Puławy 20.10.2016

Potwierdzenie wystąpienia afrykańskiego pomoru świń ;ASF w Polsce przez Europejskie Laboratorium Referencyjne (EURL) ds. ASF w Valdeolmos w Hiszpanii dla przypadku nr 42 oraz przypadków od 75 do 105 oraz ogniska nr 4 (1/2016).

ir,,rr,ll,.;:'il,, i li rj:,1!' i,,,r_i I iii, itt; i;t,r riirl:r;i r ;L::.ijr,Tr,,::,r $ MINISTERIO i;ilriir ii t r'):rl r" ,:l i¡r il li r il l : i¡j DE ECONOMÍA Y l:i ¡]" [- ! !,] ¡\ i-^ -' **-^'-***'-"-''t Instilurr: Nacio¡lal dc lnvrsrigar:irirr COMPETITIVIDAD ,l y Ir'<'nulo¡¡ia .{¡iraria y r\linrenraria

SUBDIRECCION GENERAL DE ENSAYfTS INVESTIGACIÓN Y TECNOLOGíA N" 9ó.9/LEl865 CENTRO DE INVESTIGACIÓN _ EN SANIDAD ANIMAL * Assays and activities mariett with are not supported by ENAC acueditation.

characterization

REPORT ISSUED BY THE EUROPEAN UNION REFERENCE LABORATORY FOR AFRICAN swtNE FEVER (EURL-ASF), tNtA-CrSA

DATE: September 23'o, 2016.

ARRfVAI DATE TO CISA: July 22"o,2016.

RESPONSIBTE FOR SUBMISSION: Name: Andrzej Kowalczyk Institution/Lab: National Veterinary Research Inst¡tute (NVRI) Address: 57 Partyzantow Avenue,24-tOO PULAWY Country: POTAND Tel. number: +48 81889 3047 Fax number: +48 81 886 2595

Num. ARRIVAI REGISTER CISA: REG. L44/2016.

TEST REQUESTED: AFRICAN SWINE FEVER (ASF) CONFIRMA'I-ORY DIAGNOSIS AND GENE SEQUENCING.

Num. SAMPIES RECEIVED: 86 samples from 40 wild boars comprising 20 tissues, 14 homogenates of tissues, 14 bloods and 38 DNAs.45 samples from 9 domestic pigs comprising 5 bloods, 18 tissues and 23 DNAs identified as follows (table 1):

WILD BOAR 19L2I 7 cAsE 75 I972r/t5 Bialowie2a TISSU E BONE MARROW WILD BOAR 3to7Llts 2 cAsE 79 3LO7L f{arewka TISSUE BONE MARROW WILD BOAR 32842tts 3 CASE 80 32842 llarewka TISSUE BONE MARROW WILD BOAR 3298r/L5 4 cAsE 81 3298r Biatowie2a TISSUE BONE MARROW WILD BOAR 00969/16 cAsE 85 00969 l\arewka T SSUE SPLEEN WILD BOAR 00969/16 6 cAsE 85 00969 l\arewka TISSUE LYMPHNODE WILD BOAR 00969/16 7 cAsE 85 00969 l\arewka TISSUE KIDNEY

Ctra, de Algete a El Casar, s/n Page 1 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 6202247 MINISTERIO DE ECONOMfA Y Ilrstil u¡o Nacional do lflvÉ:itigarión COMPETITIVIDAD 'li.< y rrrrlo¡¡Ía Agraria y i\lirrrcnraria i. '; $ii ? SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh CENTRO DE INVESTIGACIÓN EN SANIDADANIMAL

WILD BOAR 05778/L6 8 cAsE 89 05778 Narewka T SSUE BONE MARROW WILD BOAR 0698s/16 9 CASE 90 0698s Biatowieia TISSUE BONE MARROW WILD BOAR 0753L/L6 10 cAsE 91 0763L B¡afowieia TISSUE BONE MARROW WILD BOAR LO37s/L6 LL cAsE 93 LO375 Narewka TISSUE BONE MARROW WILD BOAR I2990t16 L2 cAsE 94 12990 Narewka TISSUE BONE MARROW WILD BOAR L40ss/16 I5 cAsE 95 14055 Biatowie2a HOMOGENATE BONE MARROW WILD BOAR t40s6/L6 L4 CASE 95 14056 B¡aiowie2a HOMOGENATE BONE MARROW DOMESTIC PIG ts337/t6 15 OUTBREAK 4 15337 Hainówka TISSUE K DNEY DOMESTIC PIG L5337 116 IO OUTBREAK 4 15337 Hajnówka TISSUE SPLEEN DOMESTIC PIG 1s338/16 L7 OUTBREAK 4 15338 Hainówka T SSUE K DNEY DOMESTIC PIG 1s338/16 18 OUTBREAK 4 15338 Hajnówka TISSUE SPLEEN DOMESTIC PIG Ls339t76 19 OUTBREAK 4 15339 Hajnówka T SSUE KIDNEY DOMESTIC PIG 15339/16 20 OUTBREAK 4 15339 Hainówka TISSUE SPLEEN DOMESTIC PIG 75340/L6 2T OUTBREAK 4 15340 Hainówka TISSUE K DNEY DOMESTIC PIG Ls34O/16 22 OUTBREAK 4 15340 Hajnówka TISSUE SPLEEN DOMESTIC PIG r534U].6 z5 OUTBREAK 4 15341 Hainówka T SSUE KIDNEY DOMESTIC PIG Ls34t/t6 24 OUTBREAK 4 15341 Hajnówka TISSUE SPLEEN DOMESTIC PIG 15553/16 25 OUTBREAK 4 15553 llainówka TISSUE KIDNEY DOMESTIC PIG 15553/16 ¿o OUTBREAK 4 IJJf,5 Hainówka TISSUE SPLEEN DOMESTIC PIG t5554/t6 27 OUTBREAK 4 15554 Hainówka TISSUE KIDNEY DOMESTIC PIG L5ss4/L6 28 OUTBREAK 4 15554 Hainówka TISSUE SPLEEN DOMESTIC PIG 15sss/16 ZJ OUTBREAK 4 1555s Hainówka T SSUE K DNEY

DOMESTIC PIG lssss/16 30 OUTBREAK 4 15555 Hainówka TISSU E SPLEEN

DOMESTIC PIG L5556/16 5l OUTBREAK 4 15556 l{ainówka TISSU E KIDNEY DOMESTIC PIG 1sss6/16 5Z OUTBREAK 4 15556 llainówka TISSUE SPLEEN WILD BOAR Ls777/L6 33 cAsE 96 L5777 Czy2e HOMOGENATE BONE MARROW WILD BOAR 15778116 34 cAsE 96 15778 Czvie HOMOGENATE BONE MARROW WILD BOAR 15779tL6 35 cAsE 96 15779 Czv2e HOMOGENATE BONE MARROW WILD BOAR r5780/L6 5b cAsE 95 15780 Czv2e HOMOGENATE BONE MARROW WILD BOAR r5780/L6 cAsE 95 15780 Czy2e TISSUE SPLEEN WILD BOAR L6O8O/16 38 CASE 98 16080 B¡afowie¿a HOMOGENATE BONE MARROW WILD BOAR 16554/L6 39 cAsE 99 16554 llainówka HOMOGENATE BONE MARROW WILD BOAR 16555/16 40 cAsE 99 16555 l-lajnówka TISSUE SPLEEN WILD BOAR L65ss/L6 4I cAsE 99 16555 llajnówka TISSUE KIDNEY WILD BOAR t6557 /t6 42 CASE 99 L6557 Hainówka T SSUE SPLEEN WILD BOAR L6557 116 43 CASE 99 16557 l-lainówka TISSUE KIDNEY WILD BOAR 16677 lL6 44 cAsE 101 L6677 TISSUE KIDNEY WILD BOAR t6677/L6 45 CASE 101 LOOt I TISSUE SPLEEN WILD BOAR L68s8/L6 46 CASE 102 16858 l-lainówka TISSUE BONE MARROW WILD BOAR 17073tt6 47 CASE 103 L7OL3 flainówka HOMOGENATE BONE MARROW WILD BOAR L70L4l16 48 cAsE 104 t7074 flainówka HOMOGENATE BONE MARROW WILD BOAR L7jLs/t6 49 cAsE 104 t7075 flainówka HOMOGENATE BONE MARROW WILD BOAR 770t6/16 50 cAsE 104 t7016 Flainówka HOMOGENATE BONE MARROW WILD BOAR 17LO7 lL6 51 CASE 105 17L07 Flainówka HOMOGENATE BONE MARROW WILD BOAR 05097/\s JZ CASE 42 05097 Gródek HOMOGENATE BONE MARROW WILD BOAR L976s-t7/rs )5 CASE 75 19765 -WB Ne 17 Krvnki BLOOD BLOOD-EDTA WILD BOAR 27L52-55tt5 54 CASE 77 27152- WB Ne 55 Gródek BLOOD BLOOD-EDTA WILD BOAR 3029GLO/Ls 55 CASE 78 30296 - WB Ne 10 S dra BLOOD BLOOD-EDTA WILD BOAR 332s6-22lLs CASE 83 33256 - WB Ne 22 Gródek BLOOD BLOOD-EDTA WILD BOAR 3324s-3/75 57 cAsE 82 33245- WB Ne 3 Narew BLOOD BLOOD-EDTA WILD BOAR 00347-22/t6 58 cAsE 84 00347 WB Ne 22 Narew BLOOD BLOOD-EDTA WILD BOAR 00969/16 59 CASE 85 00969 Narewka BLOOD BLOOD-EDTA WILD BOAR 03s18-8/16 60 CASE 86 03518 - WB Ne 8 Narewka BLOOD BLOOD-EDTA WILD BOAR o45Itt6 OI CASE 87 04513 Supraó1, BLOOD BLOOD-EDTA WILD BOAR o5432-r2lL6 62 cAsE 88 05432 WB Ne 12 B¡afow¡e¿a BLOOD BLOOD-EDTA WILD BOAR 08s90-u16 o5 cAsE 92 08590 - WB Ne r 1 Michatowo BLOOD BLOOD-EDTA

Ctra de Algete a Ef Casar, s/n Page 2 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 6202247 s.Jl, MINISTERIO DE ECONOMIA Y Ilrstiluto Nat:io¡ral r.le Invrsrigar:lnrr COMPETITIVIDAD y lccnoln¡¡ía dgraria y Alirncntaria

SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGÍA CENTRO DE I¡¡VESTIGACIÓ¡. EN SANIDAD¡,NIMAL * Assays and activities marked with ore not supported' by ENAC accreditotion.

DOMESTIC PIG Ls337/16 64 OUTBREAK 4 15337 Hainówka BLOOD BLOOD-EDTA DOMESTIC PIG 1s338/16 OUTBREAK 4 15338 Hainówka BTOOD BLOOD-EDTA DOMESTIC PIG L5339/16 66 OUTBREAK 4 15339 Hainówka BLOOD BLOOD.EDTA DOMESTIC PIG r534O/L6 67 OUTBREAK 4 15340 Hainówka BLOOD BLOOD-EDTA DOMESTIC PIG t534r/L6 68 OUTBREAK 4 t5347 Hajnówka BLOOD BLOOD-EDTA WILD BOAR L5887-3t16 69 cAsE 97 15887 WB Ne 3 Narewka BLOOD BLOOD-EDTA WILD BOAR L6565-7/t6 70 cAsE 100 16565 WB Ne 7 Hainówka BLOOD BLOOD-EDTA WILD BOAR 16677 /L6 7T cAsE 101 LOOT I Narewka BLOOD BLOOD-EDTA WILD BOAR L9IzL 72 cAsE 75 L9L2L|15 Bialowie2a DNA BONE MARROW WILD BOAR 3707U15 73 cAsE 79 3to77 Narewka DNA BONE MARROW WILD BOAR 32842/75 74 cAsE 80 32842 Narewka DNA BONE MARROW WILD BOAR 3298L1t5 75 CASE 81 3298t Biatowie2a DNA BONE MARROW WILD BOAR 33245-3/Ls 76 cAsE 82 33245- WB Ne 3 Narew DNA BLOOD-EDTA WILD BOAR 00969/16 cAsE 85 00969 Narewka DNA SI LEEN WILD BOAR 00969/16 78 cAsE 85 00969 Narewka DNA LYMPHNODE WILD BOAR 00969/16 79 CASE 85 00969 Narewka DNA KIDNEY WILD BOAR 00969/16 80 CASE 85 00969 Narewka DNA BLOOD-EDTA WILD BOAR o45r/16 81 cAsE 87 04513 SupraSl DNA BLOOD-EDTA WILD BOAR o5778/t6 82 cAsE 89 05778 Narewka DNA BONE MARROW WILD BOAR 06985/16 ¿l5 cAsE 90 06985 Biatow¡eia DNA BONE MARROW WILD BOAR 0763LlL6 84 cAsE 91 0763L Biatowie2a DNA BONE MARROW WILD BOAR 10375/L6 85 cAsE 93 10375 Narewka DNA BONE MARROW WILD BOAR L299O/t6 86 CASE 94 L2990 Narewka DNA BONE MARROW WILD BOAR 140551t6 87 cAsE 95 14055 B¡afowie2a DNA BONE MARROW WILD BOAR r40s6/16 88 cAsE 95 14056 Biaiowieia DNA BONE MARROW DOMESTIC PIG Ls337 /L6 89 OUTBREAK 4 15337 Hajnówka DNA BLOOD-EDTA DOMESTIC PIG 15337 /16 90 OUTBREAK 4 15337 Hainówka DNA KIDNEY DOMESTIC PIG rs337 /L6 91 OUTBREAK 4 15337 Hainówka DNA SPLEEN DOMESTIC PIG 15338/16 92 OUTBREAK 4 15338 Hainówka DNA BLOOD-EDTA DOMESTIC PIG 1s338/16 93 OUTBREAK 4 15338 Hainówka DNA KIDNEY DOMESTIC PIG 1s338/16 94 OUTBREAK 4 15338 Hajnówka DNA SPLEEN DOMESTIC PIG 15339/16 >f, OUTBREAK 4 1s339 Hainówka DNA BLOOD-EDTA DOMESTIC PIG L5339116 96 OUTBREAK 4 15339 Hainówka DNA K DNEY DOMESTIC PIG Ls339/t6 97 OUTBREAK 4 15339 Hainówka DNA SPLEEN DOMESTIC PIG r$40/L6 98 OUTBREAK 4 15340 Hainówka DNA BLOOD.EDTA DOMESTIC PIG Ls34O/16 99 OUTBREAK 4 15340 Hainówka DNA KIDNEY DOMESTIC PIG Ls34O/L6 100 OUTBREAK 4 15340 Hainówka DNA SPLEEN DOMESTIC PIG L534Ut6 101 OUTBREAK 4 15341 Hainówka DNA BLOOD-EDTA DOMESTIC PIG r534t/16 LO2 OUTBREAK 4 15341 Hajnówka DNA KIDNEY DOMESTIC PIG r534rl16 103 OUTBREAK 4 15341 Hainówka DNA SPLEEN DOMESTIC PIG L5553/L6 104 OUTBREAK 4 15553 Hainówka DNA KIDNEY DOMESTIC PIG 15553/16 105 OUTBREAK 4 15553 Hainówka DNA SPLEEN DOMESTIC PIG L5554/L6 106 OUTBREAK 4 15554 Hainówka DNA KIDNEY DOMESTIC PIG tsss4/L6 LO7 OUTBREAK 4 15554 Hainówka DNA SPLEEN DOMESTIC PIG 15555/16 108 OUTBREAK 4 1s555 Hainówka DNA KIDNEY DOMESTIC PIG 15555/16 109 OUTBREAK 4 15555 Hajnówka DNA SPLEEN DOMESTIC PIG 15556/16 110 OUTBREAK 4 1f,f,f,o Hainówka DNA K DNEY DOMESTIC PIG 1s556/16 111 OUTBREAK 4 15556 Hainówka DNA SPLEEN WILD BOAR 15777 lL6 LL2 cAsE 96 r5777 Czv2e DNA BONE MARROW WILD BOAR L5778/L6 LL3 cAsE 96 L5778 Czy2e DNA BONE MARROW WILD BOAR 15779/76 tL4 CASE 96 15779 Czv2e DNA BONE MARROW WILD BOAR r5780/16 115 cAsE 96 15780 DNA BONE MARROW WILD BOAR L57801t6 116 CASE 96 15780 Czv2e DNA SPLEEN WILD BOAR 16080/16 tt7 cAsE 98 16080 Biafowie¿a DNA BONE MARROW WILD BOAR !6s54/t6 118 cAsE 99 16554 Hainówka DNA BONE MARROW WILD BOAR 16555/16 119 cAsE 99 165s5 Hainówka DNA SPLEEN

Ctra. de Algtete a El Casar, s/n Page 3 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 620 22 47 # MINISTERIO DE ECONOMíA Y InstiluLo Nacional dc lnlestigarri(:n COMPETITIVIDAD y Tecrrologia A¡¡raria y,A,lilnrnr aria

SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGiA CENTRO DE INVESTIGACIÓN EN SANIDADANIMAL

WILD BOAR 16sss/16 L20 CASE 99 16555 Hainówka DNA K DNEY WILD BOAR 16557/t6 t2t CASE 99 16557 Hainówka DNA K DNEY WILD BOAR L6557/t6 L22 cAsE 99 L6s57 Hainówka DNA SPLEEN WILD BOAR L6565-7/76 L23 CASE 1OO 16565 - WB Ns 7 Hajnówka DNA BLOOD-EDTA WILD BOAR L6677/L6 LZ+ cAsE 101 t6677 DNA BLOOD-EDTA WILD BOAR t66771t6 L25 cAsE 101 L6677 DNA KIDNEY WILD BOAR L6677 116 126 cAsE 101 LOO/ I DNA SPLEEN WILD BOAR r68s8/L6 t27 CASE 102 16858 Hainówka DNA BONE MARROW WILD BOAR L70L3lL6 t28 cAsE 103 L7OL3 Hainówka DNA BONE MARROW WILD BOAR L70t4/16 L29 cAsE 104 L7OL4 Hainówka DNA BONE MARROW WILD BOAR t70r5t16 130 cAsE 104 L70t5 Hajnówka DNA BONE MARROW WILD BOAR r7ot6/L6 131 cAsE 104 L70t6 Hajnówka DNA BONE MARROW WILD BOAR 17tO7 It6 L32 cAsE 10s L7TO7 Hainówka DNA BONE MARROW

EXECUT|ON DATE: From July, 26th to September t2th,2016

ASF DIAGNOSTIC TESTS PERFORMED: t. ASF virus detection: o. ASFV genome detect¡on. A tOo/o (w/v) clarified homogenized t¡ssue suspensions have been

prepared in phosphate-buffered salin€ ler,rrTcrsA/ppA/MuEsrRAs/rt. The DNA has been extracted directly from the 34 tissue homogenates and 14 bloods from the 40 wild boar and from 18 tissue homogenates and 5 bloods from the 9 domestic pigs received using the High Pure

PCR Template Preparation Kit [Rer. 117s6828001 (RocHE)] following the standardised procedure

tpNr/crsA/ppA/ExrRAccróN ¡or/rl. For amplification of the ASFV DNA, the UPL real-time PCR

(Fernández-Pinero et ol.,20t3l lpNr/crsA/ppA/pcn/:t have been performed in the DNAs extracted from blood, tissue homogenates and from the 61 extracted DNAs directly received from the NRL.

b. ASF virus isolotion and hoemadsorption (HAD) assay* rpNr/crsA/ppA/vrl1t was performed on porcine blood monocytes (PBM) according is described in the OIE Manual (OlE 20L2). The PBM were inoculated at a multiplicity of infection (moi) 1:10 with selected PCR positive

samples (8 wells/per sample; 10 pl inoculum per well) in which was obtained a positive PCR result. After inoculation, a preparation of !% homologous red blood cells in phosphate- buffered sal¡ne was added to each well and incubated at 95% relative humidity with 5% COz, at 37"C. The plates were examined for haemadsorption over a period of seven days. The samples have been blind passaged three times.

2. ASF serological díagnosis. For ASF antibody detection, 65 samples included 23 tissue exudates, 19 blood and24 bone marrow homogenates were tested using the indirect immunooeroxidase

technique (lPT)* lpur/crs¡/pprlrprlr] in two fold dilutions starting from L/2O (in case of bone marrow starting from 1/5).

Ctra de Algete a El Casar, s/n Page 4 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 6202247 i ;ii' l! -, r i::i" r:l,] v rr; 'i1i:fr iii¡l;', '.i:i i,); I ,:,,:lii, )j! l:.i si.tij,r!;1r,i:jii MINISTERIO :ri qi--,nA

DE ECONOMíA Y is .. Instil urr¡ Nar-ir¡llal rlr' Invr.srigarlérr COMPETITIVIDAD ?1 -:.. 1' li'r:rrolo¡1ia A¡¡raria y .,\linrcrrtaria

SUBDIREccIÓN GENERAL DE rNVESTrcAcróN y rEcNoLoGh :l-1r: It,.: J]3 E NSAYOS iIijl; . g6s/LF.r R6s cENTRo oe l¡¡veslc¡clótr EN SANIDAD ANIMAL

Assays and dctivities mdrked with * ore not supported by ENAC accreditation,

3. ASFV moleculdr choracterization*. Genetic characterization of ASFV from 23 wild boar and 2 domestic pig has been performed by PCR and further sequencing throughout the analysis of three independent regions of the ASFV genome which comprises; i) the C- terminal end of Vp72 coding protein gene, which differentiates up to 23 distinct genotypes (Boshoff et al., 2OO7; Achenbach JE et al., 2016l, ii) the intergenic region located between the t73R and t3291genes and characterized by the presence of TRS (Gallardo et al., 2OL4) and iii) the central variable region(CVR)withintheB602L-gene(Gallardoetol.,2OLL). Wehavecomparedthenucleotide sequences obtained with those of previously described representative genotype ll ASFVs isolated since April 2007 up to September 2OL6 from wild and domestic pigs in Eastern and Central European countries (Lithuania, Poland, Latvia, Estonia, Belarus, Ukraine, Armenia, Azerbaijan, Georgia and Russia Federation).

RESULTS

1. ASF VIRUS DETECTION:

1.1.WltD BOAR SAMPLES ) Positive ASFV result has been obtained by the UPL real-time PCR in 79 out of 86 samples tested from wild boar confirming the presence of ASFV in 34 out of the 40 wild boar received (85%). The weak positive result (CD35) obtained in the samples identified as 58 and 60 (lD CISA) was confirmed in a second PCR amplification run in which the extracted DNA per each sample was analysed by duplicate. After three passages in PAM, the

ASFV has been isolated only from 2 wild boar identified as 32981 (rD crsA4, case 81) and 07631 (ro crs¡

10, case e1) showing the classical haemadsorbing pattern of the ,ASFV circulating in Europe. The results obtained in virus detection are summarized in the table i2.

L cAsE 75 r9r27/75 TISSU E BONE MARROW 28.35 POSTTTVE NEG cAsE 79 3LO7t TISSUE BONE MARROW 32."t5 POSTTTVE NT 3 CASE 80 32842 TISSUE BONE MARROW 25.56 POStTtVE NEG 4 cAsE 81 32981 TISSUE BONE MARROW 25.ti2 POStTtVE POS l cAsE 85 00969 T SSUE SPLEEN 2r.it4 POSTTTVE NEG 6 CASE 85 00969 TISSUE LYMPHNODE 32.4 POStTtVE NT CASE 85 00969 TISSUE K DNEY 24.12 POSITIVE NT 8 CASE 89 o5778 T SSUE BONE MARROW 25.98 POSTTtVE NEG 9 CASE 90 06985 TISSUE BONE MARROW 27.03 POSITIVE NEG 10 cAsE 91 0763L TISSUE BONE MARROW 26.01 POSITIVE POS TL cAsE 93 70375 T SSUE BONE MARROW 23.01 POStTtVE NEG

Ctra de Algete a El Casar, s/n Page 5 of 13 28130 Valdeolmos MADRID TEL: 91 6202300 FAX: 91 6202247 MINISTERIO DE ECONOMÍA Y Instiluto Nacio¡¡al rk: lnvrsti¡lacirin COMPETITIVIDAD y'licru'rlogia A¡iraria y r\lhncnr ari:t

SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh CENTRO DE INVESTIGACIÓN EN SANIDAD ANIMAL * Assays and activities marked with are not supported by ENAC accreditotion.

T2 cAsE 94 L2990 TISSUE BONE MARROW 25.L3 POSTTtVE NEG IJ cAsE 95 14055 HOMOGENATE BONE MARROW L4.79 POSITIVE NT L4 CASE 95 14056 HOMOGENATE BONE MARROW 18.59 POSITIVE NT 33 cAsE 96 L5777 HOMOGENATE BONE MARROW 29.38(1) POSITIVE NT 34 cAsE 95 L5778 HOMOGENATE BONE MARROW 30.s POSITIVE NT 35 cAsE 96 L5779 HOMOGENATE BONE MARROW 31.34 POSITIVE NT 50 cAsE 96 15780 HOMOGENATE BONE MARROW 19.36 POStTtVE NT 37 cAsE 96 15780 TISSUE SPLEEN 24.85 POStTtVE NT 38 CASE 98 16080 HOMOGENATE BONE MARROW 2L.96 POSITIVE NT 5v cAsE 99 16554 HOMOGENATE BONE MARROW 33.87 POSITIVE NT 40 cAsE 99 16555 TISSUE SPLEEN 25.81 POStTtVE NEG 4L cAsE 99 16555 T SSUE K DNEY 30.L2 POSITIVE NT 42 cAsE 99 16557 TISSUE SPLEEN 24.94 POSITIVE NT 43 cAsE 99 ro55 / T SSUE KIDNEY No ct NEGATIVE NT 44 cAsE 101 L6677 TISSUE K DNEY 26.5 POSITIVE NT 45 CASE 101 L6677 TISSUE SPLEE N 26.24 POSITIVE NEG 46 cAsE 102 168s8 TISSUE BONE MARROW 24.6 POSITIVE NEG 47 cAsE 103 170t3 HOMOGENATE BONE MARROW 23.85 POSTTtVE NEG 48 CASE 104 170I4 HOMOGENATE BONE MARROW 23.2 POStTtVE NEG 49 cAsE 104 17015 HOMOGENATE BONE MARROW 26.LL POSITIVE NT )U CASE 104 170t6 HOMOGENATE BONE MARROW 304 POSITIVE NT 51 cAsE 105 t7LO7 HOMOGENATE BONE MARROW 30.57 POSTTtVE NT 52 CASE 42 0s097 HOMOGENATE BONE MARROW 28.74 POSITIVE NEG 53 cAsE 76 19765 -WB Ne17 BLOOD BLOOD-EDTA No ct(1) NEGATIVE NT 54 CASE 77 27152- WB Ne 55 BLOOD BLOOD-EDTA No ct NEGATIVE NT 55 CASE 78 30296- WB Ne 10 BLOOD BLOOD-EDTA No ct NEGATIVE NT 5b CASE 83 33256- WB Ne 22 BLOOD BLOOD-EDTA No ct NEGATIVE NT )t CASE 82 33245- WB Ne 3 BLOOD BLOOD-EDTA 26.7L POSITIVE NT 5ó cAsE 84 00347 WB Ne 22 BLOOD BLOOD-EDTA 36.53(r) 36.731 36.64 WEAK NT 59 cAsE 85 00969 BLOOD BLOOD-EDTA 20.4t11 POSTTtVE NT 60 CASE 86 03518 - WB Ne 8 BLOOD BLOOD-EDTA 37.95 38.8É; 39.64 WEAK NT 61 cAsE 87 04513 BLOOD BLOOD-EDTA 19.155 POStTtVE NT o¿ cAsE 88 05432 WB Ne 12 BLOOD BLOOD-EDTA No ct NEGATIVE NT 63 cAsE 92 08590 - WB Ne 1 BLOOD BLOOD-EDTA No ct NEGATIVE NT 69 cAsE 97 15887 WB Ne 3 BLOOD BLOOD-EDTA 26.57 POSITIVE NT 70 cAsE 100 16565 WB Ne 7 BLOOD BLOOD-EDTA 28.25 POSTTtVE NT 7t cAsE 101 LOOt I BLOOD BLOOD-EDTA 23.88 POSITIVE NT 72 cAsE 75 L9L2r/rs DNA BONE MARROW 26 POSITIVE NT 73 cAsE 79 3to7r DNA BONE MARROW 29.t7 POSITIVE NT 74 CASE 80 32842 DNA BONE MARROW 2L.L4 POSITIVE NT IJ cAsE 81 32981 DNA BONE MARROW 25.16 POStTtVE NT 76 cAsE 82 33245- WB Ns 3 DNA BLOOD-EDTA 26.2a POSITIVE NT 77 CASE 85 00969 DNA SPLEEN 20.43 POSITIVE NT 78 CASE 85 00969 DNA LYMPHNODE 20.9 POStTtVE NT 79 cAsE 85 00969 DNA KIDNEY 2r.84 POSITIVE NT 80 CASE 85 00969 DNA BLOOD-EDTA 19.1 POSITIVE NT 81 cAsE 87 04513 DNA BLOOD-EDTA 19.87 POSTTtVE NT 82 cAsE 89 05778 DNA BONE MARROW 27.76 POSTTTVE NT 83 cAsE 90 06985 DNA BONE MARROW 29.35 POSITIVE NT 84 CASE 91 0763r DNA BONE MARROW 29.7L POSTTTVE NT 85 cAsE 93 10375 DNA BONE MARROW 24.72 POSITIVE NT 86 cAsE 94 t2990 DNA BONE MARROW 25.53 POStTtVE NT 87 CASE 95 14055 DNA BONE MARROW 18.29 POSITIVE NT 88 cAsE 95 14056 DNA BONE MARROW 18.94 POStTtVE NT tr2 CASE 95 L5777 DNA BONE MARROW 27.2t POSTTTVE NT I15 cAsE 96 L5778 DNA BONE MARROW 22.57 POSTTTVE NT

Ctra. de Algete a El Casar, s/n Page 6 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 6202247 MINISTERIO DE ECONOMÍA Y Insliluto Nac'iu¡¡al de lnvmriEar irln COMPETITIVIDAD ñ r, 'lix.-nologÍa ,{¡¡raria y Alinrcnraria

SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh I ¡v¡ CENTRO DE INVESTIGACIÓII ti,ii,'i r-l 1-',.,-a-',.,.,....:.,,._.,... -t+'l EN SANIDADANIMAL * Assoys and activities marked with are not supported by ENAC acueditation.

rt4 CASE 96 r5779 DNA BONE MARROW 25.77 POSTTTVE NT 115 cAsE 96 1s780 DNA BONE MARROW 20.tt POSITIVE NT rlo cAsE 96 15780 DNA SPLEEN 20.51 POSITIVE NT TT7 CASE 98 16080 DNA BONE MARROW 20.76 POSITIVE NT 118 cAsE 99 16554 DNA BONE MARROW 31.4 POStTtVE NT 119 cAsE 99 rb555 DNA SPLEEN 19.46 POSITIVE NT 720 CASE 99 16555 DNA KIDNEY 24.38 POSITIVE NT I2L CASE 99 L6557 DNA KIDNEY 27.L4 POSITIVE NT L22 cAsE 99 16557 DNA SPLEEN 20.34 POSIT VE NT L23 CASE 1OO 16565 - WB Ne7 DNA BLOOD-EDTA 29.28 POSITIVE NT L24 CASE 101 L6677 DNA BLOOD-EDTA 2L.52 POSITIVE NT 1RR'|'' !z) cAsE 101 DNA KIDNEY 22.95 POSITIVE NT 126 CASE 101 16677 DNA SPLEEN 2l.3 POSITIVE NT r27 CASE 102 16858 DNA BONE MARROW 24.42 POStTtVE NT L28 CASE 103 L7OI3 DNA BONE MARROW 19.32 POSITIVE NT L29 CASE 104 17OT4 DNA BONE MARROW t9.97 POSITIVE NT 130 cAsE 104 L7OLs DNA BONE MARROW 2L.25 POSITIVE NT 131 cAsE 104 L7OL6 DNA BONE MARROW 28.9 POStTtVE NT 132 cAsE 105 t7ro7 DNA BONE MARROW 24.48 POStTtVE NT NT

(a) UPL-rcdl t¡me PCR ) Reol time PcR test described by Fernóndez-P¡nero et ol.. 2073 bosed on the IJniversal Probe Librcry (UpL). tpNT/ctsA./ppA/pcR/3l (b) Vl-HADt ) Virus ¡solotion ond hoemodsorpt¡on test on PBM cells os ¡s descilbed ¡n the OIE Monuot of diagnosis for ASF (Chopter 2.8.J.. OtE seventh ed¡tion 20121,

1.2.DOMEST|C PIG SAMPTES ) Positive ASFV result has been obtained by the UPL real-time PCR in the 43 domestic pig samples tested, conf¡rm¡ng the presencre of I\SFV inr all domest¡c p¡gs rece¡ved. After three passages in PAM, the ASFV has been isolated from 2 domestic pigs identified as 15553 (rDcrsA26)and 15555 (rDcrsA30)showing the classical haemadsorbing pattern of the ASFV circulating in Europe. The results obtained in virus detection are summarized in the table 3.

lD tube Sample Type Ct volue Result lf, OUTBREAK 4 L5337 TISSUE K DNEY 28.87 POStTtVE NEG 16 OUTBREAK 4 75337 TISSUE SPLEEN 27.53 POSITIVE NEG L7 OUTBREAK 4 15338 TISSUE K DNEY 28.18 POSITIVE NT 18 OUTBREAK 4 15338 T SSUE SPLEEN 22.36 POSITIVE NT 19 OUTBREAK 4 15339 TISSUE K DNEY 28.L4 POSITIVE NT ¿v OUTBREAK 4 15339 TISSUE SPLEEN 25.53 POSITIVE NT 27 OUTBREAK 4 15340 T SSUE K DNEY 25.4L POSITIVE NT 22 OUTBREAK 4 15340 TISSUE SPLEEN 24.L5 POSITIVE NT 23 OUTBREAK 4 15341 TISSUE KIDNEY 23.38 POSITIVE NT 24 OUTBREAK 4 15341 TISSUE SPLEEN 24.91 POSITIVE NT 25 OUTBREAK 4 15553 T SSUE K DNEY 28.53 POStTtVE NT 20 OUTBREAK 4 15553 TISSUE SPLEEN 23.53 POSITIVE POS 27 OUTBREAK 4 15554 TISSUE KIDNEY 23.79 POSITIVE NT 28 OUTBREAK 4 15554 TISSUE SPLEEN 22.O4 POSITIVE NT 29 OUTBREAK 4 15555 TISSUE KIDNEY 25.54 POStTtVE NT 30 OUTBREAK 4 L5555 TISSUE SPLEEN 2?.84 POSITIVE POS 2l OUTBREAK 4 15556 TISSUE K DNEY 21.8t POSTTTVE NT

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N INVESTIGACIÓN Y TECNOLOGh t E¡¡SAYOS CENTRO DE INVESTIGACIÓN i:,: ,ir 'lí_ 1\i. 9691LE1865 ; i;lli'r i-rr- l ...i EN SANIDAD ANIMAL * Assays ond activities marked with are not supported by ENAC accreditation.

32 OUTBREAK 4 15556 T SSUE SPLEEN 22.Or POfiITIVE NT 64 OUTBREAK 4 t5337 BLOOD BLOOD-EDTA 2L.74 PO$tTtVE NT OJ OUTBREAK 4 15338 BLOOD BLOOD-EDTA 23.51 POStTtVE NT 66 OUTBREAK 4 15339 BLOOD BLOOD-EDTA NT NT NT 67 OUTBREAK 4 15340 BLOOD BLOOD-EDTA NT NT NT 68 OUTBREAK 4 L534I BLOOD BLOOD-EDTA NT NT NT 89 OUTBREAK 4 15337 DNA BLOOD-EDTA 20.49 POSITIVE NT 90 OUTBREAK 4 15337 DNA KIDNEY 25.53 PO$tTtVE NT 91 OUTBREAK 4 15337 DNA SPLEEN 21.t2 POSITIVE NT J¿ OUTBREAK 4 1s338 DNA BLOOD-EDTA 20.36 POSITIVE NT 93 OUTBREAK 4 1s338 DNA KIDNEY 22 POStTtVE NT 94 OUTBREAK 4 1s338 DNA SPLEEN 20.26 POStTtVE NT 95 OUTBREAK 4 1s339 DNA BLOOD-EDTA 26.92 POSTTTVE NT 96 OUTBREAK 4 15339 DNA KIDNEY 28.96 POSITIVE NT 97 OUTBREAK 4 1s339 DNA SPLEEN 22.35 POSITIVE NT 98 OUTBREAK 4 15340 DNA BLOOD-EDTA 20.5 POSITIVE NT 99 OUTBREAK 4 15340 DNA K DNEY 22.A3 POStTtVE NT 100 OUTBREAK 4 1s340 DNA SPLEEN 18.9 POSITIVE NT 101 OUTBREAK 4 15341 DNA BLOOD-EDTA 23.35 POStTtVE NT ro2 OUTBREAK 4 15341 DNA K DNEY 2t.8 POSTTTVE NT 103 OUTBREAK 4 L5341 DNA SPLEEN 20.53 POSITIVE NT 104 OUTBREAK 4 15553 DNA KIDNEY 24.O4 POSITIVE NT 105 OUTBREAK 4 15553 DNA SPLEEN 20.88 POSITIVE NT 106 OUTBREAK 4 15554 DNA KIDNEY 22.74 POSTTTVE NT LO7 OUTBREAK 4 15554 DNA SPLEEN 20.33 POStTtVE NT 108 OUTBREAK 4 15555 DNA KIDNEY 25.36 POSITIVE NT 109 OUTBREAK 4 15555 DNA SPLEEN 20.72 POSITIVE NT 110 OUTBREAK 4 15556 DNA KIDNEY 22.36 POSITIVE NT LL7 OUTBREAK 4 15556 DNA SPLEEN 19.33 POSITIVE NT NT ) Somples not tested. o) UPL-rcol t¡me PCR ) Reol t¡me PCR test described by Fernóndez-Pinero et ol., 20L3 bosed on the lJniversol Probe L¡brory (UPL). IPNT/CISA,/PPAlPCR/3I b) V|-HAD* ) v¡tus ¡solot¡on ond hoemodsorpt¡on test on PBM cells as ¡s described ¡n the OtE Monuol of diognosis for ASF (Chopter 2,8.7. OIE seventh edít¡on 2012).

2. ASF ANTIBODY DETECTION: 2.1.WltD BOAR SAMPLES á By IPT*, the presence of ant¡bodies was detected in L4 out of the 44 samples tested, which corresponds to 12 out of the 40 l30%l wild boar receíved (table a).

lD tubr: Sample TvDe Titer Result L cAsE 75 L972t/15 TISSUE BONE MARROW NEG NEG NEGATIVE 2 cAsE 79 3to7r TISSUE BONE MARROW NEG NEG NEGATIVE 5 cAsE 80 32842 TISSUE BONE MARROW 1/20 WEAK WEAK CASE 81 3298L TISSUE BONE MARROW NEG NEG NEGATIVE 5 cAsE 85 00969 TISSUE SPLEEN NT NT NT 6 cAsE 85 00969 T SSUE LYMPHNODE NT NT NT 7 CASE 85 00969 TISSUE K DNEY NT NT NT 8 cAsE 89 05778 TISSUE BONE MARROW NEG NEG NEGATIVE 9 CASE 90 0698s TISSUE BONE MARROW NEG NEG NEGATIVE 10 cAsE 91 07631 T SSUE BONE MARROW NEG NEG NEGATIVE LL cAsE 93 10375 TISSUE BONE MARROW 7/LO WEAK WEAK

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SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh CENTRO DE INVESTIGACIÓN EN SANIDAD ANIMAL

Assays dnd activities marked with * dre not supported by ENAC occreditation.

HOMOGENATE

NT=NOT TESTED; WEAK= IPT TiÜe < (o) URL-IPT ) tndirect ¡mmunoperoxidose on ETGMS ¡nfected cells us¡ng the prctocol stondordized ond volidoted by the Eurcpeon Un¡on Reference Loborotory (EUR L).

2.2.DOMESTIC PIG SAMPLES By IPT*, the presence of antibodies was detected in 19 out of the 22 samples tested which ' to 8 out of the 9 (88.8%) domestic pig received (table 5).

Ctra. de Algete a El Casa¡, s/n Page 9 of 13 28130 Valdeolmos MADRID TEL: 91 620 23 00 FAX: 91 6202247 MINISTERIO DE ECONOMÍA Y lnsrilut<¡ Nacional tle lnvlsrigaciórr COMPETITIVIDAD 1,'l'c'cnolo¡¡ia Agraria y r\linrentaria

SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh CENTRO DE INVESTIGACIÓI\ EN SANIDAD ANIMAL Assay$t

¿J OUTBREAK 4 15 1 T SSUE KIDNEY r1320 POS POSITIVE 24 OUTBREAK 4 t1 TISSUE SPLEEN Llt60 POS POSITIVE z) OUTBREAK 4 15 TISSUE KIDNEY rl80 POS POSITIVE zo OUTBREAK 4 15 TISSUE SPLEEN Ll80 POS POSTTtVE 27 OUTBREAK 4 15 ;4 T SSUE KIDNEY r1640 POS POSITIVE 28 OUTBREAK 4 15 ;4 T SSUE SPLEEN ut28O POS POSITIVE ¿J OUTBREAK 4 15: iq TISSUE K DNEY Ll640 POS POSITIVE 5U OUTBREAK 4 15: iq TISSUE SPLEEN tl640 POS POSITIVE 5I OUTBREAK 4 15t )o TISSUE KIDNEY tl160 POS POSITIVE 52 OUTBREAK 4 15t )o TISSUE SPLEEN tlt60 POS POSITIVE 64 OUTBREAK 4 15 17 BLOOD BLOOD-EDTA r1320 POS POSTTTVE OUTBREAK 4 15 |8 BLOOD BLOOD.EDTA NEG t\Eu NEGATIVE 66 OUTBREAK 4 15 |9 BLOOD BLOOD-EDTA Llst2o POS POSITIVE OT OUTBREAK 4 I] IU BLOOD BLOOD-EDTA Ll32O POS POStTtVE 68 OUTBREAK 4 153 1 BLOOD BLOOD-EDTA LlL280 POS POSITIVE URL',PT ) tndhed immunoperoxidose technique on E70-Ms inÍected cells using the üotocol standod¡zed ond vol¡doted by the Europeon Union Reference Loborotory GURL).

3. ASFV MOIECUIAR CHARACTERIZATION* ) The expected =478 bg of the C-terminal p72-Eene has been obtained in all samples selected (table 6). The PCR amplicons were sequenced in order to place the ASFV Poland viruses in one of the defined p72 genotypes. The wild boar ASF Poland viruses clustered within p72 genotype ll and showed 100% nucleotide identity with all compared ASFV isolates from Eastern and Central Europe across the 478-bp C-terminal p72 gene.

Poll5/WB-CASE 42 Wild boar Gródek Lsl03/2015 05097lrs 52 Pol15/WB-CASE 75 W¡ld boar B¡alow¡e2a t3/o8/20ts T9L2I I Pol15/WB-CASE 79 Wild boar Narewka 27/ttl2oLs 3L07tlrs 2 Pol15/WB-CASE 80 Wild boar Narewka t6/L2t20Ls 328421Ls 5 Pol15/WB-CASE 81 Wild boar Biaiowieia 2Ut2/20t5 3298L/Ls 4 Pol16/WB-CASE 82 Wild boar Narew 2slr2/20Ls 33245-3175 t0 Pol16/WB-CASE 85 Wild boar Narewka 20/0r/2ot6 00969/t6 5v Pol16/WB-CASE 87 Wild boar Su praó1. 09/o3/20L6 o4f/16 OI Pol16/WB-CASE 89 Wild boar Narewka 22/03/20L6 os778h6 8 PoIX6/WB.CASE 90 Wild boar B¡atowie2a tt/04/20L6 0698s/16 9 PolI6/WB-CASE 91 Wild boar Biatow¡e2a 7s/o4/2OL6 0763L/t6 10 Poll6/WB-CASE 93 Wild boar Narewka r2t0st20t6 LO37slL6 11 Pol16/WB-CASE 94 Wild boar Narewka 03/06/20L6 L2990/L6 t2 Pol16/WB-CASE 95 Wild boar Biafow¡e¿a L4/06/20t6 L40ss/L6 13 Pol16/WB-CASE 97 Wild boar Narewka 2s/o6120L6 L5887-31L6 AO Pol16/WB-CASE 95 Wild boar CzvZe 26/06/2016 L5780/L6 37 Pol16/WB-CASE 98 Wild boar Biaiow¡e¿a 29/O6/20L6 16080/16 J6 Pol16/wB-CASE 100 Wild boar Hainówka oLloT/2016 16565-7 116 70 Pol16/WB-CASE 99 Wild boar Hainówka 04/07/2016 16555 40 Pol16/WB-CASE 101 Wild boar Narewka os/o7/20L6 t6677 45 Pol16/WB-CASE 102 Wild boar Hainówka 06/07/2016 168s8 46 Pol15/WB-CASE 103 Wild boar Hainówka 07to1t20r6 t70t3 47 Pol15/WB-CASE 104 Wild boar Hainówka o8/o7/2016 170r4/t6 48 Pol16/DP-OuTa (16) Domestic pis Hainówka 221O6/2OL6 Ls337 /L6 16 Pol16/DP-OUr4 (26) Domestic p¡g Hainówka 23tO6/20t6 15553/16 ¿o

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Nucleotide and amino acid sequence analysis of the CVR-PCR product placed the poland wild boar viruses within the variant Gll-CVRl with the presence of L0 copies of amino acid tetramer repeats that were 100% identical and unique to those of the ASFV circulating in the Eastern countries since 2007. Finaf ly, the sequence analysis of the intergenic region between the t73R and t329L genes (lGRnentszgi revealed the presence of the nucleotide tandem repeat (TRS) insertion ( ) which is representative of all the ASFV isolates circulating within the EU countries Estonia, Latvia, Poland and Lithuania since the entrance of the disease in January 2014. This intergenic region variant named Gll-lGRz¡n-pze¡2 is also present in the Ukraine2Ot2 and 2015 isofates, in the Belarus 2013 viruses and, since 2072in Russia Federation, mainly in the Russian Tulskaya region (Gallardo et al.,2Ot4; Goller et al.,2OI5l. coNctustoN*

t. The presence of ASF has been confirmed throughout antibody andlor ASFV genome detection in all wild boar received from the cases 42 and from cases 75 to 105 which were occurred in Poland from march 2015 (case a2l up to 9 July 2016 (case 105). 2. The presence of ASF has been confirmed in all domestic pig received from the fourth outbreak occurred on June 2016. 3. The p72 genotyping of the new Poland wild boar and domestic pig ASFV clustered the viruses within p72 genotype ll circulating in the Eastern European countries since the first introduction in Georgia in2OO7. 4. The CVR subtyping placed the ASF domestic pig and wild boar Poland viruses within the CVR variant 1 identical to those of the ASFV circulating in the Eastern countries since 2007. 5. The intergenic region (rGR r73R-r32sr¡ subtyping showed than the ASF Poland viruses responsible of the wild boar cases and domestic pig outbreaks were 100% homologous to the viruses responsible of the cases occurred in wild boar and in domestic pigs in Lithuania. Latvia. Estonia and Poland since the ASFV entrance into the EU in 2014 and ¡n¡t¡af ly discovered ¡n cefta¡n regions of the Russia Federation in 2Ot2.

Ctra de Algete a El Casar, s/n Page11of13 28130 Valdeolmos MADRID TEL: 91 6202300 FAX: 91 6202247 ,t ¡l MINISTERIO dr ¡l{ DE ECONOMfA Y ii =t )"' lnsriluto Nar:ional rll lnvrsri¡¡ar irin COMPETITIVIDAD ,l il v li'r:rrolo¡¡ia Agraria y ¡\linr¿'nraria iltl ¡ilr SUBDIRECCION GENERAL DE :l¡l INVESTIGACIÓN Y TECNOLOGh II CENTRO DE INVESTIGACIÓ¡, EN SANIDAD ANIMAL 'jii * Assays and octivities marked with are not supporteal by ENAC occreditation.

The table below summar¡zes the results obtained in each wild boar combining virus and antibody detection tests, as well as the genotyping results.

1 13/o8/20ts t9L2t/75 cAsE 75 Potozelce, Bialowie¿a, POStTIVE NEGATIVE Pol1s/WB-CASE 75 Gll/cvR- u lGRr?!n.rr2er-2 2 27/rrl20L5 3t07r CASE 79 Stoczek, Narewka. POSITIVE NEGATIVE Pol15/WB-CASE 79 Gil/CVR. 1/ tGRr75R.re2eL-2 3 L6/12/20Ls 32842 cAsE 80 Olchowka, Narewka, POSITIVE WEAK2) Poll5/WB-CASE 80 Gll/cvR- t/ lGRPrR.re2er-2 4 2L/r2/20ts 32981 cAsE 81 B¡alowieza, Bialowie¿a. POSTTtVE NEGATIVE Pol15/WB-CASE 81 Gll/cvR- 1/ lGRrzs¡.r¡rr-2

5 20/0r/20L6 00969 cAsE 85 Gruszki, Narewka POSITIVE NEGATIVE Poll16/WB-CASE 85 Gll/cvR- 1/ tGRr73R.r32er-2

8 22/0312016 05778 CASE 89 Zamosze/Gluszec. Narewka POSITIVE NEGATIVE Poll16/WB-CASE 89 Gll/cvR- 1/ lGRr73n.r3zer-2 9 Ltl04/2016 0698s CASE 90 Budy,y Bialowie¿a POSITIVE NEGATIVE Poll6/wB-CASE 90 Gll/cvR- 1/ IGRER-'3rer-2 10 rs/04/2076 07637 CASE 91 Budy,y B¡¡low¡e¿a. POSTTtVE NEGATIVE Pol16/WB-CASE 91 cll/cvR- 1/ lGRrTsR-r.Er-2

11 r2los12076 10375 cAsE 93 Janowo, Na¡ewka. POSITIVE WEAK Pol16/WB-CASE 93 Gll/cvR- 1/ lGRrT3R-¡32er-2 t2 03/06/2076 L2990 cAsE 94 Krynica, Narewka POSITIVE NEGATIVE Pol16/WB-CASE 94 Gll/cvR- 1/ lGRrr3R.r32etr2 L3 t4/o6/2016 14055 cAsE 95 Teremiski, Bialowie¿a. POStTtVE NEGATIVE Pol16/WB-CASE 95 Glt/cvR- 1/ lGRr73R.r32eL-2 L4 L4/06/2016 14056 cAsE 95 Teremiski, Bialowieia. POSITIVE NEGATIVE 55 26106/2016 L5777 CASE 96 Osowka, Czyie POSITIVE NEGATIVE 35 26t06t20t6 t5779 CASE 96 Osowka, Czyie. POSITIVE NEGATIVE 50 26/06/20L6 15780 cAsE 96 Osowka, Czyie POSITIVE NEGATIVE 37 261O6/2Ot6 15780 CASE 96 Osowka, czy¿e POSITIVE NEGATIVE PoI16/WB-CASE 96 Gil/CVR- 1/ tGRrER.r34r-2 38 2910612016 16080 cAsE 98 Podcerkwy, Eialowieia POS TIVE NEGATIVE Pol16/WB-CASE 98 Gil/CVR- l/ lGRrBR.'*r-2 39 04t07/20L6 16554 cAsE 99 Sacharewo, Hajnówka POStTtVE NEGATIVE 40 04107/20L6 16555 CASE 99 Sacharewo, Hajnówka POStTtVE NEGATIVE Pol16/WB-CASE 99 Gil/CVR- 1/ lGRr73R.riler2 42 04/07/20L6 ro)) / cAsE 99 Sacharewo, Hajnówka POSITIVE NEGATIVE 44 os/o7/2016 t6677 cAsE 101 Tarnopol, Narewka, POStTtVE POSITIVE Pol16/WB-CASE 101 Gll/CVR- U lGRrr¡¡.r¡zqr-2 46 06107/2076 16858 CASE 102 Mochnate, Hajnówka POS TIVE NEGATIVE Pol16/WB-CASE 102 Gll/cvR- 1/ IGRD¡n-rszsr.2

47 07/o7/2016 17013 CASE 103 Lipiny, Hajnówka, POSITIVE NEGATIVE Pol16/WB-CASE 103 Gll/cvR- 1/ tGR¡73¡.¡32e¡-2

48 08107/20L6 170t4 CASE 104 Sacha¡ewo, Hajnówka. POStTtVE NEGATIVE Pol16/WB-CASE 104 Gll/cvR. 1/ tGRr73R{52er-2 49 08/07/2016 L7OLs cAsE 104 5acharewo, Hajnówka. POSITIVE NEGATIVE qn 08107/2OL6 L7OL6 cAsE 104 Sacharewo, HEjnówkE. POSITIVE NEGATIVE 51 09lo7/20L6 L7t07 cAsE 105 Topilo, Hajnówka POSITIVE NEGATIVE

)z L5/03120t5 0s097 CASE 42 B¡elew¡cze, Gródek. POStTtVE NEGATIVE PolIS/WB-CASE 42 Gll/cvR- 1/ tGRr738.r32er-2 )5 2UO8/20r5 19765-WB Ne 17 cAsE 76 Lap¡cze, Krynk¡. NEGATIVE POSITIVE 54 2s/tol2oLs 27L52-WB Ne 55 cAsE77 Straszewo, G.ódek, NEGATIVE POSITIVE )f 23/7L/2Or5 30296-WB Ne 10 CASE 78 Pohoranv, Sidra NEGATIVE POSITIVE qA 23t72t20t5 33256-WB Ne 22 CASE 83 Mostowlany, Gródek. NEGATIVE POSITIVE 57 2s/L2/20ts 33245-WB Ne 3 CASE 82 Kaczaly, Narew POSITIVE NEGATIVE Poll6/WB-CASE 82 Gll/cvR- 1/ lGR,Bi.,3!r-2 58 09/ou20r6 00347-WB Ne 22 cAsE 84 Waski, Narew. WEAKU) NEGATIVE 60 24/O2/2016 03518-WB Ne 8 cAsE 86 Bernacki lMost, Narewka WEAK POStTtVE OI 09/03/2076 04513 cAsE 87 SupraÉ1. POStTtVE NEGATIVE PoI16/WB-CASE 87 G[/CVR- 1/ tcR,,,".,.,",-2 62 t7/03/2016 05432-WB Ne 12 cAsE 88 Terem¡sk¡, Bialowie¿a, NEGATIVE POSITIVE o5 20/o4l2016 08590-WB Ne 1 CASE 92 Kopce, M¡chalowo. NEGATIVE POSITIVE 69 25/06t20t6 15887-WB Ne 3 cAsE 97 Bernacki Most. Narewka, POSITIVE POSITIVE Pol16/WB-CASE 97 Gil/CVR- 1/ rGR,?¡i.Eñ-2 70 oLloT/2016 16565-WB Ne 7 CASE 1OO Nieznany Bor, Hajnówka. POSITIVE POSTTTVE Pol16/WB-CASE 100 Gll/cvR- 1/ lGRrz¡n-r¡ugr-2 (1) WEAK = PCR-UPL weok (Ct=35-40) (2) WEAK = IPT weok titer < 1/80

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SUBDIRECCIÓN GENERAL DE INVESTIGACIÓN Y TECNOLOGh CENTRO DE INVESTIGACIÓT\ EN SANIDAD ANIMAL * Assays dnd activities marked with ore not supported by ENAC occreditation.

The table below summar¡zes the results obtained in each domestic pis comb¡ning virus and antibody detection tests, as well as the genotyping results.

16 22/06/2016 15337 OUTBREAK 4 Bielszczyzna, Hajnówka. POStTTVE POSITIVE Pol16/DP-OUT4 (161 GIUCVR- V lGRrz¡Hs¡r2 22/06/2OL6 t7 15338 OUTBREAK 4 Bielszczyzna, Hajnówka. POSITIVE NEGATIVE GlI/CVR- U lGRrr¡n.rszer-2 19 22/06/2076 15339 OUTBREAK 4 B¡elszczyzna, Hainówka. POSTTtVE POSITIVE Gll/CVR- U lGRr¡¡u¡¿r.-2 22/06/20t6 15340 OUTBREAK 4 Bielszczyzna, Hajnówka. POSITIVE POSITIVE Gll/CVR- 1/ lGRrzrnr:zsr-2 23 22/06/2Ot6 r534t OUTBREAK 4 Bielszczyzna, Hajnówka. POStTtVE POStTtVE GlI/CVR- 1/ lGRrz¡n-rszgr-2 z) 23/06t20r6 15553 OUTBREAK 4 Bielszczvzna, Hainówka POSITIVE POStTtVE Pol16/DP-oUT4 126) GlI/CVR- 1/ lGRrr¡n.rs:er-2 2t 23106120L6 15554 OUTBREAK 4 Bielszczyzna, Hainówka. POStTtVE POSITIVE Gll/CVR- 1/ tGRrzrn.r:zsr-2

REFERENCES

1. Achenbach JE, Gallardo C, Nieto-Pelegrín E, Rivera-Arroyo B, Degefa-Negi T, Arias M, Jenberie S, Mulisa DD, G¡zaw D, Gelaye E, Ch¡bssa TR, Belaye A, Loitsch,4 Forsa M, Yami N,1, Diallo A, Soler A, Lamien CE, Sánchez-Vizcaíno Jlvl. ldentificat¡on of a New Genotype of Afr¡can Swine Fever Virus in Domestic Pits from Elhiopia, Trunsbound Emery Dis. 20\6 May 22. doii rO.717lltbed.!25!7. 2 Boshoff,Cl, Bastos,A D., Gerber, LJ &Vosloo, W, (2007), Geneticcharacterisation ofAf¡¡can swinefevervi.usesfrom outbrcaks ¡n southernAfr¡ca (7g:3-7gggl.Vet, Microbiol. 72!,45-55. 3. Carm¡na Gallardo, lovita Fernández-P¡nero, Virg¡n¡a Pelayo, lsmail Gazaev, lwona Markowska-Dan¡el, Gediminas Pridotkas, Raquel N¡eto, Paloma Fernández-pacheco, Svetlana Bokhan, OlegNevolko, ZhannaDrozhzhe, Covadonga Pérez, Alejandro Soler, Den¡s Kolvasov, and Marisa Arias (2014). Genet¡c Var¡ation among African Sw¡ne Fever Genotype ll V¡ruses, Eastern and CeNfral Europe. Emee, lnÍect. D¡s Volume 20, Number 9-September2O14. 4 Fernández-P¡nero J, Gallardo c, Elizalde M, Robles A, Gómez c, B¡shop R, Heath L, couacy-Hymann E, Fasina Fo, Pelayo V, sole¡ A, Arias M, lvlolecular diagnosis of Afr¡can swine fever by a new real-time PCR using universal probe l¡brary (2013). fronsbound Emery Dk 60 (2013) 48-58. doi: 10.1111, 1865-18a2,20!2,Ot3l:.x Epub 20!2 Ma¡ 7.

5. Ga llardo C, Anchue lo R, Pelayo V, Poudevitne F, Leon T, Nzoussi J, B¡shop R, Pérez C, Soler A, N ieto R, Martín H, Arias M. (2011). African swine fever virus p72 genotype lX in domestic pigs, Con$o, EmerE lnfect. D¡s, AuEi 7718)17556-a 6. Goller KV, lúalogolovkin AS, Katorkin S, Kolbasov D, Titov l, Hdper D, et al Tandem repeat ¡nsertion in Afr¡can swine fever virus, Russia, 2012 fletterl. Emerg lnfect Dis. 2015 Ap¡ (date citedl. httpJ ldx.doi.o3l rO 3207/ eid2704.747792 7. OlE. N¡ay 2012, postin8 date. Chapter 2 I 1 African swine fever ln Monuol of d¡ognost¡c tests ond vocc¡nes fot teftestildl on¡mdls2o12. World Organization for Animal Health,Par¡s,France hrtp:llwww.oie.¡NT/f¡leadmin/Home/eng/Health_standards/tahm/208.01_ASF.pdf

ln Valdeolmos, Madrid (Spain), September 23'd,2OL6.

Dr. Carmina Gallardo Frontaura Dr. Marisa Arias Neira Researcher, Laboratory Coordinator Technical Director EU Reference Laboratory for ASF Reference Laboratory for ASF INIA-CISA INIA-CISA

D¡rector INIA-CISA

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