 II CVR1 IGR-2 MGF -1 Romania II CVR1 IGR-2 MGF -1 Hungary 2018 II CVR1 IGR-2 MGF -1

33 MOLECULAR CHARACTERIZATION MAIN FINDINGS 1,115 EU ASFVs characterized since 2014 from wild and domestic pigs. All of them GENOTYPE II THE SUBTYPING BY THE ANALYSIS OF VARIABLE REGIONS indicates the presence of different genotype II variants circulating within the EU countries. The genetic data joint to the epidemiological data suggest; Multiple re-introductions of the ASFV within the EU countries (such as Poland) as a result of wild boar movements. Maintenance of the ASFV within the wild boar population Association between virus circulation in wild boar population and domestic pig outbreaks.

34 1. Active assistance to EU member states in the diagnosis of ASF outbreaks, and other European countries if requested/needed Provision of emergency ASF diagnostic kits and reference material To affected EU countries EU MS COUNTRY REAGENT DESCRIPTION ASF-PC ASF-NC ASF-LC ASF-IB ASF-IPT (ml) (ml) (ml) (strips) (fixed plates) VERO cells MS cells ASFV- Ba71V/E70 (ml) (ml) (ml) LITHUANIA POLAND LATVIA ESTONIA ROMANIA CZECH REPUBLIC TOTAL IPT PLATES FOR 5,808 DETERMINATIONS

35 1. Active assistance to EU member states in the diagnosis of ASF outbreaks, and other European countries if requested/needed Provision of emergency ASF diagnostic kits and reference material To affected non-eu countries ASF reagents and commercial kits for ASF virus detection by real time PCR for the analysis of 1,250 samples: i. ASF DNA Extraction Kit 10 kits (1000 samples). ii. DNA Amplification kit (Real-Time PCR) 3 kits (1200 samples). iii. Lyophilized primers King-s/King-a 2 units (1 pair). iv. TaqMan probe 1 unit. v. ASFV isolates and reference DNAs to be used as positive controls for the extraction and/or reaction steps 2 units. vi. ASFV negative serum 50 ml ASF reagents and commercial kits for ASF antibody detection for the analysis of 264 samples: Confirmatory test for 264 samples i. ASF positive reference sera (ASF-PC) 0.1ml. ii. ASF negative reference sera (ASF-NC) 0.1ml. iii. ASF limit reference sera (ASF-LC) 0.1ml. iv. Fixed ASFV monkey stable cells 96- wells immuno-plates (ASF-IPT) 3 plates (264 samples). v. Secondary antibody protein-a peroxidase (HRP-Protein A) for antibody detection techniques 1mg. Moldova s NRL for ASF diagnosis (>1250 PCR tests and >220 serological tests).

36 2. Molecular and biological characterization of viral isolates collected in the ASF outbreaks worldwide in Genetic characterization of current ASFV isolates: FULL GENOME SEQ. In-vivo experimental studies of current circulating ASFV isolates.

37 2. Molecular and biological characterization of viral isolates collected in the ASF outbreaks worldwide in Genetic characterization of current ASFV isolates: FULL GENOME SEQ. In-vivo experimental studies of current circulating ASFV isolates.

38 2. Molecular and biological characterization of viral isolates collected in the ASF outbreaks worldwide in Genetic characterization of current ASFV isolates In-vivo experimental studies of current circulating ASFV isolates.

39 3. Prepare and distribute SOPs, reference standards and other biological reagents for ASF serological and virological diagnosis previous request. New panel of DNAs reference material for PCR amplification Isolates Country of origin Host Species Year of outbreak Town/ Province P72 genotype E70 Spain Domestic pig 1970 Pontevedra I Zsak et al 2005 Reference BF07/IpTC Burkina Faso Domestic pig 2007 Ipelce I Unpublished data INIA-CISA SS14/WB-Sassari1 Italy Wild boar 2014 Sassari I Unpublished data INIA-CISA SS14/DP-Cagliari1 Italy Domestic pig 2014 Cagliari I Unpublished data INIA-CISA Arm07 Armenia Domestic pig 2007 Dilijan II Unpublished data INIA-CISA Ukr12/Zapo Ukraine Domestic pig 2012 Zaporozhye region II Gallardo et al 2014 Ukr15/DP-Kieve 1 Ukraine Domestic pig 2015 Kiev II Unpublished data INIA-CISA LT14/1490 Lithuania Wild boar 2014 Vilnius II Gallardo et al 2014 Pol14/Krus Poland Wild boar 2014 Podlaskie II Gallardo et al 2014 Lv14/DP/Robez3 Latvia Domestic pig 2014 Dienvidlatgale II Unpublished data INIA-CISA Est14/WB-Valga-1 Estonia Wild boar 2014 Valga II Unpublished data INIA-CISA Est15/WB-Tartu14 Estonia Wild boar 2015 Tartu II Unpublished data INIA-CISA MOL16/DP-CERNO1 Moldova Domestic pig 2016 Cernoleuca II Unpublished data INIA-CISA MOL16/DP-MOSA1 Moldova Domestic pig 2016 Mosana II Unpublished data INIA-CISA Moz64 Mozambique Domestic pig 1964 NK V Gallardo et al 2009 MwLil 20/1 Malawi Tick 1983 Chalaswa VIII Complete genome Ken11/KisP52 Kenya Domestic pig 2011 Kisumu IX Unpublished data INIA-CISA Ken06.Bus Kenya Domestic pig 2006 Busia IX Gallardo et al 2009 Ken08Tk.2/1 Kenya Tick 2007 Kapiti X Gallardo et al 2011 UG10/Tk3.2 Uganda Tick 2010 Mburu X Unpublished data INIA-CISA Eth13/1505 Ethiopia Domestic pig 2013 Bishoftu XXIII Achenbach et al 2016 New genotype II ASFV isolates from Moldavia, Estonia and Ukraine, genotype I from Sassari and the recently discovered genotype XXIII from Ethiopia.

40 3. Prepare and distribute SOPs, reference standards and other biological reagents for ASF serological and virological diagnosis previous request. EU MS COUNTRY ASF REFERENCE STANDARDS ASF-Ag ASF-PC ASF-NC ASF-LC ASF-IB (doses) (ml) (ml) (ml) (strips) BELGIUM CROATIA FINLAND ITALY 256, SLOVENIA SPAIN 8, UNITED KINGDOM TOTAL 264, EU MS COUNTRY ASF BIOLOGICAL MATERIAL ASF-IPT ASFV-ISOLATES (fixed plates) (ml) BELGIUM 9 - CROATIA 5 - FINLAND 6 - ITALY 5 - SLOVENIA 6 - UNITED KINGDOM - 4 NON-EU MS NORWAY 2 - TOTAL 33 4 COUNTRY URL-ASF REFERENCE MATERIAL AND STANDARDS URL-ASF reference sera URL-ASF reference samples URL-ASF reference DNAs ASF-PCR CONTROLS (nº panels) (nº panels) (nº panels) (units) AUSTRIA IRELAND BELARUS TOTAL

41 4. TECHNOLOGY TRANSFER and ASSISTANCE at the EURL October 2017; Hungary s NRL October 2017; Greek s NRL

42 4. TECHNOLOGY TRANSFER and ASSISTANCE IN AFFECTED COUNTRIES March 2017 TASK FORCE Romania (SUCEAVA). July 2017 Czeck Republic s NRL

43 5. EVALUATION OF ASF DIAGNOSTIC TECHNIQUES NEW COMMERCIAL PCR KITS IDEXX Real PCR*DNA test

44 5. EVALUATION OF ASF DIAGNOSTIC TECHNIQUES IN AGREEMENT WITH PREVIOUS STUDIES DONE AT THE EURL WAS OBSERVED A DECREASED SS WHEN SAMPLES WERE POOLED AT POOL OF SIZE >3

45 Evaluating the induced- ASFV clinical infection by the interpretation of the laboratorial diagnostic results. OBJECTIVE set up fixed criteria that could determine the timing of the ASF clinical infection in the hosts, mainly in the wild boar, based on laboratory diagnostic results. METHODOLOGY 1,295 samples (497 bloods and 798 tissues) collected from animals experimentally infected with P72 genotype II ASFVs of different virulence (highly virulent to attenuated strains) Parallel diagnosis by real time PCR and by indirect immunoperoxidase test (IPT)

46 497 bloods parallel diagnosis

47 497 bloods parallel diagnosis Acutely infected pigs PCR positives (66%) but negatives in IPT. Only 34% resulted virus and antibody positive

48 497 bloods parallel diagnosis ACUTE = 134 IPT TITER PCR+/IPT - =88 PCR+/IPT + =46 IPT+/PCR- =0 TOTAL DPI/DPE Nº % DPI/DPE Nº % DPI/DPE Nº % NEG % % % - 0 0% 0 0% % - 0 0% 0 0% % - 0 0% 1 1% % - 0 0% 8 6% % - 0 0% 10 7% % - 0 0% 6 4% % - 0 0% 5 4% % - 0 0% 4 3% % - 0 0% 4 3% % - 0 0% 2 1% % - 0 0% 1 1% % - 0 0% 1 1% % - 0 0% 4 3% % - 0 0% 0 0% % - 0 0% 0 0% % - 0 0% 0 0% % - 0 0% 0 0% % - 0 0% 0 0% % - 0 0% 0 0% TOTAL % % 0 0% % Acutely infected pigs PCR +/IPT + antibody titers 1:5,120 in the 87% of the bloods.

49 497 bloods parallel diagnosis Chronically infected pigs 67% only IPT positive against the 31% which resulted positive by both tests. High antibody titers ( 1:5.120) were obtained in the 93.5% of them.

50 497 bloods parallel diagnosis Subacute infected pigs 87% positive by both tests with titers 1:5.120 in the majority of them (91%).

51 798 tissues parallel diagnosis CLINICAL FORM ACUTE SUBACUTE CHRONIC Nº AA Nº AA/CLINICA TISSUE % Spleen % Heart % Liver % Liung % Kidney % Diaph. Muscle % Total LA/TA % ORG. Pool % NL. Pool % Tonsila % Nº SAMPLES % Similar results were obtained in tissues.

52 CONCLUSIONS The overall analysis of the results allowed us to differentiate among acute to subacute-chronic infections combining both virus and antibody detection tests. With regards to the antibody titer the cut-off was fixed in 1:5120 when samples were PCR and antibody positives. The parameters established within this study can be extrapolated to field samples that allow the identification of chronic infections in affected animals, mainly in the wild boar population. This can be used as a new tool to gather useful epidemiological information about the evolution of the disease in endemic areas.

53 6. EURL web site and sequence data bank.

54 6. EURL web site and sequence data bank.

55 6. EURL web site and sequence data bank.

56 6. EURL web site and sequence data bank.

57 6. EURL web site and sequence data bank. PUBLICATIONS

58 NRLs of Estonia, Latvia, Poland, Lithuania, Czeck RP, Romania and Hungary THANK YOU!