Puławy, r.

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1 Puławy, r. Potwierdzenie przypadków od 15. do 38. oraz 3. ogniska ASF w Polsce przez Europejskie Laboratorium Referencyjne (EURL) ds. ASF w Valdeolmos w Hiszpanii. W dniu br. EURL potwierdziło wyniki dodatnie uzyskane przez Krajowe Laboratorium Referencyjne ds. ASF w PIWet PIB w Puławach w próbkach pochodzących od padłych dzików z przypadków znalezionych w strefie objętej ograniczeniami oraz od padłych świń z 3. ogniska ASF w Polsce.

2 DE ECONOMÍA Y i l lf ú1t ll f l0l'il't!t rriv[sllcii!r.]i, Y it{,,rj,0gl{ AüflAflrA i ALIfulttiIAiii ;ifilp(r DI t lvtstrg ci0 { EN SA Il')AD AillflAi SAI-IDA u i mfft, t015 w [nstiluto Nacir nal dl: fnvestigación y lercnologfa Agraria y Alirnenraria SUBDIREccIÓN GENERAL DE tnvesrgactón v rec ol_ooh NUh/I RTGISTRO lqa centro DE INVESTIGACIÓN EN SANIDADANIMAL Assays and activ tíes ma*ed with * are not supported by ENAC accrcditdtion. African Swine Fever (ASFI diaenosis and molecular characterization REPORT ISSUED BYTHE EUROPEAN UNION REFERENCE ABORATORY FOR AFRICAN swrne FEVER (EURL-ASF), rnta-ctsa DATE: April 6th, ARRIVAI DATE TO CISA: Febru ary 27th,2015. RESPONSIBTE FOR SUBMISSION: Name: Dr Andrzej Kowalczyk Institution/Lab: National Veterinary Research Institute (NVR ) Address: 57 Al. Partyzantow Str.; 24-L00 Pulawy Country: POLAND Tel. number: Fax number: Num. ARRIVAI REGISTER CISA: REG. 51,/20L5. TEST REQUESTED: AFRICAN SWINE FEVER (ASF) CONFIRMATORY DIAGNOSIS AND MOLECULAR CHARACTERIZATION. Num. SAMPTES RECEIVED: 94 samples from 37 wild boars (cases L5 to 38) and 5 domestic pigs from the outbreak 3. The samples were identified as is indicated in Table 1: T L2/09/2074 WILD BOAR ,51,6 case #1.5 DNA SPLEEN/KION EY 2 22/09/20L4 WILD BOAR case Sl.6 DNA BLOOD-EDTA 3 04lL0l20L4 WILD BOAR case #17 DNA LUNG/SPLEEN/KI DN EY 06/rol20L4 WILD BOAR r case #18 DNA BLOOD-EDTA 5 06tLot2014 WILD BOAR case #18 DNA BLOOD.EDTA 6 06/L0/2014 WILD BOAR case #18 DNA BLOOD-EDTA 7 061t WILD BOAR case #18 DNA BLOOD-EDTA 8 06/70/2074 WILD BOAR case #18 DNA SPLEEN 07 /1.O/20t4 WILD BOAR case #18 DNA BLOOD-EDTA ltot20l4 WILD BOAR case #18 DNA BLOOD-EDTA 1,1 07 lr0/20t4 WILD BOAR case #18 DNA SPLEEN L2 081ro12074 WILD BOAR case #17 DNA K DNEY 08/70120L4 WILD BOAR case #I7 DNA SPLEEN Page 1 of 10 Ctra. de Algete a El Casar, sin

3 DE ECONOMíA Y lili;ríilut0 lri rl0ii ',1 lltr llvlll;l0aiirlii Y ieiln0l06ia AiiffAillA Y AI-lilriENTA?i -Fr;-o4 it,, f.s. :ii.[;\j Eti q1\,lri d '.'A. NLltui, R[ü15 f RÜ lnstitulo Nar:ir lral dc Invcstigación y 'lncnolo ia Agraria y Álinrcnraria Assays and activities morked w th * are not supported by ENAC accreditat on, T4 20/LOl2OL4 WILD BOAR case #19 DNA BONE MARROW* l) 7L/LL/20L4 WILD BOAR case #20 DNA BLOOD-EDTA 27ttU2074 WILD BOAR case #21 DNA BLOOD-EDTA WILD BOAR case #21 DNA SPLEEN 18 24/ WIID BOAR case 422 DNA BLOOD-EDTA 19 30/77/2074 WILD BOAR case #23 DNA BLOOD-EDTA 20 30/17t2014 WILD BOAR case #24 DNA LU NG 21, 02/L2/20L4 WILD BOAR case #25 DNA SPLEEN 22 02tL2t20t4 WILD BOAR case #25 DNA KI DNEY 23 02/12/2014 WILD BOAR case #25 DNA SPLEEN t WILD BOAR case #25 DNA KIDNEY WILD BOAR t t> rv-rb case #28 DNA LUNG 26 06/72/2074 WILD BOAR , case #27 DNA LUNG /20L4 WILD BOAR J l) I v-ló case fi29 DNA LU NG 28 23/t2120L4 WILD BOAR case #30 DNA SPLEEN/KI DN EY 29 76t0U20t5 WILD BOAR oo case #31 DNA SPLEEN 30 26/OL/2015 DOMESTIC PIG UI5bI-I OUTBREAK 3 DNA BLOOD-EDTA 31 25lOtl207s DOMESTIC PIG o1, OUTBREAK 3 DNA BLOOD.EDTA 32 30/O7/2O7s DOMESTIC PIG OUTBREAK 3 DNA BLOOD-EDTA 33 30/01/20t5 DOMESTIC PIG ot798-4 o1,798-4 OUTBREAK 3 DNA BLOOD-EDTA 34 30/ot/20L5 DOMESTIC PIG s OUTBREAK 3 DNA BLOOD-EDTA 35 02/02/20L5 WILD BOAR case #32 DNA SPLEEN/KI DN EY 36 02/02120Ls WItD BOAR case #32 DNA BLOOD.EDTA 29/0L120t5 WILD BOAR case #33 DNA BLOOD (clot) 38 03l02/2O7s WILD BOAR o case #34 DNA K DNEY 39 os/ WILD BOAR 0223r case #35 DNA BONE MARROW+ 40 0s/02/201s WILD BOAR case #35 DNA BONE MARROW* 41, 0s/02/207s WILD BOAR case #35 DNA BONE MARROW* 42 os/02/2015 WILD BOAR case #35 DNA SPLEEN 43 os/02/20l5 WILD BOAR o case f35 DNA SPLEEN 44 t3/02/2or5 WILD BOAR case #36 DNA BONE MARROW+ 7s/02/2075 WILD BOAR case #37 DNA BONE MARROW* 46 76/02/2075 WILD BOAR case #38 DNA BONE MARROW{ 47 22/ WILD BOAR case #16 BLOOD BLOOD-EDTA 48 06/70/2074 WILD BOAR case #18 BLOOD BTOOD-EDTA /2074 WILD BOAR case #18 BLOOD BLOOD-EDTA 50 06/70t20L4 WILD BOAR case #18 BLOOD BLOOD-EDTA J] 06/LO/20t4 WILD BOAR case #18 BLOOD BLOOD-EDTA 52 06tL0t20t4 WILD BOAR case #18 BLOOD BLOOD-EDTA 53 o7lto/2ot4 WILD BOAR case #L8 BLOOD BLOOD-EDTA tl0t20t4 WILD BOAR case #18 BLOOD BLOOD-EDTA 55 2u7u2O74 WILD BOAR , case #21 BLOOD BLOOD-EDTA 56 24t7u2074 WILD BOAR 300r case #22 BLOOD BLOOD-EDTA 57 30/7t12074 WILD BOAR case #23 BLOOD BTOOD-EDTA 58 osl72/2074 WILD BOAR case #26 BLOOD BLOOD-EDTA /2014 WILD BOAR case #30 BLOOD BLOOD-EDTA 60 02/02/20L5 WILD BOAR case #32 BLOOD BLOOD-EDTA 61 t2/09/2ot4 WILD BOAR 2t516 21,51,6 case #15 TI55U E SPLEEN 62 t2l09l20t4 WILD BOAR 2r51,6 21,51,6 case #15 TISSU E K DNEY 63 04tL0t20L4 WILD BOAR case #17 T SSUE KI DNEY t4 WILD BOAR case #17 T SSUE SPLEEN 65 0uru20t4 WILD BOAR case #17 TISSU E LUNG /2074 WILD BOAR case #1.8 T SSUE SPLEEN t70t2074 WILD BOAR case #18 TISSU E SPLEEN 68 08/L0/20L4 WILD BOAR L2 case S17 TI55U E KIDNEY 69 08/LO/20t4 WILD BOAR 2461,2 246L2 case S17 TISSU E SPLEEN 70 20/LO/20L4 WILD BOAR case #19 T SSUE BONE MARROW' 7I 2tlLt/20L4 WILD BOAR case #21 TISSU E SPLEE N 72 30/LLl20L4 WILD BOAR case #24 TISSU E LUNG 73 02tt2t2074 WILD BOAR case #25 TISSU E SPLEEN O74 WILD BOAR case #25 TISSUE KI DNEY 75 02/t WILD BOAR case #25 TISSUE SPLEEN 76 ouru20t4 WILD BOAR case #25 TISSU E KI DNEY L4 WILD BOAR 1s19-16 case #28 TISSU E tung 78 06/12/2014 WILD BOAR 37s t9-17 case #27 TISSU E LUNG Page 2 of 10 Ctfa dealgete a El Casar, s/n

4 lr,j( i r1 lr0 {ial Ji:AL ilf ií.,ri l: rl: i,;.r1,; i ; tl:.llü_cgi r::\a4fi Y,;Lriltlil r:1 t,t {i 0 0t if!vt"(ll6 ii(i l fii sai,ilü D A,tiLlAL S,É\ l-[n,a lnstltui< Nacic lal de llrvrrstigación y'lircnología Agraria y Alimenraría N ij I'l illiils.rr0 Assays and octivit es marked with * are not supported by ENAC accreditation /L2/20t4 WILD BOAR case #29 TISSU E LUNG 80 23tL2t2074 WILD BOAR case #30 TISSUE SPLEEN 81 23/t2/2074 WILD BOAR case #30 TISSU E K DNEY 82 L5/01/20rs WILD BOAR oo case f31 TISSUE SPLEEN 83 02/02/20L5 WILD BOAR case #32 T SSUE SPLEEN 84 02/o2/20t5 WILD BOAR case #32 TISSU E KI DNEY 85 29/0U207s WILD BOAR case #33 BLOOD BLOOD (clot) 86 03l02/2075 WILD BOAR o2064 o2064 case #34 TISSUE KI DN EY 87 05/o2/2OL5 WILD BOAR case fi35 T SSUE BoNE MARRoW' 88 05/02/2015 WILD BOAR case #35 TISSU E BONE MARROW{ 89 0s/02/2o7s WILD BOAR case #35 TISSUE BoNE MARRoW* 90 05/02/207s WILD BOAR case #35 TISSU E SPLEEN s WILD BOAR case #35 T SSUE SPLEEN 92 L3/02/2O7s WILD BOAR case #36 TI55U E BONE MARROW* 93 Ls/o2/2075 WILD BOAR case #37 TISSU E BONE MARROW /02/2OLs WILD BOAR case #38 T SSUE BoNE MARRow* EXECUTION DATE: From Februa ry,27th to March, 3O'h, ASF DIAGNOSTIC TESTS PERFORMED: 1. ASF virological diagnosis: t. ASF genome detection. A to% (w/v) clarified homogenized tissue suspensions (spleen, kidney, lung, and bone marrow+) were prepared in phosphate-buffered saline tpnr/crsa/ppav/muesrras/1l.the DNA was extracted from the tissue homogenates and blood samples using the High Pure PCR Template Preparation Kit [Rer. 117e682800r (RocHE)l following the standardised procedure [pnr/crsa7ppa7exrraccrón ADN/1]. For amplification of the ASFV genomic DNA, the OIE-real time rpnr/crsa/ppa/pc /2t PCR (OlE 20!2) and the UPL real-time PCR* (Fernández-Pinero et a.,2013) were performed in the undiluted DNA extracted and received per each sample. NOTE: Blood somples ld CISA 58 to uvnr cose 26, on mot 311s1) ond ld CISA 60 (D NVR: cose 32, on mat 01sr-l,) were not tested by PCR due to the few omount ol sample received. ASF virus isolation and haemadsorption (HAD) assdy* [pnr/crsa/ppa/vr/1] was performed on porcine blood monocytes (PBM) according is described in the OIE Manual (OlE 2012). The PBM were inoculated at a multiplicity of infection (moi) 1:10 with the homogenized tissue suspensions (8 wells/per sample; 10 pl inoculum per well). After inoculation, a preparation of 1% homologous red blood cells in phosphate-buffered saline was added to each well and incubated al 95% relative humidity with 5% COr, at 37"C. The plates were examined for haemadsorption over a period of seven days. Samples were blind passaged three times. 2. ASF serological diagnosís*) For ASF antibody detection, the spleen, kidney, lung and lymph node tissue exudates and the plasma from blood samples received were tested using the indirect * lprur/c sn/ppa/tpr/tl in two fold dilutions starting 1/20. Page 3 of 10 Ctra. de Algete a El Casar, s/n

5 DE ECONOMíA Y Instil uto Nac:ic nal clr lrrr.cstigar'irirr, lbcntrlogía Agraria y ñlinrurrraria SUBDIREccIÓN GENERAL DE INVESTIGACIÓN Y TECNoLoGh CENTRo DE INVESTIGACIÓN EN SANIDADANIMAL Assays and activities marked with * are not supported by ENAC accreditotion. 3. ASFV moleculor characterization*) Genetic characterization of ASFV was performed from 32 wild boars representatives of cases 15, 16, L7, 18, L9, ZO, 2!, 22, 23, 24, 25, 27,29, 30, 3I, 32, 33, 34, 35,36,37 and 38, and from the 5 positive domestic pigs obtained during the outbreak 3 (table 2). The genotyping was achieved by PCR and further sequencing, analysing three independent regions of ASFV genome. This comprises, i) the C- terminal end of VP72 coding protein geng, which differentiates up to 22 distinct genotypes (Boshoff et ol., 2007), ii) the intergenic region located between the l73r and 1329L genes and characterized by the presence of TRS (Gallardo et at.,20!41 and iii) the central variable region (CVR) within the gene (Gallardo et ot., 20tI). We compared the nucleotide sequences obtained with those of previously described representative genotype ll ASFVs isolated since April 2007 up to February 2015 from wild and domestic pigs in Eastern and Central European countries (Lithuania, Poland, Latvia and Estonia). Pol14lWB-21516#15 Podlaskie Bialostocki Zaleszant M chalowo L2t09t20L4 WILD BOAR OI ZIJ Ib case #15 Poll4/WB-23704#16 Podlaskie Bialostock SÍa5 ewo, Gródek 22l09l2014 WILD BOAR case #16 Polr4lw i17 Podlaskie Bialostockí WILD BOAR case #17 PolT4lWg-246t2-fl17 Podlaskie Bialostocki 08/ro/2074 WILD BOAR case #17 Pol14/WB-23943#18 Podlaskie Bialostocki Grodek/ M ch lowo Nowos dr 06/L0/20L4 WILD BOAR oo case #18 PotL4lWS lflta Podlaskie Bialostocki P l iowszcryzna, Gródek 06/ WILD BOAR case #18 Pol'4lW fr18 Podlaskie Bialostock P l towsz( y na, Gródek 06/10/2014 WILD BOAR case #18 Poll4lWS *18 Podlaskie Bialostocki PilatowszczEna, cródek WILD BOAR 5U case #18 PolT4lW #L8 Podlaskie Bialostocki Pil towszc.yrna, Gródek 06/L0/2074 WILD BOAR 5l case #18 Pol14/WB #18 Podlaskie Bialostocki Pol14/WB fi18 Podlaskie Bialostocki Gródek, l ich lowo Nowos dy Koloni o7/l0/20l4 WILD BOAR case #18 Gródek, M chalowo Nowos dy o7 lt,/20l4 WILD BOAR IA case #18 Polt4lWg-25744#L9 Pod ask e Bialostock WILD BOAR case #1.9 Pol'4lWS-28374#.20 Podlaskie Bialostocki Koloniá les any, Michálowo 77/7L/2074 WILD BOAR IJ case #20 Poll4lWS-2912L#2L Podlaskie Bialostock Wiejki, Gródek 2Ut7/2074 WILD BOAR 77 J52t case #21 Pol14/WB-30011#22 Podlaskie Sokolski O5kow Polldniowy & corany, Krynki 24/LU2Ot4 WILD BOAR case #22 Pol14/WB-30258#23 Podlaskie Bialostocki Gródek, Pil towszczvrna. Mieleszki 3017L120L4 WILD BOAR case #23 Poll4/WB-30420#24 Podlaskie Bialostocki Gródek, Pilatowszc y n, Miele5 ki 3017L/2014 WILD BOAR case #24 Pol14/WB-30459#25 Podlaskie Sokolski Pod lazn isko, 5z ud2ialowo 02/L WILD BOAR case #25 Pol14/WB-3(M53#25 Podlaskie Sokolski Podla n sko, tudz alowo 02/L2/2074 WILD BOAR case #25 Polf4lWB-lLslg-L7fl27 Podlaskie Bialostocki Gródek, Gzybowce,: 06/L2/20L4 WILD BOAR a/ case #27 Pol15/WB #28 Podlaskie Bialostocki Nowosadtr Michálowo 06t1,2t2014 WILD BOAR case #28 PolL4lW #30 Podlaskie Sokolski Podlaznisko, Szudzialowo 23/72/2074 WILD BOAR case S30 Poll5/WB-00847#31 Pod ask e Bialostocki Radunln, Grodek L6lOLl20t5 WILD BOAR a case #31 Pol15/WB-01903#32 Podlaskie Sokolski Z dwoa y, 5okótk 02/021201s WILD BOAR case #32 Poll5/WB #33 Podlaskie Bialostocki PilJtows?c y n,grddek 29/0L12075 WILD BOAR case #33 Pol15/WB-020ilü34 Podlaskie Bialostocki Cisowka, lvlichalowo 03/02/201s WILD BOAR case #34 Poll5/WB-02231#35 Podlaskie Bialostocki BUdLM chalowo 05102/20Ls WILD BOAR case S35 Poll5/WB-02232f35 Podlaskie Bialostock Bud%Mich lowo os/02/2075 WILD BOAR óó case #35 Poll5/WB-02234#35 Podlaskie B alostocki 0s/021207s WILD BOAR case #35 Pol15/WS-02809É36 Podlaskie Sokolski Wiezchlesie, Szudzi lowo 73/02/2O7s WILD BOAR M case #36 Pol15/WB-02976#37 Podlaskie Bialostocki Bielewic e, Gródek t5l02l207s WILD BOAR case #37 Pol15/WB-02978fi3E Podlaskie Bialostocki Kondrátki, Michalowo 76t02t20L5 WILD BOAR case #38 Pol15/DP fl3 Podlaskie Sokolski Puc lki,sokófká 26/OU2ots DOMESTIC PIG 30 urjot-t OUTBREAK 3 Poll5/DP #3 Podlaskie Sokolski P!cilki,Sokólk 2610r/20L5 DOMESTIC PIG OUTBREAK 3 Poll5/DP f3 Podlaskie Sokolski P!cilki sokólk 30/Ot/2O7s DOMESTIC PIG OUTBREAK 3 Pol15/DP #3 Podlaskie Soko sk Pucilki,sokólk. 30/or/207s DOMESTIC PIG 33 0L798-4 OUTBREAK 3 Poll5/DP #3 Podlaskie Sokolski Puc k Sokó ka 30t01,t2075 DOMESTIC PIG OUTBREAK 3 Page 4 of 10 Ctra de Algete a El Casar, s/n

6 DE ECONOMíA Y li'isfiil-it0 i;il0ijal i: irilil;i iii.(, iii Y ii."nc'tc(jia AGIARIA Y t iliiiiai],i úini?0 lf flr sl tii,;iti l Ür s r,ird10 ;t tlar I-:,ALIDA - I I I L i" lnstiluto N'acirllal tlr. Inr,r.;tigar ion v Ir'<'ntrlo Ía l\graria y ;\linrcrrraria Assays and dctivities marked with * are not supported by ENAC accreditation. RESULTS 1. ASF virus detection ) A positive result was obtained by the UPL-real time PCR* in all samples tested from wild boars and domestic pigs. Using the OIE real-time PCR, the sample ld CISA 87 gave negative although in the DNA received (sample ld CISA 39) from the same animal the presence of the ASFV genome was confirmed by both PCRs. The virus was isolated in PBM cells after three passages from the wild boar identified as (spleen, lung), 246t2 (spleen) belonging both (spleen, gg), (bone marrow, case 19), (spleen, case21), (spleen, case ]g), (spleen, ql!), the animals identified as (kidney) and O2234 (spleen) belonging to cases 34 and 35 respectivelv, showing the character st c ASF positive haemadsorption pattern*.the detailed ASF virus detection results are summarized in the table 3. and ld tube Sample Type Ct vatue Result Ct value Result 1 21,516 DNA SPLEEN/KIDN EY 2r.t2 POS POS NI DNA BLOOD POS POS NT DNA LU NG/5PLEE N/KIDN EY 23.O2 POS POS NT DNA BLOOD POS L9.26 POS NT DNA BLOOD 2L.O2 POS POS NT DNA BLOOD POS PO5 NT DNA BLOOD POS PO5 NT DNA SPLEEN PO POS NT DNA BLOOD POS POS NT DNA BLOOD 22.tt POS zt.?5 POS NT LI DNA SPLEEN POS POS NT ,2 DNA KI DNEY POS POS Nf 1J DNA SPLEEN 20 s7 PO5 t9.74 POS NT DNA BONE MARROW POS POS NT '1, DNA BLOOD POS POS NT DNA BLOOD POS t7.t7 POS NT DNA SPLEEN P POS NI DNA BLOOD POS POS NT DNA BLOOD POS POS NT DNA LU NG 3t.97 POS POS NT DNA SPLEEN POS L8.79 POS NI DNA KI DNEY POS POS NT DNA SPLEEN POS POS NT DNA KI DNEY PO5 24.O1 POS NT DNA LU NG 3t.97 POS POS NT DNA LU NG 24.9r POS 24.OO POS NT DNA LUNG POS 3T.LL POS NT DNA SPLEE N/KI DN EY P POS NT 29 oo847 DNA SPLEEN POS POS NT DNA BLOOD POS 20.t6 POS NT DNA BLOOD POS 2L.O9 PO5 NT t DNA BLOOD PO POS NT 33 o1,798-4 DNA BLOOD POS POS NT 34 o DNA BLOOD POS 2?.76 POS NT DNA SPLEEN/KI DN EY 2L.96 POS 21.2L POS NT DNA BLOOD POS PO5 NT DNA BLOOD (clot) POS POS NT Page 5 of 10 Ctra. de Algete a El Casar, s/n FAX:

7 DE ECONOMíA Y Instiltrrr Nac'iulal rlt Int'r,stigacit]lt v It'r'rrulogfa Agraria y Alinrcnraria INVESTIGACIÓN Y TECNOLOGÍA Assays and activities marked with * are not supported by ENAC accreditation, DNA KI DN EY POS 23.t9 POS NT DNA BONE MARROW POS POS /vr DNA BONE MARROW* POS 27.0L POS NT DNA BONE I\4ARROW* POS 27.2? POS NT DNA SPLEEN PO5 t9.25 POS NT DNA SPLEEN 22.L5 POS 2r.40 POS NT DNA BONE MARROWl POS to.l7 POS NT DNA BONE MARROW* POS 29.L4 POS NT DNA BONE MARROW* PO POS NT BLOOD BLOOD 22,96 PO POS NT BLOOD BLOOD 21,73 POS 20,44 POS N BLOOD BLOOD zo,t2 POS POS NT BLOOD BLOOD 2L,63 POS 2L,5 POS NI BLOOD BLOOD 24,57 POS 23,48 POS NT BLOOD BLOOD 35,77 POS 33,93 POS NI , BLOOD BLOOD 27,96 POS 26,95 POS NT BLOOD BLOOD 18,93 POS 18,9 POS NI 2932r BLOOD BLOOD 18,68 POS t7,88 POS NT BLOOD BLOOD 25,7 POS 20,39 POS NT BLOOD BLOOD 27,94 POS 27,O3 POS NT SLOOD BLOOD 30,4s POS 28,97 POS NT 61 21,5L6 TISSU E SPLEEN 20,62 POS 22.L5 POS NEG 62 21,51,6 TISSU E KI DNEY 26,13 POS POS NEG TISsUE KI DNEY 29,3t POS POS NEG TISSU E SPLEEN 2L,41 POS POS PO TI55U E LU NG 25,2 POS POS PO T SSUE SPLEEN 28,84 POS POS POS TISSUE SPLEEN 25,66 POS POS NEG !2 TISSU E KI DNEY 28,88 POS POS NEG TISSU E SPLEEN 19,55 POS POS PO TISSU E BONE MARROW* 26,L6 POS POS POS 7I 2932t TISsUE SPLEE N 18,95 POS L9 57 POS POS TISSU E LUNG 28,93 POS 29.L0 POS NEG TISSU E SPLEEN 26,!L POS POS NEG T 55UE KI DNEY 29,96 P05 28.L6 POS NT TISSUE SPLEEN 26,67 P L POS NEG TISSU E KI DNEY 30,66 POS POS NT TISSU E LU NG 29,1 PO POS NEG 78 37s19-t7 TISSU E LU NG 29,36 POS 28.t6 POS NEG TISSU E LUNG 32,42 POS POS NEG ftssue SPLEEN 2L,8L POS 22.O2 POS POS TISSUE KI DNEY 30,01 POS 26.Ot PO5 NT 82 oo847 T SSUE SPLEE N 19,59 POS POS POS TISSU E SPLEEN 26,74 POS POS NEG TISSU E KI DN EY 24,3 POS 22.5L POS NT BLOOD BLOOD {CIOT) 31,5 POS POS NT TISSU E KIDNEY 25,55 POS POS NEG TI55UE BONE MARROW- NO CT NEG POS NT TISSU E BONE MARROW- 31,8 POS POS NEG TISSU E BONE MARROW} zast POS PO5 NEG TISSUE SPLEE N L9,26 POS POS POS TISSU E spleen 24,65 POS POS NEG TISSU E BONE MARROW* 34,54 POS POS NEG TISSUE BONE MARROW+ 33,94 POS POS NEG T SSUE BONE MARROW* 33,21 POS POS NT (o) o,e-reol time PCR ) Reol time PCR (K ng et dl., 2003) techn que os is descríbed in the ote Manuol of diognosis for ASF (b) (c) (Chopter OIE seventh edition 2O12) pnt tclstlpptlpcnlzl UPL+eol time PCR ) Real t me PCR test descilbed by Fernóndez et al., 207j bosed on the ljniversal Probe Librory (UPL). V -HAD )V rus isolation ond haemadsorption test on PBM cells os is described in the OIE Monual of diagnosis for ASF (ChoDtet OIE seventh edit on 2012). (1) The result us ng the OIE-real time PCR corresponds to that obto ned in the diluted DNA dilution. NT=NOT TESTED (1/10) Page 6 of 10 Ctra de Algete a El Casar, s/n TEL:

8 DE ECONOMÍA Y ln.jtiiutii l1^tlci,lal DE ilivesii0aili0i! ;l Lsi L0Gr iilafra r A-ltlllifAr:i {]illii. CE II\IESiI6A;iÓN in SANiDIiD Ai{1I,IAI SA[-Ir)A NUful R[GiSlRO Instilulo Na<'iolral de lnt'c'stigación y 1'ccruf ogía \Braria y Alinrcnraría Assoys and activities marked with * are not supported by ENAC occredítation. 2. ASF antibody detection* ) Results obtained in antibody detection by lpt* in exudate tissues and blood are showed n the table 4. Positive antibody detection result was obtained in the wild boar identified as , , 3LL9, 246L2, 3o42o, , 3Ls]rg-17, and case 16 23t04 BLOOD BLOOD NEG NEG NEGATIVE 48 case t BLOOD BLOOD NEG NEG NEGATIVE 49 case BTOOD BLOOD NEG NEG NEGATIVE 50 case BLOOD BLOOD NEG NEG NEGATIVE 51 case BLOOD BLOOD Ll40 POS POSITIVE 52 case BLOOD BLOOD NEG NEG NEGATIVE )5 case s-1. BLOOD BLOOD NEG NEG NEGATIVE 54 case BLOOD BLOOD NEG NEG NEGATIVE 55 case t BLOOD BLOOD NEG NEG NEGATIVE 56 case BLOOD BLOOD NEG NEG NEGATIVE 57 case BLOOD BLOOD tl40960 POS POSITIVE )ó case BLOOD BLOOD Ll2s60 POS POSITIVE 5v case /5 BLOOD BLOOD NEG NEG NEGATIVE bu case BLOOD BLOOD t\eu NEG NEGATIVE ol case 15 rf,10 T SSUE SP LEE N NEG NEG NEGATIVE o case 15 ff,ro T SSUE KI DN EY NEG NEG NEGATIVE 63 case T SSUE KIDNEY NEG NEG NEGATIVE o+ case TISSU E S PLEE N NEG NEG NEGATIVE 65 case L Z TISSU E LUNG NEG NEG NEGATIVE 58 case TISSUE KIDNEY NEG NEG NEGATIVE case l TISSU E S PLEEN Ll40 POS POStTtVE 77 case IISSU E SPLEEN NEG NEG NEGATIVE case TISSU E LUNG Ll2s6O POS POStTtVE t5 case TISSU E SP LEE N NEG NEG NEGATIVE 74 case TISSU E KI DN EY NEG NEG NEGATIVE 75 case TISSUE SPLEEN tl20 NEG POSITIVE case T SSUE K DNEY NEG NEG NEGATIVE 7l case s T SSUE LUNG rll28o POS POStTtVE /a case 27 JlJl>-r / T SSUE LUNG tl640 POS POStTtVE 79 case TISSU E LUNG Llt024O POS POSITIVE 80 case /3 TISSUE SPLEEN NEG NEG NEGATIVE 81 case TISSU E KIDNEY NEG NEG NEGATIVE 82 case 31 oo847 TISSU E S P LEEN NEG NEG NEGATIVE 83 case TISSU E S P LEEN NEG NEG NEGATIVE 85 case 33 o BLOOD BLOOD (CLOr) NEG NEG NEGATIVE on case TISSU E SPLEEN LlsO POS POSITIVE 91 case 35 o2237 TISSU E S PLEE N NEG NEG NEGATIVE (o) URL-PÍ ) tndircd mmunopercxidose techn que on E7j-VERO nfected cells using the protocol stondord zed and volidoted by the Europeon Union Refercnce Loboratory (URL). 3. ASFV moleculor chorocterizatíon* ) The expected =478 bp of the C-terminal p72-gene was obtained in all positive samples. The PCR amplicons for each selected positive an mal were directly sequenced in order to place the ASFV Polish isolates in one of the defined p72 genotypes. In addition, and for easy genotyping on the basis of different-sized PCR products, the CVR within the 86021gene and the intergenic region between the /73R and Page 7 of 10 Ctra. de Algete a El Casar, s/n

9 DE ECONOMÍA Y E N S AY O S lnstituto Nac'io ral dr, lnvt:stigar k)rr y li'r-rurlo.qía r\graria y \linrcnraria EN SANIDADANIMAL Assays and activities mctrked with * are not supported by ENAC accreditation. Pol14/WB- casé#32 Poll4ME- ceseé33 Pol14ruB- cas#34 Poll4lWB- case#35 Poll4/WE- casd36 Poll lwb- caae*37 Ll14/Wg V lnius 1-12 Ll14MB-A rlus 1-22 Ltl4/WB Roki*ki61 13 Lll4PBlgnaliñ 1-11 CheOT Lt14/DP-Rok k s 1 Ltl4lDPfonal ná 1-19 Ll14&B Kup'sk 61'2 Lll 4n tb"kaunas-1 Lt1ryB-Kupiskis 3-7 IngOa Kalmykis09 tumd A 088 Lt15NB-Alytus 2].26 LT1s/WB-Zares i 1-2 LT14/ L'f 1414A2 B 113/Grodno ArmOT Ltl4/DP Utena Pol14/OP- outbreak 1 Pol14/DP Outbreak 2 Poll UDP- Outbreak 3 Pol14/WB- oa#1 Poll4MB,cs#2 Pol14rug- casd3 Pol14ruB-cas& Pol14ñB-EaE# Pol14)WB- c6d6 TAN/1O/Kyelo StP lo9 Poll{MB- ca#7 Poll4lSz 1t3 TAN/11/Chunya OronOB Lv14/OPlRobé 1,3 Lv14/OP/Pied n Lv14MB/Kerc 1-3 I Lv14^tE llndras 1 2 Po[4,^y'y'8 ca6*7 Pol14ñB cas# Poll44VA- ca6dg O Poll4MB- oabdlo a Pol14lW6-ca6d11 l PollaryVB- ca6d12 O E6t14^r'v8-vil O Esr14/W8-Velga Ukrl2tZaW O Pot14ñB-cas*13 a Poll4MB-cas t14 a Poll4mB- casell6 O Potl4/wB- case*16 a PotlffiB- case#17 a Potl4NB- casefle a Por14ruB- casdl9 a Pot1ffiB- c se#20 a Porl4frB- case#2r a Pot14&B- case422 fwro3l forjo Tvéds 1 1/Torio Rostovog Tvéó31?/Novo NOOA/Av Poll4NB- cas#23 O Poll4NB- casd2 O Poll4ME- cas#6 la Pol14/WB- ca5 #7 Pol14&8. c se#za Poll4&B- case O O Poll4MB- caser3l Tv ó71 ZL s genes were also amplified. The new Polish ASFV isolates, clustered, as expected, within p72 genotype ll and showed i.00% nucleotide identity with all compared ASFV isolates from Eastern and Central Europe across the 478-bp C-terminal p72 gene (figure 1). We obtained the same result by sequencing the CVR within the 8602L gene, revealing 10 copies of amino acid tetramer repeats that were 100% identical and unique to those of the ASFV circulating in the Eastern countries since More accurate subtyping by the analysis of the intergenic region between the l73r and lj29l genes showed that the new GEñorvpErr ASF viruses from Poland, have a nucleotide tandem repeat (TRS) insertion (ccnenreral identical to that present in the previous ASFV isolates obtained from wild boar and domestic pigs in the country as well as identical to the Latvia, Estonia and Lithuania viruses obtained during 2014 (Gallardo et al.,2ot4\. 4. Figure 1. Phylogenetictree based on the C-terminal end of the p72 prote n of new Poland wild boar (WB) and domestic pig (DP) viruses (o) grouped in cases (WB) and outbreak (DP) illustrating the genetic relationship with other ASF viruses belonging to ASFV p72 genotypes ll including previous Poland (o) viruses and representatives from Latvia (r), Estonia ( ) and Lithuania (r) and with Eastern European ASFV isolates collected since 2007 to 2013 (r) including the Belarus 2013 isolate (Bel13/Grodno). Page 8 of 10 Ctra dealgete a El Casar, s/n

10 DE ECONOMÍA Y 1 f i{ttl i-!i(] l\t.{l0iial Dt ihvesiilj/.., Lll Y i:il ijl0üí/t AGRABiA y Ar-ltfEf,JTAiitr l]i rtir0 NE tr\tvit iigai ói{ fil sartl0 D,4ititlAl S.ALID,4 i'j''liü ii[üis'ird.. llrstituto Narional cle Invcstigación y'lee:nología Agraria y Alinrcnraria EN SANIDADANIMAL ' Assays ond activities morked wíth * ore not supported by ENAC acueditation. concluston* L. The presence of AsF has been conf rmed in the wild boar samples rece ved from the OIE reported cases 15 to 38 occurred in Poland from September 2014 to February 20L5. 2. The presence of ASF has been confirmed in the samples received from the third domestic pig outbreak occurred in poland on 26th and 3oth January The p72 genotyp ng of the wild boar and domestic pig Polish viruses clustered the viruses within p72 genotype ll circulating in the Eastern European countries since the first introduction in Georgia in 2OO7. 4. The subtyping by the analysis of the intergenic regions between the l73r and 1329L genes ASFV genome showed than the ASF Polish viruses responsible of the wild boar cases and responsible to the second domestic pig outbreak, are to}oa homologous to the viruses responsible of the cases occurred in wild boar and in domestic pigs in lithuania, latvia, Estonia and Poland during 2014 and The table below summarizes the results obtained in each animal combining virus and antibody detection tests, as well as the genotyping results. zrsro case#15 t2l09l20t4 POStTtVE NEGATIVE PolL4lWg-2t5t6#ls case#16 22/09/2074 POSITIVE NEGATIVE Polt4lWB-23704ÉLG 1o0% HOMOTOGY 8e113/Grodno case#17 04t10t2074 POs T VE NEGATIVE Pol14lWB-2t947-2#L7 tl case#17 08/ POStTtVE POSITIVE PolL4lWB-24612üt7 1m% HOMO OGY 8e113/Grodno case#18 06t70t20L4 POS T VE NEGATIVE Pol14lWB-23943# l case#18 06/70120L4 POStTtVE NEGATIVE tl lm% HOMOLOGY 8e113/crodno case#18 05/rol20!4 POSITIVE NEGATIVE Pol14/wB #18 ll lfi'% HOMOLOGY Bell3/Grodno case#18 06/t0/2014 POSITIVE NEGATIVE Polt4lW É18 tl l(xl% HOMOLOGY Bel13/Grodno case# POSITIVE POStTIVE Poll4/WB #18 ll 1ür% HOMOTOGY 8e113/Grodno L case# /20rs POSITIVE NEGATIVE 1ü)% HOMOLOGY Bell3/Grodno case#18 07/LO/20t5 POStTtVE NEGATIVE PolL4lWB-243os-2#LA HOMOLOGY Bel13/Grodno case#18 07 llgl20t5 POSITIVE NT case#19 201t0/2014 POSITIVE NT Pol14lwg-25744#.L9 l(x % HOMOTOGY Sel13/Grodno case#20 L7/7t/2074 POSITIVE NT Polt4lW fl20 100% HOMOLOGY gel13/grodno case#21 2rlnl2074 POSITIVE NEGATIVE Polt4lWg-29lZL*21 1(xr% HOMOLOGY Bel13/Grodno case#22 24/1Lt20L4 POS T VE NEGATIVE Pol14/WB-30011#22 1(xD6 HOMOLOGY Bel13/Grodno case#23 30/rr/2014 POStTtVE POSITIVE Pol14lWB-30258f23 1ü)% HOMOLOGY 8e113/crodno case#24 30trLt20t4 POSITIVE POStTtVE Polr4lw8'-30420É'24 tl 100% HOMOLOGY Bel13/Grodno case# /2O74 POStTtVE NEGATIVE Pol14lWB-30459f25 1(x)% HOMOLOGY Bel13/Grodno case#25 02/72/2014 POSITIVE POSITIVE Polr4lWS #25 1fi'% HOMOLOGY Bell3/Grodno case#25 os/72/2074 NT POSITIVE 5IJJJ-I / case#27 06/72/2014 POStTtVE POSITIVE Poll4lW #27 lq% HOMOLOGY Bell3/Grodno 5A>t9-fO case#28 06/L2120L4 POStTtVE POStTtVE Pol14lWB #28 1ür% HOMOLOGY Bell3/Grodno casef29 06/L2/20L4 POSITIVE POStTTVE case#30 231L2120t4 POS T VE NEGATIVE Pol14lWS ,0 1(x)% HOMOTOGY Bel13/Grodno case#31 L6lO!/207s POSITIVE NEGATIVE Pol15/W # case#32 02t02/2015 POS T VE NEGATIVE Poll5/WB-01903#32 1(x)% HOMOLOGY Bell3/Grodno case#33 29/OU20t5 POSITIVE NEGATIVE Polt'lWB-02O62-2fi33 1(N'% HOMOLOGY Bell3/Grodno case#34 03l02l207s POSITIVE NT PoILSIW #34 Page 9 of 10 Ctra. de Algete a El Casar, s/n 28'130 Valdeolmos

11 DE ECONOMíA Y NU l tltgistr 0.. fia S lrr"st luto Nac;iolral de lnvnstigae:ión y'lix;nología r\ raria y Alinrenraría Assays ond activities marked with'* are not supported by ENAC acueditotion case#35 05t02t20rs POSITIVE NT Pol15/wB-0223lf 35 ll HOMOLOGY Bell3/Grodno case#35 osl02/207s POSITIVE NT Pol15/WB-02232fl35 l0 HOMOLOGYBell3/crodno case#35 0s/02/20Ls POStTTVE POStTtVE Pol15/WB-0223/t#35 I 100% HOMOTOGY 8e113/crodno case#35 oslo2l20t5 POSITIVE NT case#3s 05102/2075 POSITIVE NEGATIVE case#36 13tozt2075 POStTtVE NT Poll5/WB-02809#36 I l(xi% HOMOLOGY Bell3/Grodno case#37 75/O2/zOLs POSITIVE NT PolLSíWB-O297ü37 lüm HOMOLOGY Bel13/crodno case#38 76/O2l20tS POStTtVE NT Pol15/WB-02978#38 I 1ür% HOMOTOGY 8e113/crodno OUTBREAK L12015 POStTtVE NT Pol15/DP fi3 100% HOMOLOGY Bel13/crodno OUTBREAK u207s POSITIVE NT Pol15/DP #3 lql% HOMOLOGY 8e113/Grodno OUTBREAK 3 30t07t20t5 POS T VE NT Pol15/DP f3 lfx % HOMOLOGY Bel13/Grodno ot79a-4 OUTBREAK 3 30/oU20L5 POSITIVE NT Pol].5lDP-079A-4#3 I 10o% HOMOTOGY gel13/crodno OUTBREAK t1207s POSITIVE NT Pol15/DP #3 1(xt% HOMOLOGY Bell3/Grodno NT = not tested REFERENCES 1 Boshoff,C,Bastos,A.D,Gerber,L I &Vosloo,W (2007) Geneticcharacterisat onofafricanswinefevervirusesfromoutbreaks nsouthernafrica( Vet. M crob o\.121, Carmina Gal ardo, Jov ta Fernández-Pinero, V rginia Pelayo, lsmail Gazaev, lwona Markowska-Daniel, cedim nas Pr dotkas, Raque Nieto, paloma Fernández- Pacheco, Svetlana Bokhan, OlegNevolko, ZhannaDrozhzhe, Covadonga Pérez, Alejandro Soler, Denis Kolvasov, and Marisa Arias (2014) cenetic Var at on 3 amongafricanswinefevergenotypellv ruses,easternandcentraleurope Emerg,tnfect.D s,volume20,number9-septembe12014 GallardoC,AnchueloR,PelayoV,PoudeviSneF,LeonT,Nzouss J,BishopR,Pérezc,SolerA,NietoR,Man nh,ariasm.(2011) Afr cansw nefeverviruspt2 genotype lx n domest c p gs, Congo. Eme g /rfect D 5, Augr 17(8): Fernández-P neroj,gallardoc,eliza dem,roblesa,gómezc,bishopr,heathl,couacy-hymanne,fas nafo,pelayov,solera,ariasm Moleculard agnosis ofafricanswinefeverbyanewreal-timepcrus nguniversalprobel brary(2013) TtunsboundoryondEmergingD seoses 60(2013)48-58.do :IOLII7I f2.O!377.x Epub 2012 Mar OIE May2012,postin8date.Chapter2.8.1.Afr canswinefever, nmonuolofd ognost ctestsondvoccínesforteftestriolon mols2\72 WorldOrgan zationfor 6 Animal Health, Par s, France. th_standards/tahm/2.08 o1_asf pdf K ng DP, Re d SM, Hutchings GH, Grierson SS, Wi k nson Pl, Dixon LK, Bastos AD, Drew TW (2003) Development of a TaqMan pcr assay with internal ampl f tion control fo. the detection ofafrican sw ne fever v rus J V rol.methods Jan:107(1): In Valdeolmos, Madrid (Spain), April 6th, Dr. Carmina Gallardo Frontau Researcher, Laboratory EU Reference Laboratory for INIA-CISA Technical Director EU Reference Laboratory for ASF INIA-CISA 1,.'r, tt'$ \i.,.. ' t l:1,4-*:; Approvar Dr. Víctor Briones Dieste Director INIA-CISA Th s report shall not be reproduced, except in its entirety, without the written approval of lnla-clsa. The results provided are only related to the samples received. lnia-cisa accepts no responsibility for improper use of this report. Page 10 of 10 Ctra de Algete a El Casar, TEL: 9'l

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