Post on 15-Jan-2016
description
Aspectos virológicos y epidemiológicos de los nuevos virus A(H1N1)v en España
INMACULADA CASAS
Laboratorio de Gripe y Virus RespiratoriosÁrea de Virología
CNM, ISCIII
- Múltiples hospedadores, Aves acuáticas son los reservorios naturales-Transmisión desde aves a mamíferos- Desarrollo de pandemias humanas por transmisión directa o indirecta desde aves- Cerdo es susceptible de infección por virus aviares y porcinos- Han existido reagrupamientos genéticos en cerdo- Se conocía al cerdo como “vasija mezcladora” de virus
Ecología de los virus de la Gripe A
A/NewCaledonia/20/99
A/Brisbane/59/20077 AA mutados
1. DERIVA GENÉTICA “drift”
necesidad de actualización anual de las vacunas antigripales
Mecanismos de adaptación de los virus
H1N1 estacional2008-2009
2. REAGRUPAMIENTO DE GENES DE VIRUS DIFERENTESCambio antigenico mayor “shift”
GRIPE A: POTENCIAL PANDÉMICO
A/H5N1
Nuevo virus
Mecanismos de adaptación de los virus
CAMBIO ANTIGÉNICO MAYOR “GENETIC SHIFT” o REAGRUPAMIENTO
'mixing vessels theory’
PANDEMIAS HUMANAS POR GRIPE A
1918“Gripe Española”H1N1, 20-40 millon
1957“Gripe Asiática”H2N2, 1-2 millon
1968“Gripe HongKong”H3N2, 700.000
1977“Gripe Rusa”H1N1, No pandémica
2009-VIRUS EPIDÉMICOS
Evolución antigénica de los virus8 cambios del componente vacunal H1
Classical sw H1N1
European sw H1N1
Maximum likelihood tree of concatenated genome sequences (54 whole genomes) of European and classical swine H1N1 IAVs.
•genetically and antigenically stable viral lineage •emerged by transfer of the human 1918 pandemic virus to swine •spread to swine in America and other parts of the world, including Europe (1976).
•novel lineage of avian-like H1N1 swine IAV •emerged in Europe in 1979 that essentially replaced classical swine IAV•is enzootic in swine-producing regions of Western Europe, where it cocirculates with swine IAVs of the H3N2 and H1N2 subtypes
H1 N1M
PB2, PB1, PANS, NP
Origin of A(H1N1)v
Contemporary human From 1918 A(H1N1)
1998
Occasionally isolated from humansLimited human-to-human trnsmission
July-August 2008?Where?
Host and lineage origins for the gene segments of the 2009 A(H1N1) virus
8 Centros Colaboradores de la OMS
CDCAtlanta
MRCLondres
CSLMelbourne
NIIDTokio
110 Centros Nacionales de Gripe: Laboratorios de Virología
MADRID, VALLADOLID, BARCELONA
• Laboratorios de Virología de las CCAAs• Epidemiología
Médicos centinelas: recogida de muestras clínicas
A1
St. Jude Children'sMemphis
Institut Pasteur
Paris
2000-20012008-2009
CIRCUITO DE INFORMACIÓN ANTE DECLARACIÓN DE UNA ALERTA POR GRIPE AVIAR EN HUMANOS
Red de Vigilancia de Gripe en las CCAAs• Epidemiología• Laboratorios de Virología
“CASO SOSPECHOSO HUMANO”según los criterios de OMS
CNMCNE
ALERTA
MSPS
ISCIIILaboratorio de Referencia OMSLaboratorio de Virología
OMS y ECDC
Procesamiento de muestras clínicas en el laboratorio de bioseguridad de nivel 3 (CNM, ISCIII)
Recepción y registro de muestras
Procesamiento e inactivación de las muestras
Salida de las muestras del laboratorio P3
y
reparto a los Servicios y Laboratorios del CNM
Diagnóstico• Específico de Gripe aviar-Gripe estacional
• Diferencial con el resto de patógenos
RT-nested PCR genérica para detección de gripe A+B+CGen NP
Influenza ABC NP
New
SW clas
pacopozo@isciii.es
A particular emphasis was put on the suitability of
this method for the detection of both the Eurasian
and the American lineages of H5 viruses.
Thirteen different reference strains of subtypes
H5N1, H5N2, H5N3, H5N8 and H5N9 were used for
the evaluation of the PCR method.
Sensitivity of the assay was estimated by testing
serial tenfold dilutions of H1, H3 and H5 specific
plasmids. Detection levels were 10 copies.
Phylogenetic analysis of the second amplification
product nucleotide sequences of H5 viruses (Kimura
2-parameter ) would fit a particular strain
into one of the two previously described lineages. MEGA v3.1
Implementation of a Sensitive Diagnostic and Surveillance System to Detect Emerging Influenza Strains
Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos III, 28220 Majadahonda, Madrid
Thirteenth International ConferenceNegative Strand Viruses 2006
Salamanca, Spain, June 17th - 22nd, 2006
Immediate knowledge about human infection with the subtype H5 and then with the
currently circulating influenza A subtypes H1 and H3 would be desirable for timely
epidemiological investigations and, if possible, infection control.
Nevertheless, next pandemic might be caused by a different subtype of influenza virus. In
fact, three other avian influenza viruses (H9N2, H7N7 and H7N3) have caused illness in
humans.
As a result, diagnostic
laboratories receiving requests to test specimens from patients with an influenza-like illness
are being encouraged to the implementation of rapid and sensitive procedures not only for
typing influenza A virus but also for subtyping at least H1, H3 and H5 viruses directly from
clinical specimens.
Strategies for containing
.
Since their reemergence in late 2003, the establishment of the highly pathogenic avian
influenza (HPAI) A subtype H5N1 as an endemic virus in poultry in several countries of Asia
and the recent extensive geographical spread to Africa, Europe and the Middle East, in
addition with its repeated interspecies transmission to humans are giving this virus increased
opportunities to improve their transmissibility in humans, and thus develop into a pandemic
strain,
an emerging influenza pandemic or at
least attempting to slow down its spread worldwide will depend on several factors but the
prompt laboratory identification of the first cases would be unanswerably the starting point
being evident that H5N1 virus poses a continuing global human public health risk.
H5N9 -A/ma llard/Sweden/8 0/0 2
H5N6-A/mallard/Sweden/40/02
H5N2-A/duck/Mongolia/54/01
H5N1-A/Hanoi/30 408/05
H5N1-A/chicken/HK/728 /97
H5N9 -A/ch icken/Italy/9 097/97
H5N8-A/turkey/Ireland/137 8/83
H5N3-A/duck/Miyagi/54/76
H5N6-A/duck/Potsd am/2216-4/84
H5N3 -A/tern/South Afri ca/61
H5N1-A/chicken/Scotland/5 9
H5N2-A/ck/Quereta ro/7 653-20/95
H5N7-A/sh orebird/Delawa/75/0 4
H5N8-A/duck/NY/191255-59/02
H5
H2N3 -A/ma llard/Alb erta/226/98
H2N5-A/mallard/Alberta/ 202/96
H2N9 -A/gull/New Jersey/75/ 85
H2N1-A/ruddy/Delaware/81/93
H2N2 -A/pintail/Praimor/625/76
H2N2 -A/Berli n/3/64
H2N2-A/Japan/305/57
H2
H1N5-A/pintail duck/Alberta/6 3
H1N4-A/teal/Alberta/ 141 /92
H1N6 -A/ma llard duck/Alberta/4 2
H1N1 -A/swine/Netherlands/3 /80
H1N3-A/duck/New Zealand/160/76
H1N2-A/swin e/Minneso/55551/00
H1N2-A/duck/NC/91347/0 1
H1N1-A/swine/Wisconsin/168/97
H1N9-A/NWS-G70c /70
H1N1-A/USSR/90/77
H1N2-A/swine/England/690421/95
H1N1-A/Taiwan/603/05
H1N2-A/Yo kohama/22/02
H1N1-A/New Caledon ia/20/99
H1N2-A/England/2/02
H1N2 -A/New York/217/02
H1
H11N9-A/pintail/Alberta/84/00
H11N3-A/duck/Alberta/797/83
H11 N2-A/malla rd/Netherl/7/99
H11N6-A/du ck/England /56
H11
H16N3-A/gull/ Sweden /2/9 9
H16N3-A/herring/Delawar/712/88 H16 H13N2-A/whale/Maine/328 HN/84
H13N9-A/shorebird/DE/68/04
H13N8-A/gull/Netherlands/1/00
H13N6-A/gull/Astrakan/22 7/84
H13
H6N8 -A/ma lla rd/Alb erta/761/78
H6N1-A/chicken/Hong Kong/17/77
H6N5-A/duck/Australia/4045/80
H6N2-A/turkey/Canada/63
H6N4-A/shorebird/DE/194/98
H6N7-A/goose/HK/W222/97
H6N2-A/ck /California/905/01
H6N9 -A/duck/HK/182/77
H6N3-A/mallard/Alberta/ 253/90
H6N6-A/widgeon/Alberta/256/82
H6
H8N4 -A/ma llard/Alb erta/283/77
H8N4-A/malla rd/Alberta/ 194 /92 H8 H12 N5-A/teal/Alberta /199/91
H12N4-A/ruddy/DE/67/98
H12 N5-A/duck/Alberta/6 0/7 6
H12N1-A/mallard/Alberta/342/83
H12
H9N2 -A/turkey /Wisconsin/66
H9N2-A/ch icken/Shan dong/6 /96
H9N1-A/duck/Shantou/1588/00
H9N3-A/duck/Vietnam/3 40/01
H9N6-A/duck/Hong Kong/147/77
H9N1-A/mallard/Alberta/506/83
H9N3 -A/ma llard/Alberta/11/91
H9N5-A/rudd y/Delaware/2576/87
H9N6-A/rudd y/Delaware/510/88
H9
H4N8-A/pintail/Alberta/2 07/ 99
H4N1-A/malla rd/Alberta/47/98
H4N5-A/teal/Alb erta/103/90
H4N2-A/mallard/Alberta/630/84
H4N6-A/swine/Ontari/01911-1/99
H4N3-A/mallard/Alberta/300/77
H4N5-A/seal/MA/133/82
H4N6-A/duck/New Zealand/31/76
H4
H14-A/malla rd/Gurjev/263/82
H14-A/mallard/Gurjev/244/82 H14 H3N8-A/equine/Romania/80
H3N8-A/equine/Uruguay/1/63
H3N8 -A/canine/Io wa/13 628/05
H3N3-A/se al/MA/3911/9 2
H3N8-A/mallard/Alberta/117/97
H3N6-A/chick/Nan chang/7-010/00
H3N2 -A/swine/Sp ain/39139/02
H3N2-A/New York/357/05
H3N1-A/swine/Taiwan/0408/04
H3N2-A/England/72
H3N2 -A/Hong Kong /1/68
H3
H10N1-A/pintail/A lberta/129/93
H10 N6-A/duck/Alberta/4 0/8 4
H10N3-A/teal/Alberta/778/78
H10N7-A/chicken/Germany/N/49
H10 N4-A/mink/Swede n/8 4
H10
H15N8-A/du ck/Australia/341/83
H15 N9-A/shearwat/Austr/2576/79 H15 H7N2 -A/chicken/NJ/15814-9/99
H7N3 -A/ck /British Col/GSC/04
H7N7-A/eq/Cambridg e/1/63
H7N7 -A/FPV/Dutch/27
H7N4-A/chicken /NSW/1/97
H7N7-A/avian/Netherl/127/03
H7N1 -A/ostrich/Zimbabwe/222/ 96
H7N3-A/turkey/England /63
H7
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
9 9
1 0 0
1 00
1 0 0
1 0 0
1 00
1 0 0
9 9
9 9
1 0 0
1 0 0
8 9
10 0
1 0 0
9 9
10 0
1 0 0
5 9
9 2
9 9
9 9
10 0
8 0
6 1
74
1 0 0
4 7
5 5
9 9
9 9
1 0 0
1 0 0
0 .1
Strains used for the evaluation of the multiplex PCR method
subtype strain kindly provided by
H5N1 A/Hong Kong/486/97
A/Vietnam/1194/04
A/Vietnam/1203/04
A/chicken/Scotland/59
A/chicken/Cambodia/7/04
CDC (USA)
MRC (UK)
MRC (UK)
PASTEUR (FRANCE)
PASTEUR (FRANCE)
H5N2 A/turkey/England/N28/73
A/chicken/Italy/8/98
A/chucker/Minnesota/14591-7/98
CISA (SPAIN)
HIPRA (SPAIN)
CDC (USA)
H5N3 A/tern/South Africa/61
A/duck/Singapore/3/97
A/turkey/California/6878/79
ISCIII (SPAIN)
MRC (UK)
CDC (USA)
H5N8 A/turkey/Wisconsin/68 CDC (USA)
H5N9 A/ratite/New York/12716/94 CDC (USA)
Primers of the second
round PCR were selected from highly conserved regions of each of the
hemagglutinin gene segments using multiple alignments built with available
sequences obtained from databases, and were designed to render
amplification products of different size for subtypes H1 (1009 bp), H3 (847
bp), H5 (591 bp). An internal control, consisting of 100 molecules of a cloned
DNA fragment, was included in the extraction buffer to detect false negative
results.
847 bp591 bp
350 bp IC
H5H3
1009 bp H1
104 103 102 10 1 104 103 102 10 1 104 103 102 10 1MW
Serial 10-fold dilutions of H1, H3 and H5 specific plasmids
p
A s e co n d
round PCR assay using degenerate deoxyinosine-
substituted rimers was developed for reliable detection
of any of the 16 types of hemagglutinin in clinical
specimens.
As for the first round PCR, primers (PHA2 sense and
antisense) were selected from very highly conserved
regions evidenced by the alignment of multiple sequences
very different each other in terms of not only
hemagglutinin type but also host, country and isolation
date of the virus.
Direct sequencing of the second amplification product,
fragments 450-500 base pairs in length, and s
Nucleotide sequences were aligned using the program
Clustal X v1.81. Phylogenetic tree was infered using the
Kimura 2-parameter algorithm included in the program
MEGA v3.1 and reconstructed by the Neighbour-Joining
method. Statistical significance was evaluated by
bootstrap resampling of sequences 1,000 times.
ubsequent
phylogenetic analysis of nucleotide sequences enabled
hemagglutinin classification.
nt 14nt 1 nt 1100 nt 1125
GGATGGACAGGAATGATAGATGGATG H1N1 A/swine/Wisconsin/168/97-----------G-----------G-- H1N1 A/swine/Netherlands/3/80------CA-------G-T-----T-- H2N2 A/pintail/Praimoric/625/76------CA-------G-T-----T-- H2N2 A/Berlin/3/64------GA---------------T-- H3N3 A/seal/MA/3911/92--C---GAG------G-T-----G-- H3N8 A/equine/Romania/80--C---CA---TC----T-----T-- H4N6 A/swine/Ontario/01911-1/99--C---CA---CC-A--T-----G-- H4N6 A/duck/New Zealand/31/76------CAG------G----C--T-- H5N8 A/turkey/Ireland/1378/83------CAG------G-------T-- H5N1 A/Hanoi/30408/05-----------C-------------- H6N8 A/mallard duck/Alberta/761/78--------T--------------G-- H6N1 A/chicken/Hong Kong/17/77------GA---TC----T-------- H7N1 A/ostrich/Zimbabwe/222/96------GA---TC--G-T-------- H7N3 A/turkey/England/63------T-T--G-----T-----G-- H8N4 A/mallard duck/Alberta/283/77------T-T--G-----T-------- H8N4 A/mallard/Alberta/194/92--T---C-----C--G-T-C------ H9N5 A/ruddy turnstone/Delaware/2576/87--T---T----GC-AG-C-C---T-- H9N2 A/chicken/Shandong/6/96------GA-------G----C--C-- H10N7 A/chicken/Germany/N/49------GA-------G----C--C-- H10N4 A/mink/Sweden/84------C----TT-A--CA----T-- H11N9 A/pintail/Alberta/84/00--G---C----GC-T--CA------- H11N2 A/mallard/Netherlands/7/99------C----GC-AG-G-C---T-- H12N1 A/mallard duck/Alberta/342/83------C-----C-AG-G-C---T-- H12N5 A/green-winged teal/Alberta/199/91--T---C-----T-A---A----T-- H13N8 A/black-headed gull/Netherlands/1/00--T---C----GT-A---A----T-- H13N2 A/pilot whale/Maine/328 HN/84--T---CA---CC----T-----G-- H14N5 A/mallard/Gurjev/263/82--T---CA---CC----T-----G-- H14N6 A/mallard/Gurjev/244/82------GA----C-C--T-------- H15N8 A/duck/Australia/341/83------GA---GC-C--T-------- H15N9 A/shearwater/West Australia/2576/79------C-C--GT-A--CA------- H16N3 A/black-headed gull/Sweden/2/99--G---C-T--TC-A--TA-C----- H16N3 A/herring gull/Delaware/712/88
CCIACCIKWCCIKACYAICKRCCWAC PRIMERS
AGCAAAAGCAGGGG----------------------------------------------------------------------------------A---------------
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------
GGGGTTAGCAAAAGCAGGRG5’- -3’ -5’3’-
Fusion peptide
S S-
HA1 (nt 1-1063 GenBank AB239125) HA2 (nucleotides 1064-1776)
The proposed
procedure have been designed in such a way that the first round
amplification product, obtained using degenerate deoxyinosine-
substituted primers, was shared by both the diagnostic (H1-H3-H5
multiplex PCR) and surveillance (H1-16 PCR) methods.
To develop diagnostic and surveillance molecular
tools for the rapid and reliable detection of any of the hemagglutinin
types of influenza A virus directly in clinial specimens.
To develop diagnostic and surveillance molecular
tools for the rapid and reliable detection of any of the hemagglutinin
types of influenza A virus directly in clinial specimens.
H5N1 A/Thailand/2-SP-33/2004
H 5N 1 A/chicken/Vietnam/HD1/2004
H5N1 A/Thailand/1-KAN-1/2004
H5N1 A/chicken/Thailand/Tak-01/2004
H5N1 A/chicken/Thailand/Nakornsawan
H5N1 A/chicken/Thailand/Bangkok/01
H 5N 1 A/VietNam/3046/2004
H5N1 A/Hanoi/03/2004
H5N1 A/VietNam/3062/2004
H 5N 1 A/bird/Thailand/3.1/2004
H5N1 A/whitepeafo/Thailand/CU-11/04
H5N1 A/VietNam/1194/2004
H 5N 1 A/VietNam/1203/2004
H 5N 1 A/chicken/HK/2133.1/2003
H 5N 1 A/chicken/Indonesia/4/2004
H5N1 A/chicken/Indonesia/2A/2003
H5N1 A/chicken/Guangdong/174/04
H5N1 A/duck/Novosibirsk/56/2005
H 5N 1 A/chicken/Crimea/1/2005
H5N1 A/domestic cat/Iraq/820/06
H5N1 A/chicken/Nigeria/641/2006
H 5N 1 A/mallard/Bavaria/1/2006
H5N1 A/mallard/Italy/332/2006
H 5N 1 A/chicken/Egypt/960N3-004/2006
H5N1 A/chicken/Jil in/9/2004
H 5N 1 A/duck/HongKong/2986.1/2000
H5N1 A/chicken/HongKong/31.4/02
H5N1 A/duck/Shanghai/35/2002
H5N1 A/duck/Anyang/AVL-1/2001
H5N1 A/duck/Fujian/01/2002
H5N1 A/environ/HongKong/437-10/99
H5N1 A/chicken/HongKong/317.5/2001
H 5N 1 A/swine/Shandong/2/03
H5N1 A/goose/Guangdong/1/9 6
H 5N 1 A/HongKong/481/97
H 5N 1 A/HongKong/156/97
H5N1 A/HongKong/97/98
H5N1 A/duck/HongKong/p46/97
H5N1 A/chicken/HongKong/1203/97
H 5N 1 A/HongKong/486/97
H5N2 A/chicken/Italy/312/97
H5N2 A/chicken/Italy/8/98
H5N2 A/guineafowl/Italy/330/97
H5N3 A/duck/Singapore/3/97
H 5N 9 A/chicken/Italy/9097/97
H5N1 A/turkey/England/50-92/91
H5N2 A/duck/Potsdam/1402-6/86
H5N3 A/duck/HongKong/205/77
H5N3 A/duck/HoChiMinh/14/78
H5N8 A/duck/Ireland/113/83
H5N8 A/turkey/Ireland/1378/83
H5N6 A/duck/Potsdam/2216-4/84
H 5N 2 A/turkey/England/N28/73
H5N3 A/tern/SouthAfrica/61
H5N1 A/chicken/Scotland/59
Eurasian
H 5N 2 A/chick/Pennsylvania/1/83
H 5N 2 A/chick/Penn/1370/83
H5N3 A/ruddyturnstone/NJ/2242/00
H5N2 A/duck/Michigan/80
H5N1 A/duck/Minnesota/1525/81
H5N3 A/mallard/Wisconsin/169/75
H5N9 A/turkey/Ontario/7732/66
H5N9 A/turkey/Wisconsin/68
H 5N 1 A/gull/Pennsylvania/4175/83
H 5N 2 A/chick/Mexico/31381-Avilab/94
H 5N 2 A/chicken/Taiwan/1209/03
H5N2 A/chicken/Queretaro/7653-20/95
H5N2 A/chicken/Puebla/8623-607/94
H5N3 A/chicken/TX/167280-4/02
H5N2 A/avian/NY/31588-3/00
H 5N 8 A/duck/NY/191255-59/02
H5N2 A/turkey/Minnesota/10734/95
H5N9 A/mallard/Ohio/556/1987
H 5N 2 A/emu/Texas/39442/93
American
H2N8 A/ruddy turns/Delaware/142/98
99
42
93
31
99
34
79
95
61
9 9
9 9
9 5
0.05
A/goose/Guangdong/1/96hemagglutinin-derived cluster of HPAI viruses
Primer Sequence (5’ - 3’) Gene position(GenBank reference strain)
H1 sense CAA TAT GTA TAG GCT ACC ATG C 56-77 (AY289929)H1 antisense CCC TCA ATR AAA CCR GCA AT 1064-1045 (AY289929)H3 sense ACT GCA CAC TRA TAG ATG C 236-254 (AY531033)H3 antisense CCC TCC CAA CCA TTT TCT AT 1082-1063 (AY531033)H5 sense TTC AGR AAT GTR GTR TGG 506-523 (AB239125)H5 antisense TAT RAA ICC YGC TAT WGC 1093-1076 (AB239125)
PHA2 sense WHH TIT GGG GIR TIC AYC A 597-615 (AB239125)PHA2 antisense AAI CCW GCW ATI GCI CCR AA 1089-1070 (AB239125)
Second round PCR oligonucleotide primers
HA Subtyping
pacopozo@isciii.es
A particular emphasis was put on the suitability of this method for the detection of both the Eurasian and the American lineages of H5 viruses.
Thirteen different reference strains of subtypes H5N1, H5N2, H5N3, H5N8 and H5N9 were used for the evaluation of the PCR method.
Sensitivity of the assay was estimated by testing serial tenfold dilutions of H1, H3 and H5 specific plasmids. Detection levels were 10 copies.
Phylogenetic analysis of the second amplification product nucleotide sequences of H5 viruses (Kimura 2-parameter ) would fit a particular strain into one of the two previously described lineages.
MEGA v3.1
Implementation of a Sensitive Diagnostic and Surveillance System to Detect Emerging Influenza Strains
Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos I I I , 28220 Majadahonda, Madrid
Thirteenth International ConferenceNegative Strand Viruses 2006
Sal am an ca, Sp ain , Ju n e 17th - 22n d , 2006
Immediate knowledge about human infection with the subtype H5 and then with the
currently circulating influenza A subtypes H1 and H3 would be desirable for timely
epidemiological investigations and, if possible, infection control.
Nevertheless, next pandemic might be caused by a different subtype of influenza virus. In
fact, three other avian influenza viruses (H9N2, H7N7 and H7N3) have caused illness in
humans.
As a result, diagnostic
laboratories receiving requests to test specimens from patients with an influenza-like illness
are being encouraged to the implementation of rapid and sensitive procedures not only for
typing influenza A virus but also for subtyping at least H1, H3 and H5 viruses directly from
clinical specimens.
Strategies for containing
.
Since their reemergence in late 2003, the establishment of the highly pathogenic avian
influenza (HPAI) A subtype H5N1 as an endemic virus in poultry in several countries of Asia
and the recent extensive geographical spread to Africa, Europe and the Middle East, in
addition with its repeated interspecies transmission to humans are giving this virus increased
opportunities to improve their transmissibility in humans, and thus develop into a pandemic
strain,
an emerging influenza pandemic or at
least attempting to slow down its spread worldwide will depend on several factors but the
prompt laboratory identification of the first cases would be unanswerably the starting point
being evident that H5N1 virus poses a continuing global human public health risk.
H5N9-A/mallard/Sweden/80/02 H5N6-A/mallard/Sweden/40/02
H5N2-A/duck/Mongolia/54/01 H5N1-A/Hanoi/30408/05
H5N1-A/chicken/HK/728/97 H5N9-A/chicken/Italy/9097/97
H5N8-A/turkey/Ireland/1378/83 H5N3-A/duck/Miyagi/54/76 H5N6-A/duck/Potsdam/2216-4/84 H5N3-A/tern/South Africa/61 H5N1-A/chicken/Scotland/59 H5N2-A/ck/Queretaro/7653-20/95
H5N7-A/shorebird/Delawa/75/04 H5N8-A/duck/NY/191255-59/02
H 5
H2N3-A/mallard/Alberta/226/98 H2N5-A/mallard/Alberta/202/96
H2N9-A/gull/New Jersey/75/85 H2N1-A/ruddy/Delaware/81/93 H2N2-A/pintail/Praimor/625/76
H2N2-A/Berlin/3/64 H2N2-A/Japan/305/57
H 2
H1N5-A/pintail duck/Alberta/63 H1N4-A/teal/Alberta/141/92
H1N6-A/mallard duck/Alberta/42 H1N1-A/swine/Netherlands/3/80
H1N3-A/duck/New Zealand/160/76 H1N2-A/swine/Minneso/55551/00 H1N2-A/duck/NC/91347/01 H1N1-A/swine/Wisconsin/168/97
H1N9-A/NWS-G70c/70 H1N1-A/USSR/90/77
H1N2-A/swine/England/690421/95 H1N1-A/Taiwan/603/05 H1N2-A/Yokohama/22/02 H1N1-A/New Caledonia/20/99 H1N2-A/England/2/02 H1N2-A/New York/217/02
H 1
H11N9-A/pintail/Alberta/84/00 H11N3-A/duck/Alberta/797/83
H11N2-A/mallard/Netherl/7/99 H11N6-A/duck/England/56
H 1 1
H16N3-A/gull/Sweden/2/99 H16N3-A/herring/Delawar/712/88 H 1 6 H13N2-A/whale/Maine/328 HN/84
H13N9-A/shorebird/DE/68/04 H13N8-A/gull/Netherlands/1/00 H13N6-A/gull/Astrakan/227/84
H 1 3
H6N8-A/mallard/Alberta/761/78 H6N1-A/chicken/Hong Kong/17/77
H6N5-A/duck/Australia/4045/80 H6N2-A/turkey/Canada/63 H6N4-A/shorebird/DE/194/98
H6N7-A/goose/HK/W222/97 H6N2-A/ck/California/905/01
H6N9-A/duck/HK/182/77 H6N3-A/mallard/Alberta/253/90 H6N6-A/widgeon/Alberta/256/82
H 6
H8N4-A/mallard/Alberta/283/77 H8N4-A/mallard/Alberta/194/92 H 8
H12N5-A/teal/Alberta/199/91 H12N4-A/ruddy/DE/67/98
H12N5-A/duck/Alberta/60/76 H12N1-A/mallard/Alberta/342/83
H 1 2
H9N2-A/turkey/Wisconsin/66 H9N2-A/chicken/Shandong/6/96 H9N1-A/duck/Shantou/1588/00 H9N3-A/duck/Vietnam/340/01
H9N6-A/duck/Hong Kong/147/77 H9N1-A/mallard/Alberta/506/83 H9N3-A/mallard/Alberta/11/91 H9N5-A/ruddy/Delaware/2576/87 H9N6-A/ruddy/Delaware/510/88
H 9
H4N8-A/pintail/Alberta/207/99 H4N1-A/mallard/Alberta/47/98 H4N5-A/teal/Alberta/103/90 H4N2-A/mallard/Alberta/630/84 H4N6-A/swine/Ontari/01911-1/99
H4N3-A/mallard/Alberta/300/77 H4N5-A/seal/MA/133/82
H4N6-A/duck/New Zealand/31/76
H 4
H14-A/mallard/Gurjev/263/82 H14-A/mallard/Gurjev/244/82 H 1 4
H3N8-A/equine/Romania/80 H3N8-A/equine/Uruguay/1/63 H3N8-A/canine/Iowa/13628/05
H3N3-A/seal/MA/3911/92 H3N8-A/mallard/Alberta/117/97 H3N6-A/chick/Nanchang/7-010/00 H3N2-A/swine/Spain/39139/02 H3N2-A/New York/357/05
H3N1-A/swine/Taiwan/0408/04 H3N2-A/England/72 H3N2-A/Hong Kong/1/68
H 3
H10N1-A/pintail/Alberta/129/93 H10N6-A/duck/Alberta/40/84 H10N3-A/teal/Alberta/778/78
H10N7-A/chicken/Germany/N/49 H10N4-A/mink/Sweden/84
H 1 0
H15N8-A/duck/Australia/341/83 H15N9-A/shearwat/Austr/2576/79 H 1 5 H7N2-A/chicken/NJ/15814-9/99
H7N3-A/ck/British Col/GSC/04 H7N7-A/eq/Cambridge/1/63
H7N7-A/FPV/Dutch/27 H7N4-A/chicken/NSW/1/97 H7N7-A/avian/Netherl/127/03
H7N1-A/ostrich/Zimbabwe/222/96 H7N3-A/turkey/England/63
H 7
100
100
100
100
100
100
100
99
100
100
100
100
100
100
99
99
100
100
89
100
100
99
100
100
59
92
99
99
100
80
61
74
100
47
55
99 99
100
100
0.1
Strains used for the evaluation of the multiplex PCR method
subtype strain kindly provided byH5N1 A/Hong Kong/486/97
A/Vietnam/1194/04A/Vietnam/1203/04A/chicken/Scotland/59A/chicken/Cambodia/7/04
CDC (USA)MRC (UK)MRC (UK)PASTEUR (FRANCE)PASTEUR (FRANCE)
H5N2 A/turkey/England/N28/73A/chicken/Italy/8/98A/chucker/Minnesota/14591-7/98
CISA (SPAIN)HIPRA (SPAIN)CDC (USA)
H5N3 A/tern/South Africa/61A/duck/Singapore/3/97A/turkey/California/6878/79
ISCIII (SPAIN)MRC (UK)CDC (USA)
H5N8 A/turkey/Wisconsin/68 CDC (USA)
H5N9 A/ratite/New York/12716/94 CDC (USA)
Primers of the second round PCR were selected from highly conserved regions of each of the hemagglutinin gene segments using multiple alignments built with available sequences obtained from databases, and were designed to render amplification products of different size for subtypes H1 (1009 bp), H3 (847 bp), H5 (591 bp). An internal control, consisting of 100 molecules of a cloned DNA fragment, was included in the extraction buffer to detect false negative results.
847 bp591 bp
350 bp IC
H5H3
1009 bp H1
104103102 10 1 104103102 10 1 104103 102 10 1MW
Serial 10-f old dilutions of H1, H3 and H5 specifi c plasmids
p
A second round PCR assay using degenerate deoxyinosine-substituted rimers was developed for reliable detection of any of the 16 types of hemagglutinin in clinical specimens.
As for the first round PCR, primers (PHA2 sense and antisense) were selected from very highly conserved regions evidenced by the alignment of multiple sequences very different each other in terms of not only hemagglutinin type but also host, country and isolation date of the virus.
Direct sequencing of the second amplification product, fragments 450-500 base pairs in length, and s
Nucleotide sequences were aligned using the program Clustal X v1.81. Phylogenetic tree was infered using the Kimura 2-parameter algorithm included in the program MEGA v3.1 and reconstructed by the Neighbour-Joining method. Statistical significance was evaluated by bootstrap resampling of sequences 1,000 times.
ubsequent phylogenetic analysis of nucleotide sequences enabled hemagglutinin classification.
nt 14nt 1 nt 1100 nt 1125
GGATGGACAGGAATGATAGATGGATG H1N1 A/swine/Wisconsin/168/97-----------G-----------G-- H1N1 A/swine/Netherlands/3/80------CA-------G-T-----T-- H2N2 A/pintail/Praimoric/625/76------CA-------G-T-----T-- H2N2 A/Berlin/3/64------GA---------------T-- H3N3 A/seal/MA/3911/92--C---GAG------G-T-----G-- H3N8 A/equine/Romania/80--C---CA---TC----T-----T-- H4N6 A/swine/Ontario/01911-1/99--C---CA---CC-A--T-----G-- H4N6 A/duck/New Zealand/31/76------CAG------G----C--T-- H5N8 A/turkey/Ireland/1378/83------CAG------G-------T-- H5N1 A/Hanoi/30408/05-----------C-------------- H6N8 A/mallard duck/Alberta/761/78--------T--------------G-- H6N1 A/chicken/Hong Kong/17/77------GA---TC----T-------- H7N1 A/ostrich/Zimbabwe/222/96------GA---TC--G-T-------- H7N3 A/turkey/England/63------T-T--G-----T-----G-- H8N4 A/mallard duck/Alberta/283/77------T-T--G-----T-------- H8N4 A/mallard/Alberta/194/92--T---C-----C--G-T-C------ H9N5 A/ruddy turnstone/Delaware/2576/87--T---T----GC-AG-C-C---T-- H9N2 A/chicken/Shandong/6/96------GA-------G----C--C-- H10N7 A/chicken/Germany/N/49------GA-------G----C--C-- H10N4 A/mink/Sweden/84------C----TT-A--CA----T-- H11N9 A/pintail/Alberta/84/00--G---C----GC-T--CA------- H11N2 A/mallard/Netherlands/7/99------C----GC-AG-G-C---T-- H12N1 A/mallard duck/Alberta/342/83------C-----C-AG-G-C---T-- H12N5 A/green-winged teal/Alberta/199/91--T---C-----T-A---A----T-- H13N8 A/black-headed gull/Netherlands/1/00--T---C----GT-A---A----T-- H13N2 A/pilot whale/Maine/328 HN/84--T---CA---CC----T-----G-- H14N5 A/mallard/Gurjev/263/82--T---CA---CC----T-----G-- H14N6 A/mallard/Gurjev/244/82------GA----C-C--T-------- H15N8 A/duck/Australia/341/83------GA---GC-C--T-------- H15N9 A/shearwater/West Australia/2576/79------C-C--GT-A--CA------- H16N3 A/black-headed gull/Sweden/2/99--G---C-T--TC-A--TA-C----- H16N3 A/herring gull/Delaware/712/88
CCIACCIKWCCIKACYAICKRCCWAC PRIMERS
AGCAAAAGCAGGGG----------------------------------------------------------------------------------A--------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------
GGGGTTAGCAAAAGCAGGRG5’- -3’ -5’3’-
Fusion pep tide
S S-
HA1 ( n t 1 - 1 0 6 3 G e n B a n k A B 2 3 9 1 2 5 ) HA2 ( n u c l e o t i d e s 1 0 6 4 - 1 7 7 6 )
The proposed procedure have been designed in such a way that the first round amplification product, obtained using degenerate deoxyinosine-substituted primers, was shared by both the diagnostic (H1-H3-H5 multiplex PCR) and surveillance (H1-16 PCR) methods.
To develop diagnostic and surveillance molecular tools for the rapid and reliable detection of any of the hemagglutinin types of influenza A virus directly in clinial specimens.
To develop diagnostic and surveillance molecular tools for the rapid and reliable detection of any of the hemagglutinin types of influenza A virus directly in clinial specimens.
H5N1 A/Thailand/2-SP-33/2004 H5N1 A/chicken/Vietnam/HD1/2004H5N1 A/Thailand/1-KAN-1/2004 H5N1 A/chicken/Thailand/Tak-01/2004 H5N1 A/chicken/Thailand/NakornsawanH5N1 A/chicken/Thailand/Bangkok/01H5N1 A/VietNam/3046/2004H5N1 A/Hanoi/03/2004 H5N1 A/VietNam/3062/2004 H5N1 A/bird/Thailand/3.1/2004 H5N1 A/whitepeafo/Thailand/CU-11/04H5N1 A/VietNam/1194/2004 H5N1 A/VietNam/1203/2004 H5N1 A/chicken/HK/2133.1/2003H5N1 A/chicken/Indonesia/4/2004 H5N1 A/chicken/Indonesia/2A/2003 H5N1 A/chicken/Guangdong/174/04 H5N1 A/duck/Novosibirsk/56/2005H5N1 A/chicken/Crimea/1/2005 H5N1 A/domestic cat/Iraq/820/06 H5N1 A/chicken/Nigeria/641/2006H5N1 A/mallard/Bavaria/1/2006 H5N1 A/mallard/Italy/332/2006 H5N1 A/chicken/Egypt/960N3-004/2006H5N1 A/chicken/Jilin/9/2004 H5N1 A/duck/HongKong/2986.1/2000 H5N1 A/chicken/HongKong/31.4/02H5N1 A/duck/Shanghai/35/2002 H5N1 A/duck/Anyang/AVL-1/2001H5N1 A/duck/Fujian/01/2002 H5N1 A/environ/HongKong/437-10/99 H5N1 A/chicken/HongKong/317.5/2001H5N1 A/swine/Shandong/2/03 H5N1 A/goose/Guangdong/1/96H5N1 A/HongKong/481/97H5N1 A/HongKong/156/97 H5N1 A/HongKong/97/98 H5N1 A/duck/HongKong/p46/97 H5N1 A/chicken/HongKong/1203/97H5N1 A/HongKong/486/97H5N2 A/chicken/Italy/312/97 H5N2 A/chicken/Italy/8/98 H5N2 A/guineafowl/Italy/330/97H5N3 A/duck/Singapore/3/97H5N9 A/chicken/Italy/9097/97 H5N1 A/turkey/England/50-92/91H5N2 A/duck/Potsdam/1402-6/86 H5N3 A/duck/HongKong/205/77H5N3 A/duck/HoChiMinh/14/78 H5N8 A/duck/Ireland/113/83 H5N8 A/turkey/Ireland/1378/83 H5N6 A/duck/Potsdam/2216-4/84 H5N2 A/turkey/England/N28/73 H5N3 A/tern/SouthAfrica/61 H5N1 A/chicken/Scotland/59
Eurasian
H5N2 A/chick/Pennsylvania/1/83H5N2 A/chick/Penn/1370/83H5N3 A/ruddyturnstone/NJ/2242/00 H5N2 A/duck/Michigan/80 H5N1 A/duck/Minnesota/1525/81H5N3 A/mallard/Wisconsin/169/75 H5N9 A/turkey/Ontario/7732/66H5N9 A/turkey/Wisconsin/68 H5N1 A/gull/Pennsylvania/4175/83 H5N2 A/chick/Mexico/31381-Avilab/94H5N2 A/chicken/Taiwan/1209/03 H5N2 A/chicken/Queretaro/7653-20/95H5N2 A/chicken/Puebla/8623-607/94H5N3 A/chicken/TX/167280-4/02H5N2 A/avian/NY/31588-3/00 H5N8 A/duck/NY/191255-59/02 H5N2 A/turkey/Minnesota/10734/95H5N9 A/mallard/Ohio/556/1987 H5N2 A/emu/Texas/39442/93
American
H2N8 A/ruddy turns/Delaware/142/98
99
42
93
31
99
34
79
95
61
99
99
95
0.05
A/goose/Guangdong/1/96hemagglutinin-derived cluster of HPAI viruses
Primer Sequence (5’ - 3’) Gene position(GenBankreferencestrain)
H1 sense CAA TAT GTA TAG GCT ACC ATG C 56-77 (AY289929)H1 antisense CCC TCA ATR AAA CCR GCA AT 1064-1045 (AY289929)H3 sense ACT GCA CAC TRA TAG ATG C 236-254 (AY531033)H3 antisense CCC TCC CAA CCA TTT TCT AT 1082-1063 (AY531033)H5 sense TTC AGR AAT GTR GTR TGG 506-523 (AB239125)H5 antisense TAT RAA ICC YGC TAT WGC 1093-1076 (AB239125)
PHA2 sense WHH TIT GGG GIR TIC AYC A 597-615 (AB239125)PHA2 antisense AAI CCW GCW ATI GCI CCR AA 1089-1070 (AB239125)
Second round PCR oligonucleot ide primers
1009bp H1
847bp H3
591bp H5
350bp IC
Fusion peptideN-terminal signal
RT+common 1st amplification
H5
H1H3
pacopozo@isciii.es
A particular emphasis was put on the suitability of
this method for the detection of both the Eurasian
and the American lineages of H5 viruses.
Thirteen different reference strains of subtypes
H5N1, H5N2, H5N3, H5N8 and H5N9 were used for
the evaluation of the PCR method.
Sensitivity of the assay was estimated by testing
serial tenfold dilutions of H1, H3 and H5 specific
plasmids. Detection levels were 10 copies.
Phylogenetic analysis of the second amplification
product nucleotide sequences of H5 viruses (Kimura
2-parameter ) would fit a particular strain
into one of the two previously described lineages. MEGA v3.1
Implementation of a Sensitive Diagnostic and Surveillance System to Detect Emerging Influenza Strains
Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos III, 28220 Majadahonda, Madrid
Thirteenth International ConferenceNegative Strand Viruses 2006
Salamanca, Spain, June 17th - 22nd, 2006
Immediate knowledge about human infection with the subtype H5 and then with the
currently circulating influenza A subtypes H1 and H3 would be desirable for timely
epidemiological investigations and, if possible, infection control.
Nevertheless, next pandemic might be caused by a different subtype of influenza virus. In
fact, three other avian influenza viruses (H9N2, H7N7 and H7N3) have caused illness in
humans.
As a result, diagnostic
laboratories receiving requests to test specimens from patients with an influenza-like illness
are being encouraged to the implementation of rapid and sensitive procedures not only for
typing influenza A virus but also for subtyping at least H1, H3 and H5 viruses directly from
clinical specimens.
Strategies for containing
.
Since their reemergence in late 2003, the establishment of the highly pathogenic avian
influenza (HPAI) A subtype H5N1 as an endemic virus in poultry in several countries of Asia
and the recent extensive geographical spread to Africa, Europe and the Middle East, in
addition with its repeated interspecies transmission to humans are giving this virus increased
opportunities to improve their transmissibility in humans, and thus develop into a pandemic
strain,
an emerging influenza pandemic or at
least attempting to slow down its spread worldwide will depend on several factors but the
prompt laboratory identification of the first cases would be unanswerably the starting point
being evident that H5N1 virus poses a continuing global human public health risk.
H5N9 -A/ma llard/Sweden/8 0/0 2
H5N6-A/mallard/Sweden/40/02
H5N2-A/duck/Mongolia/54/01
H5N1-A/Hanoi/30 408/05
H5N1-A/chicken/HK/728 /97
H5N9 -A/ch icken/Italy/9 097/97
H5N8-A/turkey/Ireland/137 8/83
H5N3-A/duck/Miyagi/54/76
H5N6-A/duck/Potsd am/2216-4/84
H5N3 -A/tern/South Afri ca/61
H5N1-A/chicken/Scotland/5 9
H5N2-A/ck/Quereta ro/7 653-20/95
H5N7-A/sh orebird/Delawa/75/0 4
H5N8-A/duck/NY/191255-59/02
H5
H2N3 -A/ma llard/Alb erta/226/98
H2N5-A/mallard/Alberta/ 202/96
H2N9 -A/gull/New Jersey/75/ 85
H2N1-A/ruddy/Delaware/81/93
H2N2 -A/pintail/Praimor/625/76
H2N2 -A/Berli n/3/64
H2N2-A/Japan/305/57
H2
H1N5-A/pintail duck/Alberta/6 3
H1N4-A/teal/Alberta/ 141 /92
H1N6 -A/ma llard duck/Alberta/4 2
H1N1 -A/swine/Netherlands/3 /80
H1N3-A/duck/New Zealand/160/76
H1N2-A/swin e/Minneso/55551/00
H1N2-A/duck/NC/91347/0 1
H1N1-A/swine/Wisconsin/168/97
H1N9-A/NWS-G70c /70
H1N1-A/USSR/90/77
H1N2-A/swine/England/690421/95
H1N1-A/Taiwan/603/05
H1N2-A/Yo kohama/22/02
H1N1-A/New Caledon ia/20/99
H1N2-A/England/2/02
H1N2 -A/New York/217/02
H1
H11N9-A/pintail/Alberta/84/00
H11N3-A/duck/Alberta/797/83
H11 N2-A/malla rd/Netherl/7/99
H11N6-A/du ck/England /56
H11
H16N3-A/gull/ Sweden /2/9 9
H16N3-A/herring/Delawar/712/88 H16 H13N2-A/whale/Maine/328 HN/84
H13N9-A/shorebird/DE/68/04
H13N8-A/gull/Netherlands/1/00
H13N6-A/gull/Astrakan/22 7/84
H13
H6N8 -A/ma lla rd/Alb erta/761/78
H6N1-A/chicken/Hong Kong/17/77
H6N5-A/duck/Australia/4045/80
H6N2-A/turkey/Canada/63
H6N4-A/shorebird/DE/194/98
H6N7-A/goose/HK/W222/97
H6N2-A/ck /California/905/01
H6N9 -A/duck/HK/182/77
H6N3-A/mallard/Alberta/ 253/90
H6N6-A/widgeon/Alberta/256/82
H6
H8N4 -A/ma llard/Alb erta/283/77
H8N4-A/malla rd/Alberta/ 194 /92 H8 H12 N5-A/teal/Alberta /199/91
H12N4-A/ruddy/DE/67/98
H12 N5-A/duck/Alberta/6 0/7 6
H12N1-A/mallard/Alberta/342/83
H12
H9N2 -A/turkey /Wisconsin/66
H9N2-A/ch icken/Shan dong/6 /96
H9N1-A/duck/Shantou/1588/00
H9N3-A/duck/Vietnam/3 40/01
H9N6-A/duck/Hong Kong/147/77
H9N1-A/mallard/Alberta/506/83
H9N3 -A/ma llard/Alberta/11/91
H9N5-A/rudd y/Delaware/2576/87
H9N6-A/rudd y/Delaware/510/88
H9
H4N8-A/pintail/Alberta/2 07/ 99
H4N1-A/malla rd/Alberta/47/98
H4N5-A/teal/Alb erta/103/90
H4N2-A/mallard/Alberta/630/84
H4N6-A/swine/Ontari/01911-1/99
H4N3-A/mallard/Alberta/300/77
H4N5-A/seal/MA/133/82
H4N6-A/duck/New Zealand/31/76
H4
H14-A/malla rd/Gurjev/263/82
H14-A/mallard/Gurjev/244/82 H14 H3N8-A/equine/Romania/80
H3N8-A/equine/Uruguay/1/63
H3N8 -A/canine/Io wa/13 628/05
H3N3-A/se al/MA/3911/9 2
H3N8-A/mallard/Alberta/117/97
H3N6-A/chick/Nan chang/7-010/00
H3N2 -A/swine/Sp ain/39139/02
H3N2-A/New York/357/05
H3N1-A/swine/Taiwan/0408/04
H3N2-A/England/72
H3N2 -A/Hong Kong /1/68
H3
H10N1-A/pintail/A lberta/129/93
H10 N6-A/duck/Alberta/4 0/8 4
H10N3-A/teal/Alberta/778/78
H10N7-A/chicken/Germany/N/49
H10 N4-A/mink/Swede n/8 4
H10
H15N8-A/du ck/Australia/341/83
H15 N9-A/shearwat/Austr/2576/79 H15 H7N2 -A/chicken/NJ/15814-9/99
H7N3 -A/ck /British Col/GSC/04
H7N7-A/eq/Cambridg e/1/63
H7N7 -A/FPV/Dutch/27
H7N4-A/chicken /NSW/1/97
H7N7-A/avian/Netherl/127/03
H7N1 -A/ostrich/Zimbabwe/222/ 96
H7N3-A/turkey/England /63
H7
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
1 0 0
9 9
1 0 0
1 00
1 0 0
1 0 0
1 00
1 0 0
9 9
9 9
1 0 0
1 0 0
8 9
10 0
1 0 0
9 9
10 0
1 0 0
5 9
9 2
9 9
9 9
10 0
8 0
6 1
74
1 0 0
4 7
5 5
9 9
9 9
1 0 0
1 0 0
0 .1
Strains used for the evaluation of the multiplex PCR method
subtype strain kindly provided by
H5N1 A/Hong Kong/486/97
A/Vietnam/1194/04
A/Vietnam/1203/04
A/chicken/Scotland/59
A/chicken/Cambodia/7/04
CDC (USA)
MRC (UK)
MRC (UK)
PASTEUR (FRANCE)
PASTEUR (FRANCE)
H5N2 A/turkey/England/N28/73
A/chicken/Italy/8/98
A/chucker/Minnesota/14591-7/98
CISA (SPAIN)
HIPRA (SPAIN)
CDC (USA)
H5N3 A/tern/South Africa/61
A/duck/Singapore/3/97
A/turkey/California/6878/79
ISCIII (SPAIN)
MRC (UK)
CDC (USA)
H5N8 A/turkey/Wisconsin/68 CDC (USA)
H5N9 A/ratite/New York/12716/94 CDC (USA)
Primers of the second
round PCR were selected from highly conserved regions of each of the
hemagglutinin gene segments using multiple alignments built with available
sequences obtained from databases, and were designed to render
amplification products of different size for subtypes H1 (1009 bp), H3 (847
bp), H5 (591 bp). An internal control, consisting of 100 molecules of a cloned
DNA fragment, was included in the extraction buffer to detect false negative
results.
847 bp591 bp
350 bp IC
H5H3
1009 bp H1
104 103 102 10 1 104 103 102 10 1 104 103 102 10 1MW
Serial 10-fold dilutions of H1, H3 and H5 specific plasmids
p
A s e co n d
round PCR assay using degenerate deoxyinosine-
substituted rimers was developed for reliable detection
of any of the 16 types of hemagglutinin in clinical
specimens.
As for the first round PCR, primers (PHA2 sense and
antisense) were selected from very highly conserved
regions evidenced by the alignment of multiple sequences
very different each other in terms of not only
hemagglutinin type but also host, country and isolation
date of the virus.
Direct sequencing of the second amplification product,
fragments 450-500 base pairs in length, and s
Nucleotide sequences were aligned using the program
Clustal X v1.81. Phylogenetic tree was infered using the
Kimura 2-parameter algorithm included in the program
MEGA v3.1 and reconstructed by the Neighbour-Joining
method. Statistical significance was evaluated by
bootstrap resampling of sequences 1,000 times.
ubsequent
phylogenetic analysis of nucleotide sequences enabled
hemagglutinin classification.
nt 14nt 1 nt 1100 nt 1125
GGATGGACAGGAATGATAGATGGATG H1N1 A/swine/Wisconsin/168/97-----------G-----------G-- H1N1 A/swine/Netherlands/3/80------CA-------G-T-----T-- H2N2 A/pintail/Praimoric/625/76------CA-------G-T-----T-- H2N2 A/Berlin/3/64------GA---------------T-- H3N3 A/seal/MA/3911/92--C---GAG------G-T-----G-- H3N8 A/equine/Romania/80--C---CA---TC----T-----T-- H4N6 A/swine/Ontario/01911-1/99--C---CA---CC-A--T-----G-- H4N6 A/duck/New Zealand/31/76------CAG------G----C--T-- H5N8 A/turkey/Ireland/1378/83------CAG------G-------T-- H5N1 A/Hanoi/30408/05-----------C-------------- H6N8 A/mallard duck/Alberta/761/78--------T--------------G-- H6N1 A/chicken/Hong Kong/17/77------GA---TC----T-------- H7N1 A/ostrich/Zimbabwe/222/96------GA---TC--G-T-------- H7N3 A/turkey/England/63------T-T--G-----T-----G-- H8N4 A/mallard duck/Alberta/283/77------T-T--G-----T-------- H8N4 A/mallard/Alberta/194/92--T---C-----C--G-T-C------ H9N5 A/ruddy turnstone/Delaware/2576/87--T---T----GC-AG-C-C---T-- H9N2 A/chicken/Shandong/6/96------GA-------G----C--C-- H10N7 A/chicken/Germany/N/49------GA-------G----C--C-- H10N4 A/mink/Sweden/84------C----TT-A--CA----T-- H11N9 A/pintail/Alberta/84/00--G---C----GC-T--CA------- H11N2 A/mallard/Netherlands/7/99------C----GC-AG-G-C---T-- H12N1 A/mallard duck/Alberta/342/83------C-----C-AG-G-C---T-- H12N5 A/green-winged teal/Alberta/199/91--T---C-----T-A---A----T-- H13N8 A/black-headed gull/Netherlands/1/00--T---C----GT-A---A----T-- H13N2 A/pilot whale/Maine/328 HN/84--T---CA---CC----T-----G-- H14N5 A/mallard/Gurjev/263/82--T---CA---CC----T-----G-- H14N6 A/mallard/Gurjev/244/82------GA----C-C--T-------- H15N8 A/duck/Australia/341/83------GA---GC-C--T-------- H15N9 A/shearwater/West Australia/2576/79------C-C--GT-A--CA------- H16N3 A/black-headed gull/Sweden/2/99--G---C-T--TC-A--TA-C----- H16N3 A/herring gull/Delaware/712/88
CCIACCIKWCCIKACYAICKRCCWAC PRIMERS
AGCAAAAGCAGGGG----------------------------------------------------------------------------------A---------------
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------
GGGGTTAGCAAAAGCAGGRG5’- -3’ -5’3’-
Fusion peptide
S S-
HA1 (nt 1-1063 GenBank AB239125) HA2 (nucleotides 1064-1776)
The proposed
procedure have been designed in such a way that the first round
amplification product, obtained using degenerate deoxyinosine-
substituted primers, was shared by both the diagnostic (H1-H3-H5
multiplex PCR) and surveillance (H1-16 PCR) methods.
To develop diagnostic and surveillance molecular
tools for the rapid and reliable detection of any of the hemagglutinin
types of influenza A virus directly in clinial specimens.
To develop diagnostic and surveillance molecular
tools for the rapid and reliable detection of any of the hemagglutinin
types of influenza A virus directly in clinial specimens.
H5N1 A/Thailand/2-SP-33/2004
H 5N 1 A/chicken/Vietnam/HD1/2004
H5N1 A/Thailand/1-KAN-1/2004
H5N1 A/chicken/Thailand/Tak-01/2004
H5N1 A/chicken/Thailand/Nakornsawan
H5N1 A/chicken/Thailand/Bangkok/01
H 5N 1 A/VietNam/3046/2004
H5N1 A/Hanoi/03/2004
H5N1 A/VietNam/3062/2004
H 5N 1 A/bird/Thailand/3.1/2004
H5N1 A/whitepeafo/Thailand/CU-11/04
H5N1 A/VietNam/1194/2004
H 5N 1 A/VietNam/1203/2004
H 5N 1 A/chicken/HK/2133.1/2003
H 5N 1 A/chicken/Indonesia/4/2004
H5N1 A/chicken/Indonesia/2A/2003
H5N1 A/chicken/Guangdong/174/04
H5N1 A/duck/Novosibirsk/56/2005
H 5N 1 A/chicken/Crimea/1/2005
H5N1 A/domestic cat/Iraq/820/06
H5N1 A/chicken/Nigeria/641/2006
H 5N 1 A/mallard/Bavaria/1/2006
H5N1 A/mallard/Italy/332/2006
H 5N 1 A/chicken/Egypt/960N3-004/2006
H5N1 A/chicken/Jil in/9/2004
H 5N 1 A/duck/HongKong/2986.1/2000
H5N1 A/chicken/HongKong/31.4/02
H5N1 A/duck/Shanghai/35/2002
H5N1 A/duck/Anyang/AVL-1/2001
H5N1 A/duck/Fujian/01/2002
H5N1 A/environ/HongKong/437-10/99
H5N1 A/chicken/HongKong/317.5/2001
H 5N 1 A/swine/Shandong/2/03
H5N1 A/goose/Guangdong/1/9 6
H 5N 1 A/HongKong/481/97
H 5N 1 A/HongKong/156/97
H5N1 A/HongKong/97/98
H5N1 A/duck/HongKong/p46/97
H5N1 A/chicken/HongKong/1203/97
H 5N 1 A/HongKong/486/97
H5N2 A/chicken/Italy/312/97
H5N2 A/chicken/Italy/8/98
H5N2 A/guineafowl/Italy/330/97
H5N3 A/duck/Singapore/3/97
H 5N 9 A/chicken/Italy/9097/97
H5N1 A/turkey/England/50-92/91
H5N2 A/duck/Potsdam/1402-6/86
H5N3 A/duck/HongKong/205/77
H5N3 A/duck/HoChiMinh/14/78
H5N8 A/duck/Ireland/113/83
H5N8 A/turkey/Ireland/1378/83
H5N6 A/duck/Potsdam/2216-4/84
H 5N 2 A/turkey/England/N28/73
H5N3 A/tern/SouthAfrica/61
H5N1 A/chicken/Scotland/59
Eurasian
H 5N 2 A/chick/Pennsylvania/1/83
H 5N 2 A/chick/Penn/1370/83
H5N3 A/ruddyturnstone/NJ/2242/00
H5N2 A/duck/Michigan/80
H5N1 A/duck/Minnesota/1525/81
H5N3 A/mallard/Wisconsin/169/75
H5N9 A/turkey/Ontario/7732/66
H5N9 A/turkey/Wisconsin/68
H 5N 1 A/gull/Pennsylvania/4175/83
H 5N 2 A/chick/Mexico/31381-Avilab/94
H 5N 2 A/chicken/Taiwan/1209/03
H5N2 A/chicken/Queretaro/7653-20/95
H5N2 A/chicken/Puebla/8623-607/94
H5N3 A/chicken/TX/167280-4/02
H5N2 A/avian/NY/31588-3/00
H 5N 8 A/duck/NY/191255-59/02
H5N2 A/turkey/Minnesota/10734/95
H5N9 A/mallard/Ohio/556/1987
H 5N 2 A/emu/Texas/39442/93
American
H2N8 A/ruddy turns/Delaware/142/98
99
42
93
31
99
34
79
95
61
9 9
9 9
9 5
0.05
A/goose/Guangdong/1/96hemagglutinin-derived cluster of HPAI viruses
Primer Sequence (5’ - 3’) Gene position(GenBank reference strain)
H1 sense CAA TAT GTA TAG GCT ACC ATG C 56-77 (AY289929)H1 antisense CCC TCA ATR AAA CCR GCA AT 1064-1045 (AY289929)H3 sense ACT GCA CAC TRA TAG ATG C 236-254 (AY531033)H3 antisense CCC TCC CAA CCA TTT TCT AT 1082-1063 (AY531033)H5 sense TTC AGR AAT GTR GTR TGG 506-523 (AB239125)H5 antisense TAT RAA ICC YGC TAT WGC 1093-1076 (AB239125)
PHA2 sense WHH TIT GGG GIR TIC AYC A 597-615 (AB239125)PHA2 antisense AAI CCW GCW ATI GCI CCR AA 1089-1070 (AB239125)
Second round PCR oligonucleotide primers
HA Subtyping
pacopozo@isciii.es
A particular emphasis was put on the suitability of this method for the detection of both the Eurasian and the American lineages of H5 viruses.
Thirteen different reference strains of subtypes H5N1, H5N2, H5N3, H5N8 and H5N9 were used for the evaluation of the PCR method.
Sensitivity of the assay was estimated by testing serial tenfold dilutions of H1, H3 and H5 specific plasmids. Detection levels were 10 copies.
Phylogenetic analysis of the second amplification product nucleotide sequences of H5 viruses (Kimura 2-parameter ) would fit a particular strain into one of the two previously described lineages.
MEGA v3.1
Implementation of a Sensitive Diagnostic and Surveillance System to Detect Emerging Influenza Strains
Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos I I I , 28220 Majadahonda, Madrid
Thirteenth International ConferenceNegative Strand Viruses 2006
Sal am an ca, Sp ain , Ju n e 17th - 22n d , 2006
Immediate knowledge about human infection with the subtype H5 and then with the
currently circulating influenza A subtypes H1 and H3 would be desirable for timely
epidemiological investigations and, if possible, infection control.
Nevertheless, next pandemic might be caused by a different subtype of influenza virus. In
fact, three other avian influenza viruses (H9N2, H7N7 and H7N3) have caused illness in
humans.
As a result, diagnostic
laboratories receiving requests to test specimens from patients with an influenza-like illness
are being encouraged to the implementation of rapid and sensitive procedures not only for
typing influenza A virus but also for subtyping at least H1, H3 and H5 viruses directly from
clinical specimens.
Strategies for containing
.
Since their reemergence in late 2003, the establishment of the highly pathogenic avian
influenza (HPAI) A subtype H5N1 as an endemic virus in poultry in several countries of Asia
and the recent extensive geographical spread to Africa, Europe and the Middle East, in
addition with its repeated interspecies transmission to humans are giving this virus increased
opportunities to improve their transmissibility in humans, and thus develop into a pandemic
strain,
an emerging influenza pandemic or at
least attempting to slow down its spread worldwide will depend on several factors but the
prompt laboratory identification of the first cases would be unanswerably the starting point
being evident that H5N1 virus poses a continuing global human public health risk.
H5N9-A/mallard/Sweden/80/02 H5N6-A/mallard/Sweden/40/02
H5N2-A/duck/Mongolia/54/01 H5N1-A/Hanoi/30408/05
H5N1-A/chicken/HK/728/97 H5N9-A/chicken/Italy/9097/97
H5N8-A/turkey/Ireland/1378/83 H5N3-A/duck/Miyagi/54/76 H5N6-A/duck/Potsdam/2216-4/84 H5N3-A/tern/South Africa/61 H5N1-A/chicken/Scotland/59 H5N2-A/ck/Queretaro/7653-20/95
H5N7-A/shorebird/Delawa/75/04 H5N8-A/duck/NY/191255-59/02
H 5
H2N3-A/mallard/Alberta/226/98 H2N5-A/mallard/Alberta/202/96
H2N9-A/gull/New Jersey/75/85 H2N1-A/ruddy/Delaware/81/93 H2N2-A/pintail/Praimor/625/76
H2N2-A/Berlin/3/64 H2N2-A/Japan/305/57
H 2
H1N5-A/pintail duck/Alberta/63 H1N4-A/teal/Alberta/141/92
H1N6-A/mallard duck/Alberta/42 H1N1-A/swine/Netherlands/3/80
H1N3-A/duck/New Zealand/160/76 H1N2-A/swine/Minneso/55551/00 H1N2-A/duck/NC/91347/01 H1N1-A/swine/Wisconsin/168/97
H1N9-A/NWS-G70c/70 H1N1-A/USSR/90/77
H1N2-A/swine/England/690421/95 H1N1-A/Taiwan/603/05 H1N2-A/Yokohama/22/02 H1N1-A/New Caledonia/20/99 H1N2-A/England/2/02 H1N2-A/New York/217/02
H 1
H11N9-A/pintail/Alberta/84/00 H11N3-A/duck/Alberta/797/83
H11N2-A/mallard/Netherl/7/99 H11N6-A/duck/England/56
H 1 1
H16N3-A/gull/Sweden/2/99 H16N3-A/herring/Delawar/712/88 H 1 6 H13N2-A/whale/Maine/328 HN/84
H13N9-A/shorebird/DE/68/04 H13N8-A/gull/Netherlands/1/00 H13N6-A/gull/Astrakan/227/84
H 1 3
H6N8-A/mallard/Alberta/761/78 H6N1-A/chicken/Hong Kong/17/77
H6N5-A/duck/Australia/4045/80 H6N2-A/turkey/Canada/63 H6N4-A/shorebird/DE/194/98
H6N7-A/goose/HK/W222/97 H6N2-A/ck/California/905/01
H6N9-A/duck/HK/182/77 H6N3-A/mallard/Alberta/253/90 H6N6-A/widgeon/Alberta/256/82
H 6
H8N4-A/mallard/Alberta/283/77 H8N4-A/mallard/Alberta/194/92 H 8
H12N5-A/teal/Alberta/199/91 H12N4-A/ruddy/DE/67/98
H12N5-A/duck/Alberta/60/76 H12N1-A/mallard/Alberta/342/83
H 1 2
H9N2-A/turkey/Wisconsin/66 H9N2-A/chicken/Shandong/6/96 H9N1-A/duck/Shantou/1588/00 H9N3-A/duck/Vietnam/340/01
H9N6-A/duck/Hong Kong/147/77 H9N1-A/mallard/Alberta/506/83 H9N3-A/mallard/Alberta/11/91 H9N5-A/ruddy/Delaware/2576/87 H9N6-A/ruddy/Delaware/510/88
H 9
H4N8-A/pintail/Alberta/207/99 H4N1-A/mallard/Alberta/47/98 H4N5-A/teal/Alberta/103/90 H4N2-A/mallard/Alberta/630/84 H4N6-A/swine/Ontari/01911-1/99
H4N3-A/mallard/Alberta/300/77 H4N5-A/seal/MA/133/82
H4N6-A/duck/New Zealand/31/76
H 4
H14-A/mallard/Gurjev/263/82 H14-A/mallard/Gurjev/244/82 H 1 4
H3N8-A/equine/Romania/80 H3N8-A/equine/Uruguay/1/63 H3N8-A/canine/Iowa/13628/05
H3N3-A/seal/MA/3911/92 H3N8-A/mallard/Alberta/117/97 H3N6-A/chick/Nanchang/7-010/00 H3N2-A/swine/Spain/39139/02 H3N2-A/New York/357/05
H3N1-A/swine/Taiwan/0408/04 H3N2-A/England/72 H3N2-A/Hong Kong/1/68
H 3
H10N1-A/pintail/Alberta/129/93 H10N6-A/duck/Alberta/40/84 H10N3-A/teal/Alberta/778/78
H10N7-A/chicken/Germany/N/49 H10N4-A/mink/Sweden/84
H 1 0
H15N8-A/duck/Australia/341/83 H15N9-A/shearwat/Austr/2576/79 H 1 5 H7N2-A/chicken/NJ/15814-9/99
H7N3-A/ck/British Col/GSC/04 H7N7-A/eq/Cambridge/1/63
H7N7-A/FPV/Dutch/27 H7N4-A/chicken/NSW/1/97 H7N7-A/avian/Netherl/127/03
H7N1-A/ostrich/Zimbabwe/222/96 H7N3-A/turkey/England/63
H 7
100
100
100
100
100
100
100
99
100
100
100
100
100
100
99
99
100
100
89
100
100
99
100
100
59
92
99
99
100
80
61
74
100
47
55
99 99
100
100
0.1
Strains used for the evaluation of the multiplex PCR method
subtype strain kindly provided byH5N1 A/Hong Kong/486/97
A/Vietnam/1194/04A/Vietnam/1203/04A/chicken/Scotland/59A/chicken/Cambodia/7/04
CDC (USA)MRC (UK)MRC (UK)PASTEUR (FRANCE)PASTEUR (FRANCE)
H5N2 A/turkey/England/N28/73A/chicken/Italy/8/98A/chucker/Minnesota/14591-7/98
CISA (SPAIN)HIPRA (SPAIN)CDC (USA)
H5N3 A/tern/South Africa/61A/duck/Singapore/3/97A/turkey/California/6878/79
ISCIII (SPAIN)MRC (UK)CDC (USA)
H5N8 A/turkey/Wisconsin/68 CDC (USA)
H5N9 A/ratite/New York/12716/94 CDC (USA)
Primers of the second round PCR were selected from highly conserved regions of each of the hemagglutinin gene segments using multiple alignments built with available sequences obtained from databases, and were designed to render amplification products of different size for subtypes H1 (1009 bp), H3 (847 bp), H5 (591 bp). An internal control, consisting of 100 molecules of a cloned DNA fragment, was included in the extraction buffer to detect false negative results.
847 bp591 bp
350 bp IC
H5H3
1009 bp H1
104103102 10 1 104103102 10 1 104103 102 10 1MW
Serial 10-f old dilutions of H1, H3 and H5 specifi c plasmids
p
A second round PCR assay using degenerate deoxyinosine-substituted rimers was developed for reliable detection of any of the 16 types of hemagglutinin in clinical specimens.
As for the first round PCR, primers (PHA2 sense and antisense) were selected from very highly conserved regions evidenced by the alignment of multiple sequences very different each other in terms of not only hemagglutinin type but also host, country and isolation date of the virus.
Direct sequencing of the second amplification product, fragments 450-500 base pairs in length, and s
Nucleotide sequences were aligned using the program Clustal X v1.81. Phylogenetic tree was infered using the Kimura 2-parameter algorithm included in the program MEGA v3.1 and reconstructed by the Neighbour-Joining method. Statistical significance was evaluated by bootstrap resampling of sequences 1,000 times.
ubsequent phylogenetic analysis of nucleotide sequences enabled hemagglutinin classification.
nt 14nt 1 nt 1100 nt 1125
GGATGGACAGGAATGATAGATGGATG H1N1 A/swine/Wisconsin/168/97-----------G-----------G-- H1N1 A/swine/Netherlands/3/80------CA-------G-T-----T-- H2N2 A/pintail/Praimoric/625/76------CA-------G-T-----T-- H2N2 A/Berlin/3/64------GA---------------T-- H3N3 A/seal/MA/3911/92--C---GAG------G-T-----G-- H3N8 A/equine/Romania/80--C---CA---TC----T-----T-- H4N6 A/swine/Ontario/01911-1/99--C---CA---CC-A--T-----G-- H4N6 A/duck/New Zealand/31/76------CAG------G----C--T-- H5N8 A/turkey/Ireland/1378/83------CAG------G-------T-- H5N1 A/Hanoi/30408/05-----------C-------------- H6N8 A/mallard duck/Alberta/761/78--------T--------------G-- H6N1 A/chicken/Hong Kong/17/77------GA---TC----T-------- H7N1 A/ostrich/Zimbabwe/222/96------GA---TC--G-T-------- H7N3 A/turkey/England/63------T-T--G-----T-----G-- H8N4 A/mallard duck/Alberta/283/77------T-T--G-----T-------- H8N4 A/mallard/Alberta/194/92--T---C-----C--G-T-C------ H9N5 A/ruddy turnstone/Delaware/2576/87--T---T----GC-AG-C-C---T-- H9N2 A/chicken/Shandong/6/96------GA-------G----C--C-- H10N7 A/chicken/Germany/N/49------GA-------G----C--C-- H10N4 A/mink/Sweden/84------C----TT-A--CA----T-- H11N9 A/pintail/Alberta/84/00--G---C----GC-T--CA------- H11N2 A/mallard/Netherlands/7/99------C----GC-AG-G-C---T-- H12N1 A/mallard duck/Alberta/342/83------C-----C-AG-G-C---T-- H12N5 A/green-winged teal/Alberta/199/91--T---C-----T-A---A----T-- H13N8 A/black-headed gull/Netherlands/1/00--T---C----GT-A---A----T-- H13N2 A/pilot whale/Maine/328 HN/84--T---CA---CC----T-----G-- H14N5 A/mallard/Gurjev/263/82--T---CA---CC----T-----G-- H14N6 A/mallard/Gurjev/244/82------GA----C-C--T-------- H15N8 A/duck/Australia/341/83------GA---GC-C--T-------- H15N9 A/shearwater/West Australia/2576/79------C-C--GT-A--CA------- H16N3 A/black-headed gull/Sweden/2/99--G---C-T--TC-A--TA-C----- H16N3 A/herring gull/Delaware/712/88
CCIACCIKWCCIKACYAICKRCCWAC PRIMERS
AGCAAAAGCAGGGG----------------------------------------------------------------------------------A--------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------
GGGGTTAGCAAAAGCAGGRG5’- -3’ -5’3’-
Fusion pep tide
S S-
HA1 ( n t 1 - 1 0 6 3 G e n B a n k A B 2 3 9 1 2 5 ) HA2 ( n u c l e o t i d e s 1 0 6 4 - 1 7 7 6 )
The proposed procedure have been designed in such a way that the first round amplification product, obtained using degenerate deoxyinosine-substituted primers, was shared by both the diagnostic (H1-H3-H5 multiplex PCR) and surveillance (H1-16 PCR) methods.
To develop diagnostic and surveillance molecular tools for the rapid and reliable detection of any of the hemagglutinin types of influenza A virus directly in clinial specimens.
To develop diagnostic and surveillance molecular tools for the rapid and reliable detection of any of the hemagglutinin types of influenza A virus directly in clinial specimens.
H5N1 A/Thailand/2-SP-33/2004 H5N1 A/chicken/Vietnam/HD1/2004H5N1 A/Thailand/1-KAN-1/2004 H5N1 A/chicken/Thailand/Tak-01/2004 H5N1 A/chicken/Thailand/NakornsawanH5N1 A/chicken/Thailand/Bangkok/01H5N1 A/VietNam/3046/2004H5N1 A/Hanoi/03/2004 H5N1 A/VietNam/3062/2004 H5N1 A/bird/Thailand/3.1/2004 H5N1 A/whitepeafo/Thailand/CU-11/04H5N1 A/VietNam/1194/2004 H5N1 A/VietNam/1203/2004 H5N1 A/chicken/HK/2133.1/2003H5N1 A/chicken/Indonesia/4/2004 H5N1 A/chicken/Indonesia/2A/2003 H5N1 A/chicken/Guangdong/174/04 H5N1 A/duck/Novosibirsk/56/2005H5N1 A/chicken/Crimea/1/2005 H5N1 A/domestic cat/Iraq/820/06 H5N1 A/chicken/Nigeria/641/2006H5N1 A/mallard/Bavaria/1/2006 H5N1 A/mallard/Italy/332/2006 H5N1 A/chicken/Egypt/960N3-004/2006H5N1 A/chicken/Jilin/9/2004 H5N1 A/duck/HongKong/2986.1/2000 H5N1 A/chicken/HongKong/31.4/02H5N1 A/duck/Shanghai/35/2002 H5N1 A/duck/Anyang/AVL-1/2001H5N1 A/duck/Fujian/01/2002 H5N1 A/environ/HongKong/437-10/99 H5N1 A/chicken/HongKong/317.5/2001H5N1 A/swine/Shandong/2/03 H5N1 A/goose/Guangdong/1/96H5N1 A/HongKong/481/97H5N1 A/HongKong/156/97 H5N1 A/HongKong/97/98 H5N1 A/duck/HongKong/p46/97 H5N1 A/chicken/HongKong/1203/97H5N1 A/HongKong/486/97H5N2 A/chicken/Italy/312/97 H5N2 A/chicken/Italy/8/98 H5N2 A/guineafowl/Italy/330/97H5N3 A/duck/Singapore/3/97H5N9 A/chicken/Italy/9097/97 H5N1 A/turkey/England/50-92/91H5N2 A/duck/Potsdam/1402-6/86 H5N3 A/duck/HongKong/205/77H5N3 A/duck/HoChiMinh/14/78 H5N8 A/duck/Ireland/113/83 H5N8 A/turkey/Ireland/1378/83 H5N6 A/duck/Potsdam/2216-4/84 H5N2 A/turkey/England/N28/73 H5N3 A/tern/SouthAfrica/61 H5N1 A/chicken/Scotland/59
Eurasian
H5N2 A/chick/Pennsylvania/1/83H5N2 A/chick/Penn/1370/83H5N3 A/ruddyturnstone/NJ/2242/00 H5N2 A/duck/Michigan/80 H5N1 A/duck/Minnesota/1525/81H5N3 A/mallard/Wisconsin/169/75 H5N9 A/turkey/Ontario/7732/66H5N9 A/turkey/Wisconsin/68 H5N1 A/gull/Pennsylvania/4175/83 H5N2 A/chick/Mexico/31381-Avilab/94H5N2 A/chicken/Taiwan/1209/03 H5N2 A/chicken/Queretaro/7653-20/95H5N2 A/chicken/Puebla/8623-607/94H5N3 A/chicken/TX/167280-4/02H5N2 A/avian/NY/31588-3/00 H5N8 A/duck/NY/191255-59/02 H5N2 A/turkey/Minnesota/10734/95H5N9 A/mallard/Ohio/556/1987 H5N2 A/emu/Texas/39442/93
American
H2N8 A/ruddy turns/Delaware/142/98
99
42
93
31
99
34
79
95
61
99
99
95
0.05
A/goose/Guangdong/1/96hemagglutinin-derived cluster of HPAI viruses
Primer Sequence (5’ - 3’) Gene position(GenBankreferencestrain)
H1 sense CAA TAT GTA TAG GCT ACC ATG C 56-77 (AY289929)H1 antisense CCC TCA ATR AAA CCR GCA AT 1064-1045 (AY289929)H3 sense ACT GCA CAC TRA TAG ATG C 236-254 (AY531033)H3 antisense CCC TCC CAA CCA TTT TCT AT 1082-1063 (AY531033)H5 sense TTC AGR AAT GTR GTR TGG 506-523 (AB239125)H5 antisense TAT RAA ICC YGC TAT WGC 1093-1076 (AB239125)
PHA2 sense WHH TIT GGG GIR TIC AYC A 597-615 (AB239125)PHA2 antisense AAI CCW GCW ATI GCI CCR AA 1089-1070 (AB239125)
Second round PCR oligonucleot ide primers
1009bp H1
847bp H3
591bp H5
350bp IC
Fusion peptideN-terminal signal
RT+common 1st amplification
H5H5
H1H1H3H3
H1 H1
RT-nested PCR genérica para subtipar gripe AGen HA1
H1
H2
H3H4
H5H6
H7
H8
H9
H10
H11
H12
H13
H14
H15
H16
H1N1-A/Baleares/R3205/08
H1N1-A/Baleares/R3210/08
H1N1-A/Aragon/R3207/08
H1N1-A/PaisVasco/R3174/08
H1N1-A/Brisbane/59/2007
H1N1-A/Norway/1630/07
H1N1-A/Taiwan/603/05
H1N1-A/Hawaii/01/07
H1N1-A/Norway/2159/06
H1N1-A/SolomonIslands/3/06
H1N1-A/Singapore/19/06
H1N1-A/Texas/02/07
H1N1-A/New Caledonia/20/99
H1N2-A/Yokohama/22/02
H1N2-A/England/2/02
H1N2-A/New York/217/02
A/swine/Ontario/55383/04 H1N2
H1N1-A/USSR/90/77
H1N2-A/swine/England/690421/95
H1N9-A/NWS-G70c/70
A/swine/Ontario/11112/04 H1N1
H1N2-A/duck/NC/91347/01
A/swine/NorthCarolina/36883/02
H1N2-A/swine/Minneso/55551/00
H1N1-A/swine/Wisconsin/168/97
A/swine/Kansas/00246/04 H1N2
A/swine/Ohio/24366/07 H1N1
A/swine/Ohio/C62006/06 H1N1
A/Swine/Indiana/P12439/00 H1N2
A/Swine/Ohio/891/01 H1N2
A/Swine/Indiana/9K035/99 H1N2
A/swine/Minnesota/00194/03 H1N
H1N3-A/duck/New Zealand/160/76
H1N5-A/pintail duck/Alberta/63
H1N6-A/mallard duck/Alberta/42
H1N4-A/teal/Alberta/141/92
H1N1-A/swine/Netherlands/3/80
H1N1-A/swine/Netherlands/609/9
H1N1-A/swine/Belgium/1/98
H1N1-A/swine/Italy/110971/01
H1N1-A/swine/Spain/53207/04
H1N1-A/Switzerland/8808/02
H1N1-A/Aragon/R3218/08 H1N1-A/swine/Spain/50047/03
100
100
96
100
100
99
100
100
100
100
100
100
100
100
96
100
83
99
99
100
99
100
75
96
38
68
24
27
35
46
82
90
74
85
99
62
99
62
100
99
35
44
54
49
100
68
88
57
60
100
100
77
100
56
94
93
94
52
69
56
87
51
100
90
79
9857
7050
44
82
0.1
Human seasonal
Classical sw lineage
Euroasiaticsw lineage
450-500 bp
105
H2O
SM 104 103 102 10 1
Generic amplification of each 16 sybtypes HA
RT-nested PCR genérica para detección de los 16 subtipos de HA Gen HA1
H1H3H5
Inf A subtyping
Multiplex RESP Multiplex BRQ
Influenza ABC NP
Primer caso gripe AH1N1v en Europa Almansa (Castilla La Mancha, Spain): GP1-GP2
Detección + secuenciación del fragmento amplificado + análisis de la seq
Inicio del brote: Casos estudiados y confirmados en el Centro Nacional de Gripe (CNM, ISCIII)
Semana FechasCasos
EstudioCasos
PositivosOtros virus Gripales
encontrados
17 26 Abril- 2-Mayo 196 80 10 FluB; 3 FluA H1N1; 1 FluA H3N2
18 3-Mayo- 9 Mayo 70 29 3 FluB; 1 AH1N1
19 10 Mayo- 16 Mayo 13 3 3 FluB
20 17 Mayo- 23 Mayo 47 26 1 FluB; 1 FluA H1N1; 1 FluA H3N2
21 24 Mayo- 30 Mayo 81 43 2 FluA H3N2
22 31 Mayo- 6 Junio 154 142
23 7 Junio- 13Junio 183 152
TOTAL 744 37517 FluB; 5 FluAH1N1;
4 FluH3N2
1
0
50
100
150
200
250
40 42 44 46 48 50 52 2 4 6 8 10 12 14 16 18 20
Semanas
Nº
de
tec
cio
ne
s
BA(H1N1)A(H1)A(H3N2)A(H3)A (no subtipado)
Semana 1726 Abril- 2-Mayo
Sistema de Vigilancia de la gripe en España Temporada 2008-2009
17 18 19 20 21 22 23
Cronología de los casos según el comienzo de los síntomas
0
50
100
150
200
250
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 2 4 6 8 10 12 14 16 18 20Semanas
Cas
os
gri
pe
/100
.000
h.
0
20
40
60
80
100
% d
etec
cio
nes
vir
ales
p
osi
tiva
s
Porcentaje detecciones positivas Tasa 2008-2009
Tasa 2008-09 (a partir de semana 20/2009) Tasa 2009-2010 (a partir de semana 40/2009)
Umbral basal
Junio Julio Ago Sept Oct
Tasa de incidencia semanal de gripe y porcentaje de detecciones virales positivas.
Temporada 2009-2010. Sistemas centinela
Nula
Esporádica
Local
Situación Epidémica
Tasa de detección viral (%) y número de detecciones virales centinela. Temporada 2009-2010
63 HA1 88 NA97 M1+M2 39 NP 81 NS
26 Abril a 5 Junio
Pacientes positivos secuenciados: 157 / 365
Análisis de cada segmento secuenciado A(H1N1)v
Hemaglutinina H1 63 casos positivos
Linaje Eurasiático
A/Madrid/289/GP578/09 military A/Madrid/378/GP781/09 military A/Madrid/292/GP587/09 military A/Madrid/294/GP593/09 military A/Madrid/340/GP727/09 military A/Madrid/296/GP599/09 military A/Madrid/299/GP608/09 military A/Madrid/385/GP796/09 military A/Madrid/295/GP596/09 military A/Madrid/293/GP590/09 military A/Madrid/290/GP581/09 military A/Madrid/390/GP805/09 military A/Madrid/291/GP584/09 military A/Madrid/360/GP752/09 military A/Madrid/297/GP602/09 military A/Madrid/298/GP605/09 military A/Madrid/381/GP786/09 military A/Madrid/391/GP807/09 military A/Valencia/78/GP155/09
A/Murcia/43/GP78/09 A/Catalua/66/GP145/09 A/Catalua/9/GP25/09 A/Andalucia/126/GP242/09 A/CastillaLaMancha/129/GP248/09 A/CastillaLaMancha/1/GP12/09 A/Valencia/77/GP160/09
A/CastillaLaMancha/95/GP181/09 A/CastillaLaMancha/4/GP7/09 A/Valencia/82/GP158/09 A/Valencia/2/GP3/09 A/Andalucia/117/GP230/09 A/Valencia/264/GP494/09 A/Baleares/265/GP500/09 A/Catalua/69/GP148/09 A/Valencia/79/GP161/09 A/Andalucia/167/GP317/09 A/Madrid/37/GP65/09 A/Andalucia/119/GP234/09 A/Madrid/36/GP62/09 A/Andalucia/100/GP201/09 A/Andalucia/118/GP232/09 A/Andalucia/255/GP490/09 A/Madrid/111/GP216/09 A/Catalua/13/GP29/09 A/Catalua/19/GP35/09 A/Andalucia/130/GP251/09 A/Catalua/8/GP24/09 A/Catalua/121/GP237/09 A/Catalua/123/GP239/09 A/Catalua/15/GP31/09 A/Catalua/68/GP147/09 A/Valencia/73/GP154/09 A/Catalua/25/GP41/09 A/Valencia/72/GP153/09 A/Catalua/122/GP238/09 A/Catalua/120/GP236/09 A/Andalucia/71/GP151/09 A/Catalua/65/GP144/09 A/Catalua/63/GP142/09 A/Madrid/308/GP636/09 A/Madrid/288/GP575/09 A/Madrid/305/GP628/09 A/PaisVasco/5/GP19/09
A/Swine/Indiana/P12439/00 H1N2
A/swine/Kansas/00246/04 H1N2
A/swine/Minnesota/00194/03 H1N2
A/Swine/Ohio/891/01 H1N2
A/Swine/Indiana/9K035/99 H1N2
A/swine/Ohio/24366/07 H1N1
A/swine/Ohio/24366/07(H1N1)
A/swine/Ohio/C62006/06 H1N1
A/swine/Ohio/C62006/06(H1N1)
A/swine/NorthCarolina/36883/02 H1N1
A/swine/Korea/CAS08/2005(H1N1)
A/swine/Shanghai/3/2005(H1N1)
A/swine/Shanghai/2/2005(H1N1)
A/swine/Shanghai/1/2005(H1N1)
A/swine/Maryland/23239/1991(H1N1)
A/swine/Memphis/1/1990(H1N1)
A/swine/California/T9001707/1991(H1N1)
A/swine/Ratchaburi/NIAH550/2003(H1N1)
A/swine/Iowa/1/1986(H1N1)
A/swine/Kansas/3024/1987(H1N1)
A/swine/Ontario/11112/04 H1N1
A/swine/Tennessee/4/1978(H1N1)
A/swine/Tennessee/2/1978(H1N1)
A/swine/Kyoto/3/1979(H1N1)
A/swine/Wisconsin/30954/1976(H1N1)
A/swine/Tennessee/19/1976(H1N1)
A/swine/Tennessee/107/1977(H1N1)
A/swine/Tennessee/109/1977(H1N1)
A/swine/Tennessee/9/1978(H1N1)
A/swine/Thailand/HF6/2005(H1N1)
A/swine/Chonburi/NIAH977/2004(H1N1)
A/swine/Tennessee/7/1978(H1N1)
A/swine/Ohio/K1207/06(H1N1)
A/swine/Ohio/K1130/06(H1N1)
A/swine/Ontario/55383/04 H1N2
A/Nyiregyhaza/01/2007(H1N1)
A/swine/Tianjin/01/04(H1N1)
A/swine/Henan/01/06(H1N1)
A/swine/France/WVL8/1992(H1N1)
A/swine/Hokkaido/2/1981(H1N1)
A/swine/England/WVL7/1992(H1N1)
A/swine/Spain/WVL6/1991(H1N1)
A/swine/Hungary/19774/2006(H1N1)
A/Aragon/Spain/RR3218/2008(H1N1) A/swine/Haseluenne/IDT2617/03(H1N1)
A(H1N1)v
Linaje de la gripe clásica porcina
100
100
81
69
93
96
85
100
74
52
100
100
100
100
55
99
52
100
37
50
31
20
81
100
76
57
18
39
100
44
82
51
94
87
98
98
100
57
57
48
84
100
62
46
86
62
61
54
38
55
61
42
100
29
5
26
18
14
6
35
0.05
cambio de AA A/California/07/2009A/California/07/2009
vsvsCepas españolasCepas españolas
L58IP109S (todas)T216AD291EI338V
Virus contienen sustituciones de aa en lugares antigénicos concretos cuando se compara con las cepas H1 estacionales
Ensayos antigénicos IHA
No hay resistencias a oseltamivir H274, E119, N294
Neuraminidasa N188 casos positivos
Linaje de la gripe clásica porcina
H1 humana epidémica
Linaje Eurasiático
S247NN248DV338IS340F
cambios de AA A/California/07/2009
vsCepas españolas
No se han encontrado cambios genéticosconocidos que puedan disminuir la sensibilidad a INA
M2 presenta S31NResistencia a adamantanos
presente en todas las secuencias
Marcador molecular del linaje Eurasiático
M1T185MS224NV147M
M
Matrix97 casos positivos
M gen, codifica 2 proteinas: M1 y M2Determinante del tropismo a un determinado hospedador
cambios de AA A/California/06/2009
vsCepas españolas
Human seasonal
Euroasian sw lineage
Classical sw lineage
NP PA T373I M582L not available at momment
NP NA V100I V106I
N248D
NP NA HA1 NS1 V100I V106I S206T I123V
N248D
HA1 PA S91P S224P V323I
Mutaciones pareadas
1
2
3
4
Garten et al. Science. 22 May 2009 NP T373ICastillaLaMancha/GP369Andalucia/GP381Cataluña/GP144Cataluña/GP145Valencia/GP154Andalucia/GP196Andalucia/GP201Cataluña/GP224Andalucia/GP234Cataluña/GP237Cataluña/GP236Andalucia/GP251Valencia/GP280Madrid/GP62PaisVasco/GP20Valencia/GP4
NP NAV100I N248DMadrid/GP523Andalucia/GP527Madrid/GP216Valencia/GP272Valencia/GP278
NP NA HA1 NS1V100I N248D S206T I123V
Madrid/GP523
HAS91P
Todas las cepas españolas
Análisis de cada segmento secuenciado A(H1N1)v
Marcadores moleculares de la adaptación al hospedador• 1918 H1H1• Virus H5N1 altamente patogénico
H1N1v Avian swClassswEurop Human
1918/H5N1
PB2 627 E E E E K
PB1-F2 12 Truncated Compl
NS1 220 Truncated Compl-PDZ25 N Q N,K,R,W R,W*,L Q66 E E E K* E227 - E R,G E,G* R,del
NS2 26 E E E K* E49 V V V L* V52 M M M T* M70 G S G G* G
M1 214 H Q Q H* Q
NP 284 A A A V,I* A384 R R R K* R
* A/Aragon/R3218/Spain/08
Dunham et al. J Virol June 2009
Análisis de cada segmento secuenciado A(H1N1)v
Virus nH1N1 crecido en células MDCKServicio de Microscopia Electrónica, CNM, ISCIII