Final Total - HUG

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Influenza surveillance in Switzerland Sentinella study

Winter Season 2004 – 2005

National Centre of Influenza

Central Laboratory of Virology University Hospitals of Geneva

Geneva, Switzerland

FEDERAL OFFICE OF PUBLIC HEALTH BERNE - SWITZERLAND

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National Centre of Influenza

Central Laboratory of Virology, University Hospital of Geneva 24, rue Micheli-du-Crest, 1211 GENEVA 14 – SWITZERLAND

Dr Yves THOMAS & : +41/22 372 40 81 Fax: +41/22 372 40 88 , : [email protected]

Dr Laurent KAISER & : +41/22 372 40 96 , : [email protected]

Dr Werner WUNDERLI & : +41/22 372 40 86

, : [email protected]

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Table of content 1. Acknowledgements .....................................................................................................................4 2. Summary - Résumé - Zusammenfassung...................................................................................5

2.1. Summary...............................................................................................................................5 2.2. Résumé..................................................................................................................................6 2.3. Zusammenfassung................................................................................................................7

3.1. Introduction...............................................................................................................................8 4. Method of detection for respiratory viruses...............................................................................8

4.1. Clinical identification of influenza cases............................................................................8 4.2. Detection of respiratory viruses...........................................................................................9 4.3. Characterization of influenza viruses................................................................................11 4.4. Identification of influenza A (H5N1) virus ......................................................................12

5. Results ........................................................................................................................................15 5.1. Viral detection ....................................................................................................................15 5.2. Characterization of the influenza viruses..........................................................................18

5.2.1. Influenza A (H3N2) ....................................................................................................18 5.2.2. Influenza A (H1N1) ....................................................................................................22 5.2.3. Influenza B ..................................................................................................................24

5.3. Patients with influenza infection .......................................................................................25 5.3.1. Frequency of viruses detected in a particular age group ..........................................25 5.3.2. Clinical data of patients infected with influenza.......................................................26

5.4. Evolution of the epidemic in Europe.................................................................................28 5.5. Influenza A/H2N2 ..............................................................................................................30 5.6. Avian influenza...................................................................................................................31

5.6.1. Avian influenza epidemics in South East Asia, 2004-2005 .....................................31 5.6.2. Detection of avian influenza viruses in Switzerland.................................................32

6. Recommended composition of the 2004/05 influenza vaccine ..............................................36 7. Discussion ..................................................................................................................................37 8. Bibliography...............................................................................................................................41 Annexe 1.........................................................................................................................................43 Annexe 2 : Detection of respiratory viruses during the 2004/05 season....................................44 Annexe 3 : Antigenic analysis of influenza A (H3N2)-like strains............................................45 Annexe 4 : Antigenic analysis of influenza A (H1N1)-like strains............................................51 Annexe 5 : Antigenic analysis of influenza B-like strains ..........................................................52 Annexe 6 : Alignement de l’hemagglutinine de souches récentes influenza A (H5N1) ..........54

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1. Acknowledgements

We would like to thank all members of the Sentinella network, in particular the

practitioners who actively participated in the identification of patients, provided data,

and took samples. This report is the result of a close collaboration with the Swiss

Federal Office of Public Health and we should like to especially thank Dr Reto

Hagmann for his continued support and active participation.

Our activity is equally dependent upon a collaboration with other international

centres, notably the European Influenza Surveillance Scheme (EISS) network and

the World Health Organization (WHO), and we should like to thank Dr Alan Hay, Dr

Lin Yi Pu and Mrs Vicki Gregory, members of the WHO reference centre in London

(MRC, UK). Certain developments have been carried out in close collaboration with

Dr Olav Hungnes, Oslo, Norway. Finally, we would like to thank all members of the

Central Laboratory of Virology, University Hospitals of Geneva, in particular, Sabine

Nobs-Grunenwald for her valuable work and without whom this report would not have

been possible.

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2. Summary - Résumé - Zusammenfassung

2.1. Summary

The 2004 -2005 influenza epidemic was of moderate intensity and characterized by a

rather late onset in the season. Our surveillance activity began in September 2004

and terminated at the end of April 2005 after a period of 31 weeks. The first sporadic

cases were identified in December and the detection peak was reached at week 5 in

2005 with an epidemic peak which stretched over 7 to 8 weeks until mid-March 2005.

Eight hundred and thirty-five samples were analysed, all in patients presenting with

an influenza syndrome. Of these, 345 (41%) were positive for a respiratory virus by

cell culture. The overwhelming majority (90%) was an influenza virus in the form of

type A for 268 (87%) cases, and type B in 41 (13%). Among these influenza viruses,

73% were of subtype A (H3N2) and 11%, A (H1N1). Characterization of influenza A

viruses by hemagglutination inhibition assay showed that these viruses were

antigenically related to the influenza reference strain A/Shantou/1219/2004 (H3N2),

itself very closely related to the A/California/7/2004 (H3N2) strain. These strains are

recently-appeared variants which can be distinguished antigenically from the 2004-

2005 vaccine strain A/Fujian/411/2002 (H3N2) vaccine strain. The influenza A virus

(H1N1) which predominated this year was antigenically close to the 2004-2005

vaccine strain A/New Caledonia/20/99 (H1N1). The influenza B strains detected

during this season belonged to two groups. Forty-four percent were antigenically

close to the influenza B strain included in the 2002-2003 season vaccine, and 46%

were antigenically close to the strain included in the 2004-2005 vaccine. Finally, three

influenza B strains showed a decrease of antigenic recognition with the overall

antisera used and were unable to be definitively identified at present.

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2.2. Résumé

L'épidémie grippale 2004 – 2005 a été modérée en intensité et d'une apparition

assez tardive dans la saison. Notre surveillance a débuté en septembre 2004 pour

s'achever fin avril 2005 après une période de 31 semaines. Les premiers cas

sporadiques ont été identifiés dès le mois de décembre et le pic de détection a eu

lieu dans la semaine 5 de 2005, avec un pic épidémique qui s'est étendu sur environ

7 à 8 semaines jusqu'à mi-mars 2005. 835 échantillons ont été analysés, tous chez

des patients présentant un syndrome grippal. 345 de ces échantillons (41%) se sont

révélés positifs pour un virus respiratoire par culture. L'immense majorité, soit 90%,

était un virus grippal distribué sous forme de virus influenza de type A pour 268

(87%) et influenza de type B pour 41 (13%). Parmi les virus influenza, 73% des virus

étaient de sous-type A (H3N2) et 11% A (H1N1). La caractérisation des virus

influenza A (H3N2) par inhibition de l'hémagglutination a montré que ces virus étaient

antigéniquement proches de la souche de référence influenza A/Shantou/1219/2004

(H3N2) elle-même très proche de la souche influenza A/California/7/2004 (H3N2).

Ces souches sont des variants récemment apparus qui se distinguent

antigéniquement de la souche vaccinale 2004-2005 influenza A/Fujian/411/2002

(H3N2). Les virus influenza A (H1N1) mis en évidence cette année étaient

antigéniquement proches de la souche vaccinale 2004-2005 influenza A/New

Caledonia/20/99 (H1N1). Les souches influenza B détectées au cours de cette

saison appartenaient à deux groupes. 44% étaient antigéniquement proches de la

souche influenza B incluse dans le vaccin de la saison 2002-2003 et 46% étaient

antigéniquement proches de la souche incluse dans le vaccin 2004-2005 et

finalement 3 souches influenza B ont montré une diminution de reconnaissance

antigénique avec l’ensemble des antisera utilisés et n'ont pas pu être définitivement

caractérisés à ce jour.

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2.3. Zusammenfassung

Die Grippe Epidemie 2004/2005 war von einer mässigen Intensität und begann

ziemlich spät. Die Überwachung der Grippe wurde im September 2004 gestartet und

wurde Ende April 2005 nach 31 Wochen beendet. Die ersten sporadischen Fälle von

Grippe wurden im Dezember nachgewiesen und das Maximum der Epidemie lag in

der 5. Woche (2005). Die Epidemie dauerte etwa 7 – 8 Wochen und war Mitte März

beendet.

835 Proben von Patienten mit Symptomen einer Grippe wurden untersucht. Davon

enthielten 345 Proben (41%) ein mittels Kultur nachweisbares respiratorisches Virus.

Die grosse Mehrheit der positiven Proben, d.h. 90% enthielten ein Grippevirus wovon

268 (87%) Influenza A und 41 (13%) Influenza B enthielten. Von den Influenza A

Viren gehörten 73% zum Subtyp A (H3N2) und 11% zum Subtyp A (H1N1).

Die Charakterisierung der Influenza A (H3N2) mit dem Hämagglutinationshemmtest

ergab, dass die Mehrheit der Viren verwandt waren mit dem Referenz Stamm

Influenza A/Shantou/1219/2004. Dieser Stamm selbst ist nahe verwandt mit

Influenza A/Kalifornien/7/2004 (H3N2). Diese zwei Varianten sind erst kürzlich

aufgetreten und unterscheiden sich von dem im Impfstoff (2004/2005) enthaltenen

Influenza A/Fujian/411/2002 (H3N2).

Die diese Jahr nachgewiesenen Influenza A (H1N1) waren mit dem im Impfstoff

enthaltenen Virus Influenza A/Neu/Kaledonien/20/99 (H1N1) verwandt.

Die Influenza B Viren welche diese Saison nachgewiesen werden konnten, gehörten

zwei verschiedenen Gruppen an. 44% waren verwandt mit dem Influenza B Virus

welcher im Impfstoff der Saison 2002/2003 enthalten war und 46% waren verwandt

mit dem Stamm welcher im Impfstoff der Saison 2004/2005 enthalten war. 3

Influenza B Viren zeigten eine reduzierte Affinität mit den geprüften Antiseren. Diese

Stämme konnten bis jetzt nicht definitiv charakterisiert werden.

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3.1. Introduction

The Sentinella surveillance network relies upon more than 200 general practitioners

and paediatricians distributed throughout Switzerland to identify patients in their

practice with influenza-like illness as defined by strict clinical criteria. This definition is

based on the presence of fever associated with at least one additional respiratory

symptom. Respiratory sampling, either a pharyngeal or a nasopharyngeal swab, is

carried out weekly in a sub-group of patients and sent to the National Influenza

Centre for the identification of an eventual influenza virus by cell culture. The main

target of the virologic detection system used is influenza virus, but it is also capable

of detecting other respiratory viruses. Once identified, the influenza virus undergoes

a series of tests to determine its sub-type and antigenic characteristics, and to

determine if the circulating strains are covered by the vaccine strains.

Apart from the human circulating influenza A and B strains, the 2004-2005 season is

once again marked by the risk of an avian flu pandemic. To date, no case has been

identified outside south-east Asia, but the surveillance laboratories must be equipped

with the appropriate molecular diagnostic tools in readiness to diagnose the

emergence of a new avian influenza virus in persons returning from regions where

this virus is circulating.

4. Method of detection for respiratory viruses

4.1. Clinical identification of influenza cases

Clinical identification is based on the network of practitioners in the community who

document during their daily consultations the total number of flu syndromes observed

compared with the total number of consultations carried out. The case definition used

for a flu syndrome is the presence of fever greater than 38° with or without an

impression of sickness, myalgia, or a change of general state. In addition to fever, an

acute respiratory symptom such as cough or rhinorrhea must be present. Among the

cases which meet the clinical criteria definitions, a sub-group of practitioners select

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each week up to two cases in which a nasopharyngeal or pharyngeal swab is taken

for despatch to the reference laboratory. These samples are placed in viral transport

medium and mailed to the centre the same day. The geographical distribution of the

subgroup of practitioners who take swabs is shown in Figure 1.

4.2. Detection of respiratory viruses

Detection of respiratory viruses is carried out according to the procedure shown in

Figure 2. Three different cell lines are inoculated (MDCK, LLC-MK2 et A549) at

different temperatures (37° and 33°C). After three days of incubation, screening of

cultures by monoclonal antibodies capable of detecting respiratory viruses influenza

A and B, parainfluenza 1, 2 and 3, adenovirus and respiratory syncytial virus is

conducted. A positive result leads to further, more specific screening to identify the

virus family concerned.

Figure 1 : Geographical distribution of the participants in the Sentinella network who do the sampling. Each participant is represented by a white dot. The region is distinguished by the number and the colour. Our country is divided in 6 regions comprising different number of cantons.

I

II

III

IV

V

VI

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Patient with symptoms

Nasal and throat swab (practitioner)

Samples shipped to the laboratory

Inoculation on cell cultures

(33 and 37°C)

Day 7 Screening with

monoclonal antibodies (immunofluorescence)

Negative Positive

Characterisation with Monoclonal antibodies

influenza A, B Parainfluenza-virus 1, 2, 3, virus respiratory syncytial-virus,

adenovirus Hemagglutination Test (HA)

HA titre < 1/8 HA titre ≥ 1/8

Re-inoculation of the sample on Determination of the type (A,B) and cell culture, to increase the HA titre subtype (A/H1N1, A/H3N2); Comparison of the antigenic similarities with the vaccine strains by the hemagglutination inhibition test; Distinction of Influenza A (H1N1 and Figure 2 : Procedure applied for the detection of respiratory viruses by cell culture

H1N2) by real-time PCR

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4.3. Characterization of influenza viruses

When an influenza virus is positively identified by cell culture, it is characterized by a

hemagglutination inhibition assay technique with specific antisera from immunised

ferrets. The results from this typing will be interpreted according to an antigenic table

adapted and established at the beginning of each influenza season with the aim to

use the antisera of strains which can be reasonably assumed to be circulating in the

human population during the season under study. The choice of these antisera is

equally essential in order to be able to differentiate the sub-types circulating and their

antigenic relation with the strains included in the vaccine. The titers obtained with

each clinical strain are identified and compared with standard antisera and allow a

precise identification of its antigenic behaviour. The criteria established for the 2004-

2005 season are described in table 1. As described in the legend to the table, the

homologous titers allow a comparison with the titers obtained with the circulating

influenza strains and to define the antigenic relation of these strains. This technique

permits an identification of the antigenic variations present on the hemagglutinin (HA)

which is an essential target of the immune and vaccine response. For example, in the

table illustrating the influenza A (H3N2) virus, it can be observed that the influenza

strains A/Wyoming/3/2003, A/Wellington/1/2004 and A/California/7/2004 show an

inhibition of titers comparable with antisera targeted against the influenza strain

A/Wyoming/3/2003. However, this technique does not permit to observe antigenic

variations related to the neuraminidase (NA) and it is thus impossible to differentiate

viruses with the same HA but with a different NA (e.g., H1N1 and H1N2 viruses). This

is particularly important as these viruses have circulated in Switzerland during the

last few years. To overcome this problem, we have developed a RT-PCR capable of

identifying an influenza virus type H1 containing a NA of type N1 or N2. This tool is

now applied systematically when a type H1 virus is identified.

Real-time RT-PCR techniques available in our laboratory allow to identify influenza

viruses A and B and to discriminate viruses of types N1 and N2 (annexe 1, van

Elden, 2001). Although more sensitive than cell culture, these techniques are not

used systematically for the routine detection of influenza viruses, but only to confirm

cell culture or in specific cases. To date, even molecular techniques used for

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sequencing do not allow to discriminate precisely antigenic behaviour and thus the

phenotype of similar viruses on a phylogenetic level. Hence, the inhibition of

hemagglutination assay remains the reference technique for the identification of

antigenic variations.

4.4. Identification of influenza A (H5N1) virus

For almost 2 years, the H5N1 virus has been circulating mostly in the avian batteries

of south-east Asia and is continuing nowadays (WHO, 17.06.2005). Several tens of

human cases have been documented with a massive mortality leading to rapid death

by diffuse pulmonary attack. At present, there exists a probable case of inter-human

transmission (Ungchusak, 2005) but this phenomenon remains limited and, to date,

the virus does not appear to have acquired the essential characteristics to support an

inter-human chain of transmission to efficiently spread in man. However, the threat of

this risk has to be taken seriously in consideration of the exponential aspect of the

epidemic in the avian world, the increasing number of human cases observed, and

the increasing number of countries reporting cases coupled with the difficulty to

rapidly identify patients in regions where medical and laboratory infrastructures are

limited.

For this reason, it is essential that each reference laboratory may be able to propose

diagnostic tools to allow the rapid identification of a suspected case. RT-PCR is the

preferred technique of choice for an initial screening of a suspected case. It allows to

avoid the culture of a virus which is potentially extremely dangerous and,

theoretically, also permits a more rapid diagnosis. Cell culture as well as molecular

diagnostic techniques are confronted with several unknown factors concerning this

virus. The phenotypic and antigenic variability of the influenza A (H5N1) virus can

make its identification difficult even with established techniques, and it is therefore

essential that the international surveillance networks for influenza virus share their

most recent data. For detection by molecular tools, RT-PCR techniques require

knowledge of the sequence of the targeted genome. Hence, the genomic sequencing

of viruses detected in the various countries where cases are observed must

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Table 1 : Hemagglutination inhibition (IHA) of the titer of reference influenza strains incubated with each of the reference antisera. The IHA titer obtained after incubation of a given strain with its corresponding antiserum is mentioned in bold. This value is called the homologous titer (HT). The titer obtained with a strain isolated from a sentinella sample (Sen) is then compared with the HT titer. If the ratio Sen/HT is low, the strain is considered as antigenically related to the reference strain. If the ratio is high, the strain is considered as antigenically different from the reference strain. Les 2 lignées principales des virus influenza B sont indiquées en Bleu. Yam : Yamagata-like ; Vict : Victoria-like.

Influenza A (H3N2)

Antisera

Strains A/Panama/2007/99 A/Wyoming/3/03 A/Shantou/1219/04 A/Wellington/1/04 A/California/7/04

A/Panama/2007/99 5120 640 80 640 < 160

A/Wyoming/3/2003 640 5120 640 1280 2560

A/Shantou/1219/2004 320 640 2560 1280 320

A/Wellington/1/2004 320 5120 2560 2560 1280

A/California/7/2004 < 160 5120 2560 320 2560

Influenza A (H1N1)

Antisera

Strains A/N.Caledonia/20/99 A/Beijing/262/95 A/Madag./57794/00 A/Egypt/96/02

A/N.Caledonia/20/99 20480 640 10240 2560

A/Beijing/262/95 5120 10240 2560 1280

A/Madagascar/57794/2000 5120 320 2560 2560

A/Egypt/96/2002 (H1N2) 5120 640 5120 5120

Influenza B Antisera

Strains B/Jiangsu/ 10/2003

B/Sichuan/

379/99

B/Harbin/ 7/94

B/Beijing /184/93

B/HK/ 335/01

B/Shandong/ 7/97

B/Brisbane/32/02

B/Jiangsu/10/2003 10240 1280 640 320 160 < 160 < 160 B/Sichuan/379/99 < 160 256 1280 320 640 < 160 < 160

B/Harbin/7/94 < 160 1280 5120 640 2560 320 < 160

B/Beijing/184/93 < 160 1280 5120 2560 1280 160 < 160

B/HK/335/2001 < 160 < 160 640 < 160 2560 10240 10240

B/Shandong/7/97 < 160 < 160 < 160 < 160 320 10240 2560

B/Brisbane/32/2002 < 160 < 160 160 < 160 320 2560 2560

Yam

Vict

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imperatively be made available to all laboratories participating in global surveillance

as rapidly as possible. This is not the case at present, despite the existence of an

international influenza surveillance network under the auspices of the WHO. A further

difficulty is to maintain a register of non-infectious viral RNA types or plasmids from

the most recent cases. When faced with such an epidemic, with a virus capable of a

rapid and perpetual mutation, or capable of reassortment, it is essential to adapt

diagnostic tools as fast as possible to be able to obtain pertinent data in time. The

technique developed and perfected in our laboratory relies on a RT-PCR targeting of

the HA of the H5N1 virus. We have three types of tests at our disposal which permit

to detect avian influenza viruses having circulated recently either in the avian world or

in human cases. The controls used are DNA from RT-PCR derived from the genomic

RNA of human or avian influenza viruses and plasmids encoding for the HA of

viruses from human cases infected in Viet Nam during 2004.

To facilitate the availability of a control to allow the development of these diagnostic

tests, a bank of molecular material such as positive controls is in the process of being

set up under the auspices of the EISS. In this way, these controls can be distributed

to European laboratories participating in flu surveillance. Within this context, we have

produced plasmids based on influenza A (H5N1) virus sequences. Beyond the

technical aspects, the identification strategy of suspected cases of influenza A

(H5N1) virus is based not only on the identification of initial clinical cases, but also on

the capacity of laboratories to propose an alternative diagnosis. Therefore, we

propose to carry out a series of molecular analyses for each sporadic suspected case

which allows to identify both all influenza viruses circulating and all human respiratory

viruses, thus providing clinicians and health authorities with an alternative diagnosis

in the event of a clinical suspicion.

At last, we have at our disposal a specific antiserum targeted against the human

influenza strain A/Hong Kong/156/97 (H5N1). This antiserum has been provided by

the WHO to all national reference laboratories participating in the influenza

surveillance network. This reactive can be used on a sample which would have

escaped all recognition by standard antisera and on which would cause a problem of

suspected infection by an influenza A (H5N1) virus. The antigenic table of this

antiserum is given in Table 2.

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Table 2 : Hemagglutination inhibition titer of influenza A (H5N1) inactivated virus with standard

influenza antisera

Standard Antisera (WHO) A/Hong Kong

156/97 (H5N1) A/Moscow

10/99

(H3N2)

A/Panama 2007/99 (H3N2)

A/Hong-Kong 1550/02 (H3N2)

A/N-Caledonia 20/99

(H1N1)

A/Egypt 96/02

(H1N2)

Inactivated

A (H5N1) strain (WHO 1997)

81920

< 80

< 80

< 80

< 80

< 80

5. Results

5.1. Viral detection

Surveillance began on 18 September 2004 and ended on 22 April 2005 after 31

weeks. Eight hundred and thirty-five samples were obtained from 56 Sentinella

practitioners, and a few samples were sent in by hospital laboratories. Of these, 309

influenza viruses were detected, representing a mean positive rate of 37% for the

whole season (Figure 3a). From this, we can conclude that almost 4 of 10 suspicions

of flu infection were confirmed. At the peak of the season, the proportion of flu

infection considerably increased and reached 57%. Among the 309 influenza viruses

detected, the majority was influenza A (H3N2) virus (73%), followed by influenza B

(13%) and influenza A (H1N1) virus (11%). Eight influenza A viruses could not be

sub-typed (3%). Thirty-six respiratory viruses from a family other than influenza virus

were also detected (10%; Figure 3b). Among these other viruses, respiratory

syncytial virus was isolated 14 times, adenovirus, 12 times, parainfluenza 1, 4 times,

parainfluenza 2, 2 times, and parainfluenza, 4 times (Figure 3b). Details of these data

are shown in the green table of the annexe 2.

Among the influenza viruses, the majority detected was of type A, 268 of 309 (87%).

Two hundred and twenty-five (73%) were of sub-type A (H3N2) and 35 (11%) of sub-

type A (H1N1). Of those remaining, 41 (13%) influenza type B were detected (Figure

3c).

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a)41% positive

59% Negative

b)

3% ADV

1% PIV11 PIV2

90% Influenza

4% RSV1% PIV3

c)

73% inf. A/H3N2

13% inf. B

11% inf. A/H1N1

3% inf. A

Figure 3 : Proportion of nasopharyngeal samples positive for influenza or other respiratory viruses during the 2004/05 season (n = 835). a) Percentage of positive and negative samples received during the season. b) Percentage and type of different respiratory viruses detected. c) Percentage of type and subtype of influenza viruses detected (Inf. = influenza viruses)

345 positive

309 influenza viruses

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During the first weeks of surveillance at the end of September 2004, no influenza

virus was detected. The first cases were detected at the end of November and

followed by sporadic but regular detections until week 48. From the last weeks of

December, there was a regular increase in positive cases in parallel to an increase in

medical consultations for flu syndromes. The detection rate peak was observed

during week 5 with 45 influenza viruses detected, which corresponded to 64% of all

positive samples collected (Figure 4). The medical consultation rate for flu syndrome

reached its maximum one week later with 61.7 flu consultations for 1000 medical

consultations. This was followed by a decrease both in the detection rate and the

number of medical consultations and passed under the epidemic index threshold

during week 11, being mid-March 2005 (Figure 4). The eight-week phase considered

as epidemic was rather short with medical consultations remaining above the level of

15 out of 1000. In comparison, the mean duration of flu epidemics observed during

the past 12 years has been nine weeks.

The kinetics of the circulating viruses were recorded for all six regions of Switzerland.

Medical contacts for influenza-like illness (MC-ILI) exceeded baseline values in all

regions during weeks 2 and 3 and reached the maximum value during weeks 6 and 7

(Figure 5). The different influenza viruses were detected in all regions. However, no

influenza A (H1N1) virus was detected in region 6, and no influenza B virus was

detected in region 2. The interpretation of this observation remains difficult as the

number of samples from these regions is too small. Thus, the flu epidemic in

Switzerland has been homogenous, as observed in all European countries.

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0

10

20

30

40

50

39 41 43 45 47 49 51 53 2 4 6 8 10 12 14 16

Weeks

Num

ber

of v

irus

es (n

)

0

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40

50

60

70

MC

-ILI (

‰)

Other respiratory viruses

Inf. A (H1N1)

Inf.A

Inf. A (H3N2)

Inf. B

MC-ILI 2004/05 (‰)

Threshold (15 ‰)

Figure 4 : Type of viruses detected per week. inf. A (H1N1) : influenza A (H1N1) virus; inf. A (H3N2) : influenza A (H3N2) virus; Inf. A: influenza A viruses which were not subtyped, inf. B : influenza B virus. MC-ILI: medical contacts for influenza-like illness; threshold : percentage of medical contacts for influenza-like illness indicating the presence of an epidemic (15 ‰).

5.2. Characterization of the influenza viruses

5.2.1. Influenza A (H3N2)

Of 225 influenza A (H3N2) strains detected, only four strains showed an enhanced

recognition with the antiserum A/Wyoming/3/2003, the 2004-2005 vaccine strain

(annexe 3). In contrast, the great majority of strains demonstrated good recognition

with the antisera influenza A/California/7/2004 et A/Shantou/1219/2004 and are

therefore antigenically quite distinct from the vaccine strain (189/225, 84%). Finally,

21 influenza strains showed a decrease of the titer in inhibition of the

hemagglutination with the antisera overall. The influenza strains A/California/7/2004 and A/Shantou/1219/2004 are very close

antigenically and reactions are detected with their specific antisera (see Table 1).

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100‰ M edical consult.

IBIA/H3N2IA/H1N1IA undet.Baseline%MC 2004/05

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Region 3 (BS, BL, AG, SO) Region 4 (LU, ZG, NW, OW, UR, SZ, GL)

02468

1012141618202224262830

39 41 43 45 47 49 51 53 2 4 6 8 10 12 14


0

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02468

1012141618202224262830

39 41 43 45 47 49 51 53 2 4 6 8 10 12 14 16


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Region 5 (ZH, SH, TG, SG, AI, AR) Region 6 (TI, GR) Figure 5 : Number of influenza viruses detected per week and per region. A/H3N2 : influenza A (H3N2) viruses; IA/H1N1 : influenza A (H1N1) viruses; IA undetermined : influenza A virus that have not been subtyped; IB: influenza B viruses. ‰ MC: medical consultation for ILI

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The genetic analysis confirms this small difference : there are less than 10

nucleotides of difference between the HA sequences of these two strains (Figure 6).

It is therefore reasonable to consider them as similar.

Among the 189 influenza A (H3N2) strains close to the influenza strains

A/Shantou/1219/2004 and A/California/7/2004, three populations can be

distinguished. The first is formed of strains which demonstrate significant cross-

reactions with three antisera: A/Shantou/1219/2004, A/Wellington/1/2004 and

A/Wyoming/3/2003 (86/189). The second population shows cross-reactions with the

antisera A/Shantou/1219/2004 and A/Wellington/1/2004, but a significant decrease of

recognition with the antiserum A/Wyoming/3/2003 (34/189). Finally, the third

population shows an affinity with the influenza antiserum A/Shantou/1219/2004 only

and a significant decrease of recognition with the antisera A/Wellington/1/2004 and

A/Wyoming/3/2003 (69/189). Hence, the two latter populations show a clear

decrease of recognition with the specific antiserum of the vaccine strain. This would

suggest that the influenza A (H3N2) strains have the possibility to escape or to be

less sensitive to the immune protection conferred by the 2004-2005.

The influenza antiserum A/California/7/2004 became available during the season and

allowed to distinguish between the influenza A (H3N2) strains. Sixty-one influenza A

(H3N2) strains were able to be analysed with this antiserum. Thirty-eight of 61 strains

were close to A/California/7/2004, and 19 were close to A/Shantou/1219/2004. Four

strains showed decreased titres with all the antisera used (annexe 3). This illustrates

that although similar, these two strains correspond to variants which are antigenically

slightly different.

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Figure 6 : Phylogenetic comparison of nucleotide sequences encoding for H3 hemagglutinins

In conclusion, the influenza strains A/Shantou/1219/2004 and A/California/7/2004

represented those which predominantly circulated in Switzerland during the 2004-

2005 season. These recently-appeared variants differ antigenically from the strain

contained in the 2004-2005 vaccine, A/Wyoming/3/2003 (H3N2). Moreover, the

influenza strain A/California/7/2004 (H3N2) has been chosen to be included in the

2005-2006 influenza vaccine.

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5.2.2. Influenza A (H1N1)

Thirty-five influenza A (H1N1) viruses were detected in Switzerland during this

session. Overall, these strains were antigenically close to the vaccine strain A/New

Caledonia/20/99 (H1N1) and to the most recent influenza strain

A/Madagascar/57794/2000 (H1N1) (annexe 4). Antigenic (Table 1) and genetic

(Figure 7) analyses show that these two strains are very close. The HA sequences of

these two strains are closely related (Figure 7 red arrows) ; in fact less than 10

nucleotides separate the HA of recently-detected strains worldwide (in blue at the top

of the Figure 7) with the influenza strain A/New Caledonia/20/99 (H1N1). For this

reason, the antigenic composition of influence A (H1N1) strains to be included in the

2005-2006 vaccine has not been modified.

No influenza A (H1N1) strain was close to the former vaccine strain A/Beijing/262/95

(H1N1) which circulated in Switzerland in 1995-1996. Moreover, no influenza A

(H1N2) strain has been detected in Switzerland this year. One patient was co-infected by an influenza A and an influenza B strain. The sample

contained both an influenza A (H1N1) and an influenza B strain. Unfortunately, the

presence of two strains in the same sample did not permit the use of the reaction to

hemagglutination inhibition to determine the relationship. The clinical sample was

sent to London to be studied in more depth. A RT-PCR reaction confirmed the

presence of the two viruses, the influence A (H1N1) virus being predominant. The

latter could be typed as being antigenically close to the influenza vaccine strain

A/New Caledonia/20/99 (H1N1).

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Figure 7: Phylogenetic comparison of nucleotide sequences encoding H1 hemagglutinins of

human influenza viruses. Red arrows indicate the representative strains of influenza A (H1N1) that recently circulated.

24/54

5.2.3. Influenza B Two distinct lineages of influenza B strains have been regularly detected over the

past years in Switzerland, as in Europe : the lineage “Yamagata-like” and the lineage

“Victoria-like” (Figure 8). To be able to type circulating influenza B strains, antisera

specific for these two groups have been used (table 1).

Figure 8 : Phylogenetic comparison of nucleotide sequences encoding hemagglutinins of influenza B virus. Names in red indicate representative strains.

This year, the two lineages have been detected in equal proportions in Switzerland.

First, 44% (n=18) were antigenically close to the influenza strain B/Shandong/7/97,

itself very close to the influenza strain B/Hong Kong/330/2001 (Figure 8). This type of

strain has already circulated in Switzerland during the 2002-2003 season and has

even been included in the vaccine composition for that season. 46% (n=19) influenza

B viruses were antigenically close to influenza strains B/Jiangsu/10/2003 and

B/Sichuan/379/99, the first strain being included in the 2004-2005 vaccine (annexe

5). Among these, seven were closer to the most recent influenza strain

B/Jiangsu/10/2003 than the influenza B/Sichuan/379/99 one. Three influenza B

strains showed decreased titers with the reference antisera B/Shandong/7/97 and

Victoria-like

Yamagata-like

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B/Sichuan/379/99 and are presently undergoing molecular analysis and a more

precise typing. Finally, an influenza B strain could not be typed as the sample came

from a patient co-infected by two influenza strains: an influenza B and an influenza A

(H1N1) strain.

5.3. Patients with influenza infection

5.3.1. Frequency of viruses detected in a particular age group The number of influenza viruses detected during the season has been stratified by

age group (Figure 9). As observed during past seasons, the majority of samples were

from the age groups “active” in the population, i.e., between 10 and 49 years old. The

younger age groups (less than 20 years old) do not appear to have been especially

affected this year in comparison to the 2003-2004 season where patients from this

group represented more than 40% of all those infected. In contrast, during this

season, this age group represented only 27% of all patients. In the age group of over

70 years old, the number of samples as well as the number of viruses detected is

higher than that observed in the groups of over 50 and 60 years old. This was also

observed during the 1997-1998 season. The season saw the appearance of an

antigenic variant of the influenza A (H3N2) virus : influenza A/Sydney/5/97. Thus, the

circulation of an antigenic variation close to the influenza strains

A/Shantou/1219/2004 and A/California/7/2004 (H3N2) appears to have had a more

severe effect than usual on those over 70 years old. This observation is confirmed by

the fact that the predominant virus in the age groups over 60 years old is an influenza

A (H3N2) virus. The influenza B virus infected indifferently all age groups. However,

influenza A (H1N1) virus infected mainly those less than 50 years old (Figure 9).

26/54

0

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<= 1 2-4 5-9 10-19 20-29 30-39 40-49 50-59 60-69 70 <= ?

Age of patients

influ

enza

vir

uses

(n)

(H1N1)

(H3N2)

B

Figure 9 : Number of influenza viruses classified per type and subtype according to the different age groups

5.3.2. Clinical data of patients infected with influenza

The frequency of symptoms and clinical signs observed in patients infected by the

different viruses have been documented and are shown in Figure 10. As usually

observed, the most frequent symptoms are fever and cough, followed by headache,

myalgia and rhinitis. It is interesting to note that the symptoms observed in flu

patients are the same, regardless of the influenza virus detected.

0

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lymph

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s (%

)

A/H3N2A/H1N1

B

Figure 10 : Classification of symptoms associated with patient according to the strain of influenza virus detected5.3.3. Influence of the influenza epidemic on the mortality rate in the canton of Geneva.

27/54

The elderly (over 60 years old) represent the main population at high risk of serious

complications following an influenza infection. To evaluate the severity of the 2004-

2005 influenza epidemic, we have analysed in particular the weekly mortality rate in

this age group in the canton of Geneva and compared these Figures with the weekly

rate of MC-ILI and the number of viruses detected in the canton (Figure 11). In

addition, the 2005 mortality rate has been compared with rates registered during the

seasons 1991 to 2004 and the mean of these weekly mortality rates has been

calculated plus two standard deviations. Results of these comparisons are shown in

Figure 11.

As observed at national level, the influenza epidemic in Geneva reached a peak

during week 6 : the rate of influenza viruses detected reached a maximum of 5/7

viruses detected during that week and the MC-ILIC value culminated at 11%. A

detailed analysis of the mortality rates for the following weeks showed no significant

increase when compared with the mean calculated for 1991-2004. Therefore, no

peak in mortality was observed consecutive to the influenza epidemic during the

2004-2005 season. This was not the case for the 2003-2004 season where eight

additional deaths in the canton of Geneva alone were observed one to two weeks

after the peak of MC-ILI (Thomas et al, 2004).

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0

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MC

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irus

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eath rate (n)

Medical consultations GE (%)

Influenza viruses detected (n)

Death rate >=60 years (n)

Threshold

Death rate Mean+2sd >= 60 years 1991-2004

Figure 11 : Influence of the influenza epidemic on the mortality in people older than 60 years in the canton of Geneva during the 2004/05 season.

Medical consultations GE (%): medical contacts for influenza-like symptoms in the canton of Geneva; Influenza viruses : influenza A and B viruses detected by the Sentinel network, in the canton of Geneva. Death rate : number of weekly deaths obtained from the records of the Register Office of the Canton of Geneva published by the Public Health Authorities of Geneva; Mean +2sd: mean number of the weekly deceased people older than sixty years-old + two standard deviation registered between 1991 and 2004

5.4. Evolution of the epidemic in Europe The influenza epidemic started at different times according to the various countries.

Some western European countries were the first to report outbreaks, such as Ireland

where the epidemic started relatively early (week 52, Figure 12). A few weeks later,

the epidemic culminated in the United Kingdom, Spain and Portugal (weeks 1 and 2,

Figure 12). Other countries such as France, Switzerland and Italy followed with a

maximum activity registered during week 5 (beginning of February). Finally, countries

in the more eastern part of Europe observed an epidemic which peaked during

weeks 6 and 7 (Slovenia, The Netherlands and The Czech Republic), week 8

(Norway) and week 10 (Germany).

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Figure 12 : Detection of influenza viruses in European countries according to the week of sampling. Inf.A : influenza A viruses; Inf. A H1 (influenza A (H1N1) or influenza A (H1N2)); Inf B : influenza B virus; Inf A untyped : influenza A virus not subtyped; Inf A H3 : influenza A (H3N2). Data obtained from the European Influenza Surveillance Scheme, http://www.eiss.org.

Ireland

Spain

France

Slovenia

Norway

30/54

As in Switzerland, it was observed that several different types of influenza viruses

were circulating simultaneously: influenza A (H3N2 and H1N1) viruses and influenza

B viruses. Influenza A (H3N2) viruses circulated mainly in all European countries

apart from Slovenia where the influenza B was predominant. They were antigenically

close to the influenza A/California/7/2004 and A/Wellington/1/2004 strains. Influenza

A (H1N1) viruses were also detected in several European countries but in small

quantity. All were antigenically close to the influenza vaccine strain A/New

Caledonia/20/99 (H1N1). Influenza B viruses circulated quite significantly in almost all

European countries (Figure 12). The two main lineages of the influenza B virus were

detected in Europe during the 2004-2005 season. However, the proportions varied

according to the country. Viruses antigenically close to the influenza

B/Jiangsu/10/2003 strain and belonging to the Yamagata-like lineage (Figure 8)

mainly circulated in western and northern Europe (Portugal, France, Ireland, United

Kingdom, Norway...). In contrast, viruses antigenically close to the influenza

B/Shandong/7/97 strain belonging to the Victoria-like lineage (Figure 8) were

observed mainly in countries in central and southern Europe (Germany, Switzerland,

Italy, Poland and Romania).

5.5. Influenza A/H2N2

In February 2005, the College of American Pathologists (CAP) sent quality controls to

laboratories. For this purpose, currently circulating influenza A (H3N2 and H1N1) and

B viruses are normally used. In October 2004, a panel of controls containing

influenza A (H2N2) virus was inadvertently distributed and subsequently identified by

a reference laboratory participating to the proficiency testing. This virus was similar to

influenza A (H2N2) viruses that circulated in humans in 1957-58 and caused the so-

called Asian influenza pandemic. This influenza A (H2N2) was easily transmitted

from human to human. It continued to circulate in humans and caused annual

epidemics until 1968 when it vanished after the emergence of influenza A (H3N2)

viruses that caused the next pandemic. Therefore, persons born after 1968 are

expected to have no or only limited immunity to influenza A (H2N2). This A (H2N2)

virus is not contained in current trivalent influenza vaccines. The control panel

31/54

containing this A (H2N2) virus was sent by the CAP to more than 3750 laboratories in

eighteen countries (http://www.who.int/csr/disease/influenza/h2n2_2005_04_12/en/).

Since 8 April, WHO has been working directly with the US Department of Health and

Human Services and the Centers for Disease Control and Prevention (CDC) as well

as the relevant Canadian health agencies to assure rapid destruction of the samples

containing the influenza A (H2N2) virus. In addition, WHO through its regional and

country offices contacted the Ministries of Health of the remaining 16 involved

countries and territories to support this international effort. On 21 April 2005, 15/16

countries confirmed the destruction of the samples. No human infection of laboratory

workers has been detected until now.

5.6. Avian influenza

5.6.1. Avian influenza epidemics in South East Asia, 2004-2005

In January 2004, WHO reported officially a large number of avian influenza

epidemics in the eight countries of South East Asia. More than 100 million animals in

breeding farms either died following these epidemics or were culled as a preventive

measure to check the evolution of the epidemic. On 10 March 2004, 35 human cases

had been reported in Viet Nam and Thailand alone. Of these, 24 died following an

infection by the virus (Tran et al, 2004, de Jong et al, 2005). On 19 July 2004, after a

silence of several months, new avian influenza epidemics were observed in several

batteries of Viet Nam, followed by Thailand. Human cases have been reported in

Thailand, Viet Nam and Cambodia, close to the Vietnamese border. This re-

appearance of the virus occurred in two additional phases (Table 3). The first ranged

from mid-July to the beginning of October 2004 with nine human cases and eight

deaths in Viet Nam and Thailand. The second and last phase began in mid-

December and still persists. As at 17 June 2005, this last epidemic phase had

caused 63 cases of human infection in Viet Nam and Cambodia; to date, 22 persons

have died (Table 3). The isolated viruses, both in sick animals and human victims,

were the influenza A (H5N1) viruses, genetically close to the viruses already detected

in previous years in the same region. (Li et al., 2004).

32/54

Table 3 : Cumulative number of confirmed human cases of avian influenza A (H5N1) reported to WHO (WHO, cumulative A/H5N1 cases)

5.6.2. Detection of avian influenza viruses in Switzerland

At the Swiss National Centre for Influenza, the detection of avian influenza virus is

conducted by RT-PCR. For the detection of influenza A (H5N1) virus, we have at our

disposal specific reactives of the sub-type A/H5. These reactives must be able to

recognise the viruses despite their evolution over time. For example, the influenza A

(H5N1) viruses that circulated in 1987 in human is genetically slightly different than

the one that circulated in 2003 (Horimoto et al., 2004, Li et al, 2004). To overcome

this phenomenon, three different combinations of primers have been chosen and

developed in Geneva. The hemagglutinin H5 of the virus has been targeted. Two

combinations are constituted of primers for a detection by Real-Time RT-PCR

(orange and green combinations, Figure 13), and one combination is constituted of a

primer for a classic PCR (blue combination, Figure 13).

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Figure 13 : Schéma des régions de l’hemagglutinine de virus influenza A/H5 reconnues par les différents jeux de primers (les sondes centrales ne sont pas représentées). Les combinaisons oranges et vertes sont utilisées pour une PCR en real-time et les sondes bleues sont utilisées en PCR classique. These primer combinations are regularly tested with positive controls, if available. For

security reasons, no live influenza A (H5N1) virus strain is distributed to countries

outside the country of detection. For this reason, only the RNA and the DNA of the

viruses are distributed as positive controls. Our primer sequences are also regularly

verified by alignment with the genomic sequences of the influenza A (H5N1) virus

recently detected and available in data banks such as Genbank (National Institutes of

Health) and ISDB (Los Alamos National Laboratory). Indeed, it is essential to

guarantee the efficiency of our detection reactions despite the constant modification

of the influenza genome over time due to a well-known phenomenon of the influenza

viruses : the antigenic shift. The sequences of two influenza A (H5N1) viruses of

avian origin detected recently (January 2005) in Viet Nam have been provided by the

National Influenza Centre of the National Institute of Hygiene and Epidemiology

(NIHE) in Hanoi. First sample was collected in the South of Vietnam (annexe 6,

sequence 2) and second one in the North (annexe 6, sequence 3). The alignment of

these two sequences with other influenza A (H5N1) strains detected in humans since

1997 demonstrates a satisfactory conservation of the zones recognised by our

primers. In contrast, a site of three nucleotides is missing from the sequence of the

influenza virus sampled in the north of Viet Nam. This zone is also the target of one

of our primers, the HARev. Thus, the primer combination including the HARev may

not be able to detect these types of strains. This observation well illustrates the limits

H5 Clivage

site

34/54

of molecular technique detection. However, the two other primer combinations

demonstrate practically no difference with the new sequences. Theoretically, our set

of primers should be able to detect new strains of influence A (H5N1). However, a

more recent positive control is necessary to experiment and confirm this observation.

Unfortunately, at the present time, we have been unable to obtain such controls from

countries affected by the avian influenza epidemics.

We have also available heterogeneous positive controls to validate our reactives for a

large variety of influenza (H5N1) strains. This concerns DNA comprising the

sequence encoding pour hemagglutinin of two strains of avian origin having

circulated in 1984 and 1997 and a strain detected in a human case in Viet Nam in

2004. The results of the Real-Time RT-PCR reactions carried out on the human

strain control (Figure 14a) and on the avian strains (Figure 14b) demonstrate our

primer detection efficiency.

It is important to have available adequate, standard positive controls in sufficient

quantity to allow laboratories to develop a molecular detection test for the influenza A

(H5N1) virus. One of the tasks of the European group, EISS, is to help the

laboratories of the international surveillance network to achieve this objective. As an

active member of the EISS, Switzerland participates actively to the establishment of a

data bank of molecular material to enable access to positive controls for diagnostic

tests. Within this framework, the Central Laboratory of Virology at the University

Hospitals of Geneva, in collaboration with the Norwegian Institute of Public Health in

Oslo, has developed a positive control for PCR. This type of control can help

advance a large number of European laboratories to establish or develop a detection

technique either by classic PCR or a Real-Time RT-PCR. It can also be used to

create the standardisation of detection techniques between laboratories in the

community network and EISS (CNRL) reference laboratories (CNRL).

35/54

Combinaison 1 Combinaison 2 (HA5For, HA5Rev, HA5Pro) (H5VietFor, H5VietRev, H5VietPro) A/Duck/Potsdam/2243/84 H5N6 A/Duck/Singapore-Q/F119/97

Figure 14 : Detection of three different positive controls with two Real-Time RT-PCR primer combinations. Positive controls are constituted of plasmid containing a part of the hemagglutinin sequence of A/Vietnam/1203/2004 (H5N1) and DNA containing the encoding sequence for hemagglutinin for the A/Duck/Postdam/2243/84 (H5N6) and A/Duck/Singapore-Q/F119/97 (H5N3) viruses.

The construction consists of an amplification of the most variable part of the avian

virus genome, the entire encoded sequence of the hemagglutinin. This sequence has

been amplified from the influenza A/Duck/Singapore-Q/F119/97 (H5N3) virus. The

hemagglutinin shows a good conservation with the hemagglutinins of the avian

influenza A (H5N1) virus detected in humans. The results of the amplification

obtained by PCR were then introduced into a plasmid which allowed to largely

reproduce this construction into bacteria. A diagram of this construction is shown in

Figure 15. The next steps will be to also obtain the hemagglutinin of viruses detected

in human cases.

A/Vietnam/1203/04 (H5N1) dilutions 10-3, 10-4 et 10-5

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Figure 15 : Schéma du vecteur porteur de la séquence de l’hemagglutinine H5 du virus

aviaire A/Duck/Singapore/F119-3/97 (H5N3). En noir est représenté la séquence

codante pour l’hemagglutinine HA5

6. Recommended composition of the 2004/05 influenza vaccine

The 25th of February 2005, WHO recommended that vaccines to be used in the

2005/06 influenza season and in the northern hemisphere contain the following

(WHO, 25.02.2005) :

- an influenza A/New Caledonia/20/99 (H1N1)-like virus

- an influenza A/California/7/2004(H3N2)-like virus a

- an influenza B/Shanghai/361/2002-like virus b

a A/New York/55/2004 is available as a vaccine virus bThe currently used vaccine viruses are B/Shanghai/361/2002, B/Jiangsu/10/2003 and B/Jilin/20/2003

HA5 1750 bps

M13

T7

37/54

7. Discussion

The intensity of the influenza epidemic observed this season was medium to high.

Different influenza viruses circulated in Switzerland namely influenza A (H3N2),

influenza A (H1N1) and influenza B. During the winter an influenza A (H3N2) virus

strain appeared which was antigenically distinct from the strain that circulated in

2003-2004 and which was included in the 2004-05 vaccine. Viruses which were

detected during the 2003/2004 season were antigenically close to influenza

A/Fujian/411/2002 and A/Wyoming/3/2003. This winter however, the influenza A

(H3N2) strains which circulated most frequently in Europe and in the United States

were antigenically related to influenza A/California/7/2004 and A/Shantou/1219/2004.

This means that an antigenic drift occurred which clearly could be detected with

inhibition of the hemagglutination assays. In Geneva, some infections with this virus

were observed in immuno-compromised patients vaccinated with the 2004-2005

influenza vaccine suggesting that the protection was diminished. The phylogenetic

analysis (Figure 6) shows that influenza A/California/7/2004 and influenza

A/Shantou/1219/2004 strains are distinguishable from the vaccine strains influenza

A/Fujian/411/2002 and A/Wyoming/3/2003.

The frequency with which the different influenza A (H3N2) circulated in Switzerland

since 1995 are represented in Figure 16. During the last ten years, a particular

influenza A (H3N2) virus circulated between 2 and 3 seasons on average before a

new variant showed up. Normally the new variant and the previous strain circulated

on parallel during a season before the older one disappeared. This was the case for

example for influenza A/Sydney75/97 (H3N2) (Figure 16) which appeared in 1997. It

became the dominating strain after influenza A/Wuhan/354/95 and

A/Johannesburg/33/94. In the same manner, influenza A/Fujian/411/2002 started to

circulate at the end of the 2002/03 season and became dominant during the following

season 2003-04.

The decision on the composition of the vaccine for the northern hemisphere takes

place in February each year. If a variant shows up later on during the season, the

vaccine might protect less against the new strain circulating during the following

season. The virus can then be transmitted more easily to vaccinated people. This

38/54

might have severe consequences. Such a situation was observed during the 1997-98

season where influenza A/Sydney/5/97 (H3N2) appeared. At that time influenza

A/Wuhan/354/95 was included in the 1997-98 influenza vaccine and as a

consequence gave only partial protection against A/Sydney/5/97 (H3N2). On parallel

high rates of medical consultations for influenza-like illness were observed. As a

consequence a higher mortality rate of the more than 60 years old people was

observed in the canton of Geneva (Thomas et al., 1998).

This year, the variant influenza A/California/7/2004 (Figure 16) was predominant from

the very beginning of the season. The vaccine contained the antigens from the

influenza A virus which circulated the previous years namely influenza

A/Fujian/411/2002 (Thomas et al, 2004). Although the epidemic was less severe than

the epidemic observed in 1997-98, it still was moderate to strong.

95/9

6

96/9

7

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8

98/9

9

99/0

0

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0102030405060708090

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Influenza virus rate (%)

Influenza A (H3N2) virus

A/Thessaloniki/1/95 A/Johannesburg/33/94A/Wuhan/359/95A/Sydney/5/97A/Moscow/10/99A/Fujian/411/02A/California/7/2004

Figure 16 : Influenza A (H3N2) strains which circulated in Switzerland since 1995. Each colour represents a particular influenza strain. The values represent the rate of each strain in comparison to all influenza A strains detected during the season.

Influenza B virus were also observed this year. Two lineages of influenza B,

circulating already in previous years in Switzerland, were observed at the same

frequency this season. The two variants belonged to the Yamagata and to the

Victoria lineages. As one can see in Figure 17, influenza B strains belonging to the

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Yamagata lineage circulated since 1995. Influenza B strains close to

B/Beijing/184/93 were observed between 1995 and 1999. Influenza B close to

influenza B/Sichuan/379/99 was observed between 2001 and 2005. The Victoria-like

lineage which was present in Asia made its appearance in Europe and in Switzerland

only in the last years more precisely in 2002-03. It was an influenza B related to

influenza B/Hong Kong/330/2001 (Figure 17). After the absence during the 2003-04

season, viruses related to influenza B/Hong Kong/330/2001 started to circulate again

in Switzerland during this last season. This illustrates that this virus can be

maintained in the community and has the potential to emerge again.

1995

/96

1996

/97

1997

/98

1998

/99

1999

/200

0

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/01

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/02

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/03

2003

/04

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/05

0

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40

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Influenza virus rate (%)

Influenza B

Yamagata/16/88Beijing/184/93Sichuan/379/99Hong Kong/330/01

Figure 17 : Influenza B strains which circulated in Switzerland since 1995. Each colour represents a particular influenza strain. The values represent the rate of each strain in comparison of all influenza B strains detected during the season.

Influenza A (H1N1) strains detected this year were antigenically not different from the

vaccine strain (influenza A/New Caledonia/20/99). It stayed stable since the 2000-01

seasons. The frequency was irregular during the ten last seasons. Influenza A

(H1N1) was predominant in the 2000-01 season (Figure 18).

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1995

/96

1997

/98

1999

/200

0

2001

/02

2003

/04

0

20

40

60

80

100Influenza virus

rate (%)

Influenza A (H1N1) viruses

Singapoore/6/86Texas/36/91Bayern/7/95Wuhan/371/95New Caledonia/20/99Egypt/96/2002Madagascar/57794/2000

Figure 18 : Influenza A (H1N1) strains which circulated in Switzerland since 1995. Each colour represents a particular influenza strain. The values represent the rate of each strain in comparison to all influenza A strains detected during the season.

Recent outbreaks of avian influenza among chickens show that, the influenza A

(H5N1) virus is endemic in Asia today: The reservoir is in aquatic birds which gives

the virus the possibility to infect regularly birds with terrestrial breeding. The increase

of the number of epidemics in poultry flocks increases the risk of the passage of the

avian influenza A (H5N1) virus to man. Such events are now more frequently

described. The virus caused numerous epidemics which was not the case until

recently. Eight countries in South Asia East were affected in spring 2004. Nowadays,

avian influenza A (H5N1) virus is still causing epidemics in poultry farms in Vietnam

and in Indonesia. In total 107 human victims were proven of whom 54 died (update

from WHO: June 17th, 2005). Until now infection of humans by the avian influenza A

(H5N1) virus was only observed after exposure to infected birds. No transmission

between humans could be proven. However, the risk exists that the virus adapts to

man and starts circulating. For this reason the National Centre of Influenza must be

able to detect such viruses in man. Different combinations of primers were developed

and tested for the detection of this avian influenza A (H5N1) virus. The virus is highly

variable a fact which requires a continuous verification and adaptation of the

reagents.

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8. Bibliography

de Jong MD, Bach VC, Phan TQ, Vo MH, Tran TT, Nguyen BH, Beld M, Le TP, Truong HK, Nguyen VV, Tran TH, Do QH, Farrar J. Fatal avian influenza A (H5N1) in a child presenting with diarrhea followed by coma. N Engl J Med. 2005 Feb 17;352(7):686-91.

Horimoto T, Fukuda N, Iwatsuki-Horimoto K, Guan Y, Lim W, Peiris M, Sugii S, Odagiri T, Tashiro M, Kawaoka Y., Antigenic differences between H5N1 human influenza viruses isolated in 1997 and 2003. J Vet Med Sci. 2004 Mar; 66(3):303-5.

Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature. 2004 Jul 8;430(6996):209-13.

Thomas Y., L. Kaiser, W. Wunderli. Influenza Surveillance in Switzerland, Sentinella study. Winter 2003-04. 2004.

Thomas Y., W. Wunderli. Influenza Surveillance in Switzerland, Sentinella study. Winter 1997-98, 1998.

Tran TH, Nguyen TL, Nguyen TD, Luong TS, Pham PM, Nguyen VC, Pham TS, Vo CD, Le TQ, Ngo TT, Dao BK, Le PP, Nguyen TT, Hoang TL, Cao VT, Le TG, Nguyen DT, Le HN, Nguyen KT, Le HS, Le VT, Christiane D, Tran TT, Menno de J, Schultsz C, Cheng P, Lim W, Horby P, Farrar J; World Health Organization International Avian Influenza Investigative Team. 2004, Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med. 2004 Mar 18;350(12):1179-88. Epub 2004 Feb 25.

Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, Puthavathana P, Uiprasertkul M, Boonnak K, Pittayawonganon C, Cox NJ, Zaki SR, Thawatsupha P, Chittaganpitch M, Khontong R, Simmerman JM, Chunsutthiwat S. 2005, Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med. 2005 Jan 27;352(4):333-40

Van Elden LJ, Nijhuis M, Schipper P, Schuurman R, van Loon AM, Simultaneous detection of influenza viruses A and B using real-time quantitative PCR; J Clin Microbiol 2001 Jan;39(1):196-200

WHO, 25.02.2005. Weekly epidemiological record, Recommended composition of influenza virus vaccine composition for use in the 2005-06 influenza season, WER 80, n° 8, p71-75.

WHO, 15.04.2005. Update: International response to the inadvertent distribution of H2N2 influenza virus: Destruction of virus panels proceeding, http://www.who.int/csr/disease/ influenza/h2n2_2005_04_15/en/index.html

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WHO, 17.06.2005. Report of cumulative number of confirmed human cases of avian influenza A (H5N1) reported to WHO : http://www.who.int/csr/disease/avian_influenza/ country/cases_table_2005_06_17/en/index.html

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Annexe 1

Primers and probes used in real-time PCR for the detection and characterisation of influenza viruses

Target virus Primers/ Probes

Sequence Target gene

Forward primer 5’- GGA CTG CAG CGT AGA CGC TT -3’ Reverse primer 5’- CAT CCT GTT GTA TAT GAG GCC CAT -3’ Influenza A

Probe 5’- CTC AGT TAT TCT GCT GGT GCA CTT GCC A -3’ Matrix

Forward primer 5’- AAA TAC GGT GGA TTA AAT AAA AGC AA -3’ Reverse primer 5’- CCA GCA ATA GCT CCG AAG AAA -3’ Influenza B

Probe 5’- CAC CCA TAT TGG GCA ATT TCC TAT GGC -3’ Hemagglutinin

Forward primer 5’- ATG GTA ATG GTG TTT GGA TAG GAA G -3’ Reverse primer 5’- AAT GCT GCT CCC ACT AGT CCA G -3’ Neuraminidase 1

(N1) Probe 5’- TGA TTT GGG ATC CTA ATG GAT GGA CAG -3’

Neuraminidase

Forward primer 5’- AAG CAT GGC TGC ATG TTT GTG -3’ Reverse primer 5’- ACC AGG ATA TCG AGG ATA ACA GGA -3’ Neuraminidase 2

(N2) Probe 5’- TGC TGA GCA CTT CCT GAC AAT GGG CT -3’

Neuraminidase

HA5For 5’- CCCAAATATGTGAAATCAAACAGATT-3’ HA5Rev 5’- CAAATAGTCCTCTCTTTTTTCTTCTTCTC-3’ HA5Pro 5’- TGCGACTGGACTCAGAAATACCCCTCA-3’

Hemagglutinin

H5VietFor 5’-GGA TGG CAG GGA ATG GTA GA-3’ H5VietRev 5’-TCTATTGCCTTTTGAGTGGATTCTT-3’

H5VietPro 5’-TGGGTACCACCATAGCAACGAGCAGG-3’ Hemagglutinin

WHO H5-1 5’-GCCATTCCACAACATACACCC-3’

Influenza A/H5

WHO H5-2 5’-TAAATTCTCTATCCTCCTTTCCAA-3’ Hemagglutinin

Forward primer 5’- GGC AAC AGG AAT GAA GAA TGT TCC-3’ Reverse primer 5’- AAT CAG ACC TTC CCA TCC ATT TTC-3’ Influenza A/H7

Probe 5’ AGG CCT ATT TGG TGC TAT AGC GGG TTT CAT -3’

Hemagglutinin

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Annexe 2 : Detection of respiratory viruses during the 2004/05 season. MC-ILI: proportion of medical consultations for influenza-like illness; influ A: influenza A; not typed: influenza A which could not be sub-typed. Influ. B: influenza B; other resp. viruses: other respiratory viruses.

Week MC-ILI Samp. Influ. A Other resp. Total virus Total virus

03/04 Dates

(‰) rec. undet. H3N2 H1N1

Influ.B

viruses (n) (%)

39 18-sept-04 24-sept-04 1.1 10 1Para3 1 10

40 25-sept-04 01-oct-04 2 6 0 0

41 02-oct-03 08-oct-04 2 4 1Para1, 1ADV 2 50

42 09-oct-04 15-oct-04 1.2 11 0 0

43 16-oct-04 22-oct-04 1.2 7 0 0

44 23-oct-04 29-oct-04 1.9 6 0 0

45 30-oct-04 05-nov-04 2.1 14 0 0

46 06-nov-04 12-nov-04 2.5 22 0 0

47 13-nov-04 19-nov-04 2.8 33 1Para2 1 3

48 20-nov-04 26-nov-04 3.6 24 1 1VRS 2 8

49 27-nov-04 03-déc-04 2.4 20 1 2ADV 3 15

50 04-déc-04 10-déc-04 2.8 13 1 1Para1, 1Para3 3 23

51 11-déc-04 17-déc-04 3.2 24 1 1 4

52 18-déc-04 24-déc-04 4.3 35 2 1 5 2ADV, 1VRS 11 31

53 25-déc-04 31-déc-04 8.6 22 2 2 2 1ADV 7 32

1 01-janv-05 07-janv-05 9.5 35 12 2 2ADV, 3VRS,1Para1 20 57

2 08-janv-05 14-janv-05 9.4 33 5 3 8 24

3 15-janv-05 21-janv-05 16 42 1 19 1 2ADV, 1Para3,1VRS 25 60

4 22-janv-05 28-janv-05 25.9 61 1 22 6 2 1VRS 32 52

5 29-janv-05 05-févr-05 45.8 70 40 2 3 1Para1,1Para2 47 67

6 05-févr-05 11-févr-05 61.7 61 2 30 1 2 1VRS 36 59

7 12-févr-05 18-févr-05 59.5 71 2 33 5 1 1VRS 42 59

8 19-févr-05 25-févr-05 40.4 60 1 25 2 2 2VRS 32 53

9 26-févr-05 05-mars-05 25.8 51 1 15 4 1 1ADV 22 43

10 05-mars-05 11-mars-05 17.7 37 11 2 7 20 54

11 12-mars-05 18-mars-05 13.1 31 5 1 6 2VRS 14 45

12 19-mars-05 25-mars-05 7.2 16 1 2 5 1Para3 9 56

13 26-mars-05 01-avril-05 3.9 9 1 2 1ADV, 1VRS 5 56

14 02-avril-05 08-avril-05 2.1 5 1 1 20

15 09-avril-05 15-avril-05 0.8 1 1 1 100

16 16-avril-05 22-avril-05 2.1 1 0 0

Total 835 8 225 35

268 41

309 36 345 41 %

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Annexe 3 : Antigenic analysis of influenza A (H3N2)-like strains

Antisera

A (H3N2) strains A/Shantou A/California A/Wellington A/Wyoming

A/Shantou/1219/04 (H3N2) 256 32 128 64

A/California/4/04 (H3N2) 256 256 32 512

A/Wellington/1/04 (H3N2) 256 128 256 512

A/Wyoming/3/03 (H3N2) 64 256 128 512

Date Seq N° Typisation A/Shan A/Cal A/Well A/Wyo

08-févr-05 7336 InfA H3N2 California/7/04 512 512 256 512




22-mars-05 9034 InfA H3N2 California/7/04 256 256 128 256














14-avr-05 9799 InfA H3N2 California/7/04 128 128 64 128






27-janv-05 6811 InfA H3N2 California/7/04 64 128 32 32





27-janv-05 6801 InfA H3N2 California/7/04 32 64 64 64




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07-févr-05 7270 InfA H3N2 Shantou/1219/04 512 128 128 256

06-janv-05 5970 InfA H3N2 Shantou/1219/04 512 256 512

01-mars-05 8222 InfA H3N2 Shantou/1219/04 256 128 64 128










17-févr-05 7738 InfA H3N2 Shantou/1219/04 256 256 256



















26-nov-04 4689 InfA H3N2 Shantou/1219/04 256 128 128











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30-nov-04 4817 InfA H3N2 Shantou/1219/04 128 64 64


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23-déc-04 5676 InfA H3N2 Shantou/1219/04 128 32 64




















04-mars-05 8378 InfA H3N2 Shantou/1219/04 64 32 64















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02-févr-05 6988 Inf A H3N2 Wyoming/3/03 64 128 256

03-janv-05 5779 Inf A H3N2 Wyoming/3/03 32 64 128



22-févr-05 7932 Influenza A H3N2 : 16 32 32 32




24-janv-05 6624 Influenza A H3N2 : 32 32 64



01-févr-05 6933 Influenza A H3N2 : 32 32 64













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21-janv-05 6573 Influenza A H3N2 : 8 32 64 Samples sent to MRC London for further analysis

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Annexe 4 : Antigenic analysis of influenza A (H1N1)-like strains

Antisera

A (H1N1) strains A/New Cal. A/Beij. A/Madaga.

A/New Caledonia/20/99 2048 64 1024 A/Beijing/262/95 512 1024 256 A/Madagascar/57794 512 32 256

IHA titre Date Seq. N° Typisation

A/NC A/Beij A/Madag 11.03.2005 8719 A/Madagascar/57794/00 1024 256 256 26.03.2005 9163 A/Madagascar/57794/00 512 32 256 30.03.2005 9286 A/Madagascar/57794/00 512 64 256 06.04.2005 9497 A/Madagascar/57794/00 512 32 256 08.03.2005 8493 A/Madagascar/57794/00 512 64 256 28.02.2005 8703 A/Madagascar/57794/00 512 64 128 15.03.2005 8816 A/Madagascar/57794/00 512 32 128 22.03.2005 9066 A/Madagascar/57794/00 512 32 128 24.01.2005 6714 A/Madagascar/57794/00 256 128 512 27.12.2004 5741 A/Madagascar/57794/00 256 64 256 14.01.2005 6348 A/Madagascar/57794/00 256 64 256 27.01.2005 6889 A/Madagascar/57794/00 256 32 256 27.01.2005 7357 A/Madagascar/57794/00 256 32 256 22.12.2004 5673 A/Madagascar/57794/00 256 32 128 10.01.2005 6260 A/Madagascar/57794/00 256 32 128 04.02.2005 7332 A/Madagascar/57794/00 256 32 128 10.02.2005 7554 A/Madagascar/57794/00 256 32 128 28.02.2005 8230 A/Madagascar/57794/00 256 64 128 03.03.2005 8384 A/Madagascar/57794/00 256 16 128 25.02.2005 8219 A/Madagascar/57794/00 256 32 64 14.02.2005 7628 A/Madagascar/57794/00 256 15.02.2005 7754 A/Madagascar/57794/00 256 32 21.02.2005 7937 A/Madagascar/57794/00 256 32 14.12.2004 5365 A/Madagascar/57794/00 128 32 256 24.01.2005 6720 A/Madagascar/57794/00 128 64 256 15.02.2005 7675 A/Madagascar/57794/00 128 32 256 27.12.2004 5742 A/Madagascar/57794/00 128 32 128 10.01.2005 6188 A/Madagascar/57794/00 128 32 128 02.02.2005 7072 A/Madagascar/57794/00 128 32 128 27.01.2005 7352 A/Madagascar/57794/00 128 64 128 18.01.2005 6442 A/Madagascar/57794/00 128 16 64 24.01.2005 6734 A/Madagascar/57794/00 128 32 64 14.02.2005 7632 A/Madagascar/57794/00 128 16.02.2005 7757 A/Madagascar/57794/00 128 64 07.03.2005 8619 A/Madagascar/57794/00

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Annexe 5 : Antigenic analysis of influenza B-like strains

Antisera

B strains B/Jiang. B/Harb. B/Sich. B/HK. B/Beij. B/Shan.

B/Jiangsu/10/2003 1024 64 128 16 32 < 16

B/Harbin/7/94 < 16 512 128 256 64 32 B/Sichuan/379/99 < 16 128 256 64 32 < 16 B/Hong Kong/335/2001 < 16 64 < 16 256 < 16 1024 B/Beijing/184/93 < 16 512 128 128 256 16 B/Shandong/7/97 < 16 < 16 < 16 32 < 16 1024

Antisera Date Seq N° Typisation B/Jiang B/Har B/Sich B/HK B/Bris B/Beij B/Shan

5-janv-05 5972 InfB Shandong/7/97 <16 <16 <16 <16 32 <16 1024

2-févr-05 7046 InfB Shandong/7/97 <16 <16 <16 <16 64 <16 512






23-déc-04 5710 InfB Shandong/7/97 <16 <16 <16 <16 32 <16 512




30-mars-05 9287 InfB Shandong/7/97 <16 <16 <16 <16 16 <16 512





10-déc-04 5303 InfB Shandong/7/97 <16 <16 <16 8 32 <16 256


22-mars-05 9164 InfB Jiangsu/10/02 256 128 64 32 <16 64 <16

24-févr-05 8161 InfB Jiangsu/10/03 256 64 64 32 <16 16 <16




27-déc-04 5738 InfB Jiangsu/10/03 256 64 64 32 <16 32 <16


10-mars-05 8716 B/Sichuan/379/99 128 128 64 64 <16 32 <16

21-mars-05 9275 B/Sichuan/379/99 128 64 64 <16 <16 <16 <16

18-mars-05 9274 B/Sichuan/379/99 128 64 64 <16 <16 <16 <16

22-mars-05 9070 B/Sichuan/379/99 128 64 64 32 <16 32 <16

21-mars-05 9029 B/Sichuan/379/99 128 64 64 32 <16 16 <16

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Antisera Date Seq N° Typisation B/Jiang B/Har B/Sich B/HK B/Bris B/Beij B/Shan

16-mars-05 8907 B/Sichuan/379/99 128 64 64 32 <16 32 <16

16-mars-05 8906 B/Sichuan/379/99 128 64 64 32 <16 32 <16

16-mars-05 8903 B/Sichuan/379/99 128 64 64 32 <16 16 <16

31-mars-05 9365 B/Sichuan/379/99 128 64 64 32 <16 16 <16

9-mars-05 8623 B/Sichuan/379/99 128 16 64 32 <16 16 <16

9-mars-05 8624 B/Harbin/7/94 128 64 32 32 <16 16 <16

11-mars-05 9273 B/Sichuan/379/109 64 256 256 256 <16 32 64

20-déc-04 5579 Influenza B 128 32 32 8 <16 8 <16

10-mars-05 8710 Influenza B 64 32 32 16 <16 8 <16

10-févr-05 7490 Influenza B 64 32 32 32 <16 16 <16

7-mars-05 8619 Co-infection A/H1 and B

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Annexe 6 : Alignement de l’hemagglutinine de souches récentes influenza A (H5N1) Les séquences de souches influenza A (H5N1) d’origine aviaire détectés chez l’homme ont été

alignées, dont 2 souches provenant de cas humains du Vietnam détectés au cours de l’année 2005 :

séquence 2 (Vietnam Sud) et séquence 3 (Vietnam Nord). Les primers et les sondes centrales

utilisées pour la détection des virus influenza A (H5N1) par PCR Sont mentionnés. En bleu sont

représentés les primers de PCR classique, en vert et rouge les primers de PCR en temps réel. La

région mentionnée en orange montre une délétion dans la séquence du virus du Nord du Vietnam).

Les 2 séquences de virus humains influenza A (H5N1) du Vietnam ont été aimablement fournies par

le laboratoire National de l’Influenza, du Vietnam du NIHE (National Institute of Hygiene and

Epidemiology) de Hanoi.

Who H5-1 HA5For HA5 Pro 910 1 2 3 4 5 6 7 8 9

10

12 13 14 15 16 17 18

11

H5Viet For H5VietPro HARev 1030 1 2 3 4 5 6 7 8 9

10

12 13 14 15 16 17 18

11

H5VietRev 1150 1 2 3 4 5 6 7 8 9

10

12 13 14 15 16 17 18

11

Who H5-2 1275 1 2 3 4 5 6 7 8 9

10

12 13 14 15 16 17 18

11

Final Total - HUG

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