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Klassische und real-time RT-PCR-Teste - molekulare Diagnostik aviärer Influenzavirusinfektionen -

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Lfd-Nr Val-Nr Isolat HA-type NA-type1 H1-1 A/Fort Mammoth/1/47 H1 N12 H2-1 A/duck/Potsdam/177/83 H2 N23 H2-2 A/guinea fowl/Germany/DZ3/85 H2 N24 H2-3 A/Mallard/Germ/Wv1226-30K/03 H25 H3-1 A/duck/Ukraine/1/63 H3 N86 H3-2 A/Mallard/Germ/Wv1303-04K/03 H3 N87 H4-1 A/duck/Czechoslowakia/56 H4 N68 H4-2 A/Mallard/Germ/Wv1732-34K/03 H49 H5-1 A/tern/S.Africa/61 H5 N3

10 H5-2 A/chicken/Scotland/59 H5 N111 H5-3 A/duck/Potsdam/2216/84 H5 N612 H5-4 A/duck/Potsdam/2243/84 H5 N613 H5-5 A/duck/Potsdam/617/85 H5 N214 H5-6 A/duck/Potsdam/619/85 H5 N215 H5-7 A/duck/Potsdam/1701/85 H5 N216 H5-8 A/duck/Potsdam/1402/86 H5 N217 H5-9 A/duck/Potsdam/1403/86 H5 N218 H5-10 A/chicken/Italy/8/98 H5 N219 H5-11 A/chicken/Italy/22/98 H5 N920 H5-12 A/Hongkong/156/97 H5 N121 H5-13 A/chicken/Indonesia/R132-134/03 H5 N122 H5-14 A/mallard/Germany/Wv1310-13K/03 H5 N223 H5-15 A/mallard/Germany/Wv1349-51K/03 H5 N2?N3?N9?24 H5-16 A/teal/Germany/Wv1310-13K/03 H5 N225 H5-17 A/mallard/Germany/Wv474-77K/04 H5 N?26 H5-18 A/chicken/Vietnam/P41/05 H5 N127 H5-19 A/chicken/Vietnam/P78/05 H5 N128 H5-20 A/chicken/Vietnam/P22/05 H5 N129 H5-21 A/duck/Vietnam/TG36-H2/05 H5 N130 H5-22 A/duck/Vietnam/AG40-O2/05 H5 N131 H5-23 A/duck/Vietnam/TG24-O1/05 H5 N132 H5-24 A/chicken/GXLA/1204/05 H5 N133 H5-25 A/chicken/Indonesia/R132/1/04 H5 N134 H5-26 A/chicken/Indonesia/R132/2/04 H5 N135 H5-27 A/chicken/Indonesia/R132/3/04 H5 N136 H5-28 A/chicken/Indonesia/R60/05 H5 N137 H6-1 A/turkey/Mass/3740/65 H6 N238 H6-2 A/turkey/Grub/R41/98 H6 N539 H6-3 A/turkey/Kartzfehn/R26/99 H6 N2

Lfd-Nr Val-Nr Isolat HA-type NA-type40 H6-4 A/turkey/Heidemark/R30/99 H6 N141 H6-5 A/turkey/Poeppel/R04/02 H6 N242 H7-1 A/chicken/Brescia/1902 H7 N143 H7-2 A/FPV/dutch/27 H7 N144 H7-3 A/FPV/Rostock/34 H7 N145 H7-4 A/FPV/Rostock/45/36 H7 N146 H7-5 A/chicken/Italy/444/99 H7 N147 H7-6 A/chicken/Italy/445/99 H7 N148 H7-7 A/chicken/Germany/R28/03 H7 N749 H7-8 A/duck/Alberta/48/76 H7 N350 H7-9 A/duck/Potsdam/13/80 H7 N751 H7-10 A/duck/Potsdam/15/80 H7 N752 H7-11 A/duck/Potsdam/62/81 H7 N753 H7-12 A/duck/Potsdam/64/81 H7 N3?N7?54 H7-13 A/turkey/Ireland/PV8/98 H7 N755 H7-14 A/turkey/Italy/472/99 H7 N156 H7-15 A/chicken/Italy/473/99 H7 N157 H7-16 A/turkey/Germany/R11/01 H7 N758 H7-17 A/turkey/Italy/2043/03 H7 N359 H7-18 A/mallard/Germany/Wv1776-80K/03 H7 N3?N7?60 H7-19 A/mallard/Germany/Wv38-41K/04 H7 N161 H7-20 A/equine/Prague/1/56 H7 N762 H8-1 A/turkey/Ontario/6118/68 H8 N463 H9-1 A/turkey/Wisconsin/66 H9 N264 H9-2 A/duck/Germany/113/95 H9 N265 H9-3 A/turkey/Germany/169/95 H9 N266 H9-4 A/chicken/Germany/45/98 H9 N267 H9-5 A/Pakistan/519 H9 N268 H10-1 A/quail/Italy/1117/65 H10 N869 H10-2 A/guinea fowl/Hungary/1/69 H10 N870 H10-3 A/chicken/Germany/N/49 H10 N771 H10-4 A/Moorhen/Germ/Wv1703-04/03 H10 N472 H10-5 A/Mallard/Germ/Wv1781-82K/03 H10 N773 H11-1 A/duck/England/56 H11 N674 H11-2 A/Mallard/Germ/Wv1499-1503K/03 H11 N975 H12-1 A/duck/Alberta/60/76 H12 N576 H13-1 A/gull/Maryland/704/77 H13 N677 H13-2 A/pilot whale/Maine/328/84 H13 N278 H13-3 A/Gull/Germ/Wv1136-40KR/03 H1379 H16-1 A/BHG/Sweden/5/99 H16 N3

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Lfd-Nr Val-Nr Isolat HA-type NA-type Avian H5-PCR nach H5-new-PCRSpackman et al. (2002)

TC dRlast TC dRlast9 H5-1 A/tern/S.Africa/61 H5 N3 19,25 10300 22,07 8900

10 H5-2 A/chicken/Scotland/59 H5 N1 23,38 10900 29,11 780011 H5-3 A/duck/Potsdam/2216/84 H5 N6 21,3 6500 22,64 770012 H5-4 A/duck/Potsdam/2243/84 H5 N6 22,33 6400 24 750014 H5-6 A/duck/Potsdam/619/85 H5 N2 22,75 7000 24,13 670015 H5-7 A/duck/Potsdam/1701/85 H5 N2 25,68 6200 27,48 710016 H5-8 A/duck/Potsdam/1402/86 H5 N2 28,04 9900 25,04 880017 H5-9 A/duck/Potsdam/1403/86 H5 N2 30,24 9200 27,02 900018 H5-10 A/chicken/Italy/8/98 H5 N2 19,85 16400 >42 -19 H5-11 A/chicken/Italy/22/98 H5 N9 30,85 6000 24,67 930020 H5-12 A/Hongkong/156/97 H5 N1 18,25 16200 20,74 1390021 H5-13 A/chicken/Indonesia/R132-134/03 H5 N1 25,04 16900 20,57 1530022 H5-14 A/mallard/Germany/Wv1310-13K/03 H5 N2 26,1 17900 >42 -23 H5-15 A/mallard/Germany/Wv1349-51K/03 H5 N2?N3?N9? 23,83 15100 25,35 850024 H5-16 A/teal/Germany/Wv1310-13K/03 H5 N2 22,02 16500 >42 -25 H5-17 A/mallard/Germany/Wv474-77K/04 H5 N? 24,07 21200 28,05 960026 H5-18 A/chicken/Vietnam/P41/05 H5 N1 19,51 8200 21,24 1090027 H5-19 A/chicken/Vietnam/P78/05 H5 N1 19,2 9300 20,25 1450028 H5-20 A/chicken/Vietnam/P22/05 H5 N1 21,31 5800 22,18 1220029 H5-21 A/duck/Vietnam/TG36-H2/05 H5 N1 20,46 7800 22,23 990030 H5-22 A/duck/Vietnam/AG40-O2/05 H5 N1 20,35 7600 21,32 1240031 H5-23 A/duck/Vietnam/TG24-O1/05 H5 N1 20,29 7800 22,21 1360032 H5-24 A/chicken/GXLA/1204/05 H5 N1 21,24 17500 24,3 1240033 H5-25 A/chicken/Indonesia/R132/1/04 H5 N1 33,29 16600 23,49 1380034 H5-26 A/chicken/Indonesia/R132/2/04 H5 N1 30,09 16600 21,48 1370035 H5-27 A/chicken/Indonesia/R132/3/04 H5 N1 35,76 14100 23,71 12900

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Well Name dR Last Ct (dR) Final Call (dR)H1-1 255,091 No Ct -H6-4 12856,97 23,1 +H5-2 12879,086 24,35 +H5-12 12308,949 19,82 +H5-13 13699,598 18,67 +H5-18 6840,195 21,77 +H5-19 7025,600 22,17 +H5-20 7197,273 22,4 +H5-21 5927,357 23,22 +H5-22 5716,088 23,4 +H5-23 5984,793 23,04 +H5-24 12880,836 22,49 +H5-25 12936,201 23,34 +H5-26 13029,336 19,07 +H5-27 12905,963 21,45 +H7-1 -14,708 No Ct -H7-2 25,393 No Ct -H7-3 3771,734 31,32 +H7-4 4427,586 29,23 +H7-5 14026,579 22,01 +H7-6 14380,877 24,57 +H7-14 14870,935 19,13 +H7-15 14088,938 20,7 +H7-19 13793,344 20,31 +NTC 22,686 No Ct -

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E. Starick, RDT2005 / 2: AIV, molekulare Diagnostik

AIV: Charakterisierung mittels RT-PCRund Sequenzierung

Signalpeptid Transmembranregion

HA0-Spaltstelle

HA1 HA2NH2-- --COOH

S S


AIV, Charakterisierung: Subtypspezifische RT-PCR-Teste

H1H2H3H4H5H6H7H8H9H10H11...H15

HA1 HA2S S

Lee, M.-S. Et al., J Virol Meth 97 (2001), 13-22

hohe Spezifität, gute Sensitivität

>130 Isolatevon Geflügel, Wild-und Ziervögelnergänzt HAH (Monitoring-Programme!)

ermöglicht Sequenzierung / ersteCharakterisierung (Verwandschaft)

HA1 HA2

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AIV, Charakterisierung: RT-PCR-Teste für H5 und H7

Signalpeptid Transmembranregion

HA0-Spaltstelle

HA1 HA2NH2-- --COOH

S S

H5-LeeH5-JNH5HA1 (+S)H5HA2

H7-JNH7HA-1 (+S)H7HA-2


AIV, Charakterisierung: RT-PCR-Teste für H5

Herkunft Isolat H5-Ja/Nein H5-Lee H5HA-1 H5HA-2 Material

Indonesien R60/05 + + + + AF Vietnam R75/05 + + + + R76/05 + + + + R76/05 + + + + R77/05 + + + + R78/05 + + + + R79/05 + + + + R80/05 + + + + China R81/05 + + + + Indonesien R133/03 + (+) + neg Organe R134/03 + (+) + + 1-7 neg 3/7 + 1/1 + nd Tupfer WV Länder R121/2/05 + + neg nd Tupfer + + + + AF

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AIV, Charakterisierung: Sequenzierung H5-Isolate

OIE, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 2.7.12 :

LPAIV Nicht-H5,-H7; gering- pathogen für Hühner

LPNAI H5, H7; IVPI<1.2 und HA0-Spaltstellewie für gering-pathogene Viren

HPNAI H5, H7 u.a.; IVPI>1.2 oder HA0-Spaltstelletypisch für HPAIV



A/ch/Italy/08/98 V P Q R R R K K R * G L F A/ch/Italy/22/98 V P Q K E T . . R * G L FA/dk/Potsd/619/85 V P Q R E T . . R * G L F

WV 1349-51/03 V P Q R E T . . R * G L FWV R121/2/05 V P Q R E T . . R * G L F

A/ch/Vietnam/P78/05 Q R E R R . R K R * G L FA/ch/Indonesia/R60/05 Q R E R R R K K R * G L F

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AIV, Charakterisierung: Sequenzierung H5-IsolatePhylogenetic tree of avian influenza virus isolates from Indonesia(raw data from GCG, Wisconsin Package Version 10.2)

Isolates from IndonesiaA/chicken/Indonesia/R134/03 („H5INDO03“)A/chicken/Indonesia/R60/05 („H5INDO05“)

Sequences from GenBankAY651321 A/ch/Indonesia/BL/2003AY651325 A/ch/Indonesia/5/2004AY651332 A/goose/Thailand/79/2004AY555150 A/Thailand/1(KAN-1)/2004DQ076201 A/ch/Thailand/73/2004AY834279 A/tiger/Thailand/SPB-1AY575876 A/ch/HongKong/61.9/02AY770079 A/ch/Hubai/489/2004AY609312 A/ch/Guangdong/174/04

A/ch/Indonesia/R134/03 and A/ch/Indonesia/R69/05 show a homology of 98.58% on nucleotide level (1482 bp). The homology between the Indonesian sequences from GenBankand A/ch/Indonesia/R60/05 amounts 98.28 and 98.56%, respectively.Compared to the isolates from China, the homology is about 97.5%,and with the sequences from Thailand it is about 96.7%.No differences are seen with regard to HA cleavage site, potential glycosilation sites and receptor binding sites between the own Indo-nesian sequences and those from GenBank.



AIV, H5: Phylogenetischer Baum ausgewähltereuropäischer, asiatischer und amerikanischer Isolate(Rohdaten; GCG, Wisconsin Package Version 10.2)

AF082043 A/gull/Pennsylvenia/4175/83 H5N1, lpAF082040 A/duck/Minnesota/1525/81 H5N1, lp

X07826 A/chicken/Scotland/59 H5N1, hpAF082041 A/duck/Potsdam/2216-4/84 H5N2, lpAF082042 A/duck/Potsdam/1402-6/86 H5N2, lpS68489 A/turkey/England/50-92/91 H5N1, hp AJ305306 A/chicken/Italy/8/98 H5N2, hp

AY651332 A/goose/Thailand/79/2004 H5N1, hpDQ076201 A/chicken/Thailand/73/2004 H5N1, hpAY651321 A/chicken/Indonesia/BL72003 H5N1, hpAY651325 A/chicken/Indonesia/5/2004 H5N1, hpAY609312 A/chicken/Guangdong/174/04 H5N1, hpDQ212792 A/goose/Novosibirsk/4/2005 H5N1, hpAY77079 A/chicken/Hubai/489/2004 H5N1, hp

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AIV: Charakterisierung mittels RT-PCRund Sequenzierung

HA1 HA2

H5- und H7-PCR wenn H5- oder H7-positiv:Geflügelpestverdacht(bei Sekundärausbrüchen Bestätigung GP)

direkte Sequenzierung LPAIV oder HPAIVggf. Bestätigung Geflügelpest

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