aspectos virológicos y epidemiológicos de los nuevos virus a(h1n1)v en españa

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

[email protected]

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


H6N7-A/goose/HK/W222/97

H6N2-A/ck /California/905/01

H6N9 -A/duck/HK/182/77


H6N6-A/widgeon/Alberta/256/82

H6


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


H9N3 -A/ma llard/Alberta/11/91

H9N5-A/rudd y/Delaware/2576/87


H9

H4N8-A/pintail/Alberta/2 07/ 99

H4N1-A/malla rd/Alberta/47/98

H4N5-A/teal/Alb erta/103/90


H4N6-A/swine/Ontari/01911-1/99


H4N5-A/seal/MA/133/82


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


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


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.




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


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


H 5N 1 A/swine/Shandong/2/03

H5N1 A/goose/Guangdong/1/9 6

H 5N 1 A/HongKong/481/97


H5N1 A/HongKong/97/98

H5N1 A/duck/HongKong/p46/97

H5N1 A/chicken/HongKong/1203/97


H5N2 A/chicken/Italy/312/97


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


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

[email protected]

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


Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos I I I , 28220 Majadahonda, Madrid


Sal am an ca, Sp ain , Ju n e 17th - 22n d , 2006






humans.





clinical specimens.


.






strain,





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


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


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)



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



AGCAAAAGCAGGGG----------------------------------------------------------------------------------A--------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------


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.


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


99

42

93

31

99

34

79

95

61

99

99

95

0.05


Primer Sequence (5’ - 3’) Gene position(GenBankreferencestrain)



Second round PCR oligonucleot ide primers

1009bp H1

847bp H3

591bp H5

350bp IC

Fusion peptideN-terminal signal

RT+common 1st amplification

H5

H1H3

[email protected]

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


Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos III, 28220 Majadahonda, Madrid


Salamanca, Spain, June 17th - 22nd, 2006






humans.





clinical specimens.


.






strain,





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



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


H1N2-A/swin e/Minneso/55551/00

H1N2-A/duck/NC/91347/0 1


H1N9-A/NWS-G70c /70

H1N1-A/USSR/90/77



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


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


H6N7-A/goose/HK/W222/97

H6N2-A/ck /California/905/01

H6N9 -A/duck/HK/182/77


H6N6-A/widgeon/Alberta/256/82

H6


H8N4-A/malla rd/Alberta/ 194 /92 H8 H12 N5-A/teal/Alberta /199/91

H12N4-A/ruddy/DE/67/98



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


H9N3 -A/ma llard/Alberta/11/91



H9

H4N8-A/pintail/Alberta/2 07/ 99

H4N1-A/malla rd/Alberta/47/98

H4N5-A/teal/Alb erta/103/90


H4N6-A/swine/Ontari/01911-1/99


H4N5-A/seal/MA/133/82


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


H3N6-A/chick/Nan chang/7-010/00

H3N2 -A/swine/Sp ain/39139/02


H3N1-A/swine/Taiwan/0408/04

H3N2-A/England/72

H3N2 -A/Hong Kong /1/68

H3

H10N1-A/pintail/A lberta/129/93



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


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)



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



AGCAAAAGCAGGGG----------------------------------------------------------------------------------A---------------

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------


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.







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


H 5N 1 A/bird/Thailand/3.1/2004

H5N1 A/whitepeafo/Thailand/CU-11/04


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/duck/Shanghai/35/2002

H5N1 A/duck/Anyang/AVL-1/2001

H5N1 A/duck/Fujian/01/2002

H5N1 A/environ/HongKong/437-10/99


H 5N 1 A/swine/Shandong/2/03

H5N1 A/goose/Guangdong/1/9 6



H5N1 A/HongKong/97/98

H5N1 A/duck/HongKong/p46/97

H5N1 A/chicken/HongKong/1203/97




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


H5N3 A/duck/HongKong/205/77

H5N3 A/duck/HoChiMinh/14/78

H5N8 A/duck/Ireland/113/83

H5N8 A/turkey/Ireland/1378/83


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


99

42

93

31

99

34

79

95

61

9 9

9 9

9 5

0.05


Primer Sequence (5’ - 3’) Gene position(GenBank reference strain)



Second round PCR oligonucleotide primers

HA Subtyping

[email protected]

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


Pozo, Francisco; Casas, Inmaculada; Ruiz, Guillermo and Pérez-Breña, PilarRespiratory Viruses Laboratory. Instituto de Salud Carlos I I I , 28220 Majadahonda, Madrid


Sal am an ca, Sp ain , Ju n e 17th - 22n d , 2006






humans.





clinical specimens.


.






strain,





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


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


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)



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



AGCAAAAGCAGGGG----------------------------------------------------------------------------------A--------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..............--------------------------------------------------------


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.



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


99

42

93

31

99

34

79

95

61

99

99

95

0.05


Primer Sequence (5’ - 3’) Gene position(GenBankreferencestrain)



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/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


A/swine/Ontario/55383/04 H1N2

H1N1-A/USSR/90/77


H1N9-A/NWS-G70c/70


H1N2-A/duck/NC/91347/01

A/swine/NorthCarolina/36883/02

H1N2-A/swine/Minneso/55551/00


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


H1N5-A/pintail duck/Alberta/63

H1N6-A/mallard duck/Alberta/42


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/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/Tennessee/4/1978(H1N1)


A/swine/Kyoto/3/1979(H1N1)

A/swine/Wisconsin/30954/1976(H1N1)





A/swine/Thailand/HF6/2005(H1N1)

A/swine/Chonburi/NIAH977/2004(H1N1)


A/swine/Ohio/K1207/06(H1N1)

A/swine/Ohio/K1130/06(H1N1)


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


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


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