INFLUENZA AND PRODUCTION OF VACCINES AGAINST INFLUENZA VIRUSES
Influenza virus surveillance in Switzerland Season 2014 2015...Viren, die diese Saison zirkulierten,...
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Influenza virus surveillance in Switzerland Season 2014 – 2015 National Reference Centre of Influenza Laboratory of Virology University Hospitals of Geneva, 4, Rue Gabrielle Perret-Gentil 1211 GENEVA 14 – SWITZERLAND © Pascal Cherpillod
Transcript of Influenza virus surveillance in Switzerland Season 2014 2015...Viren, die diese Saison zirkulierten,...
Influenza virus surveillance in Switzerland
Season 2014 – 2015
National Reference Centre of Influenza
Laboratory of Virology
University Hospitals of Geneva,
4, Rue Gabrielle Perret-Gentil
1211 GENEVA 14 – SWITZERLAND
© Pascal Cherpillod
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Contacts
Dr Samuel Cordey Tel: +41/22 372 40 79 Fax: +41/22 372 49 90 : [email protected]
Dr Ana Rita Gonçalves Tel: +41/22 372 40 81 Cabecinhas Fax: +41/22 372 49 90 : [email protected] Mme Patricia Suter-Boquete Tel : +41/22 372 40 81 Fax: +41/22 372 49 90 : [email protected] Prof. Laurent KAISER Tel: +41/22 372 98 01 Fax: +41/22 372 40 97 : [email protected]
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Table of Contents
CONTACTS ........................................................................................................................................................ 2
TABLE OF CONTENTS ........................................................................................................................................ 3
ABBREVIATIONS ............................................................................................................................................... 5
ACKNOWLEDGEMENTS .................................................................................................................................... 6
RESUME – ZUSAMMENFASSUNG – SUMMARY ................................................................................................ 7
1 INTRODUCTION ..................................................................................................................................... 13
2 THE FLU/INFLUENZA VIRUS ................................................................................................................... 14
3 METHODOLOGY .................................................................................................................................... 15
3.1 CLINICAL IDENTIFICATION OF INFLUENZA CASES .................................................................................................. 15
3.2 VIROLOGICAL DETECTION OF INFLUENZA VIRUSES ............................................................................................... 16
3.3 ANTIGENIC AND GENETIC CHARACTERIZATION OF INFLUENZA VIRUSES ..................................................................... 17
3.3.1 Hemagglutination inhibition assay ............................................................................................... 20
3.3.2 Genetic characterization ............................................................................................................... 22
3.3.3 Antiviral resistance ........................................................................................................................ 22
4 RESULTS OF THE 2014-2015 FLU/INFLUENZA SEASON ........................................................................... 23
4.1 DETECTION OF INFLUENZA VIRUSES IN NASOPHARYNGEAL SAMPLES ........................................................................ 23
4.2 CHARACTERISTICS OF INFLUENZA VIRUSES DETECTED BY THE SENTINEL NETWORK ..................................................... 25
4.2.1 Stratification by age ...................................................................................................................... 25
4.2.2 Stratification by flu/influenza vaccination status .......................................................................... 26
4.3 ANTIGENIC AND GENETIC CHARACTERIZATION OF INFLUENZA VIRUSES ..................................................................... 27
4.3.1 Characterization of influenza A/H3N2 .......................................................................................... 28
4.3.2 Characterization of influenza A/H1N1pdm09 viruses ................................................................... 31
4.3.3 Characterization of influenza B viruses ......................................................................................... 33
4.4 ANTIVIRAL RESISTANCE.................................................................................................................................. 38
5 USING THE FLUSURVER TOOL FOR RAPID IDENTIFICATION OF KNOWN ANTIVIRAL RESISTANCE MUTATIONS ................................................................................................................................................... 40
6 WHO RECOMMENDATION FOR THE COMPOSITION OF INFLUENZA VIRUS VACCINES FOR THE 2015-2016 FLU/INFLUENZA SEASON ................................................................................................................................ 41
7 HUMAN INFECTION WITH ANIMAL INFLUENZA VIRUSES ...................................................................... 42
7.1 SURVEILLANCE OF SWINE-TO-HUMAN FLU/INFLUENZA VIRUSES TRANSMISSION IN SWITZERLAND ................................. 42
7.2 OTHER INFLUENZA A SUBTYPES (NON-SENTINEL DATA)........................................................................................ 43
8 AVIAN INFLUENZA A IN ANIMALS (CURRENT UPDATE) ......................................................................... 46
9 DISCUSSION .......................................................................................................................................... 47
10 REFERENCES .......................................................................................................................................... 51
ANNEX 1: WEEKLY REPORT OF INFLUENZA VIRUS DETECTION AND VIRUS CHARACTERISTICS ........................ 56
ANNEX 2A: HEMAGGLUTINATION INHIBITION OF INFLUENZA A/H3N2 VIRUSES ........................................... 57
ANNEX 2B: HEMAGGLUTINATION INHIBITION OF INFLUENZA A/H3N2 VIRUSES ........................................... 58
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ANNEX 3: HEMAGGLUTINATION INHIBITION OF INFLUENZA A/H1N1 PDM09 VIRUSES .................................. 59
ANNEX 4A: HEMAGGLUTINATION INHIBITION OF INFLUENZA B VIRUSES ..................................................... 60
ANNEX 4B: HEMAGGLUTINATION INHIBITION OF INFLUENZA B VIRUSES ...................................................... 61
ANNEX 5: ANTIGENIC ANALYSES OF INFLUENZA A/H3N2 VIRUSES (WITH 20NM OSELTAMIVIR) 2015-02-24, MRC-NIMR ..................................................................................................................................................... 62
ANNEX 6: ANTIGENIC ANALYSES OF INFLUENZA A/H1N1PDM09 VIRUSES (2015 01 28), MRC-NIMR .............. 63
ANNEX 7: ANTIGENIC ANALYSES OF INFLUENZA B VIRUSES (YAMAGATA LINEAGE) 2015-01-28, MRC-NIMR . 64
ANNEX 8: PHYLOGENETIC COMPARISON OF INFLUENZA A/H1N1PDM09, HA GENES, MRC-NIMR .................. 65
ANNEX 9: PHYLOGENETIC COMPARISON OF INFLUENZA A/H1N1PDM09, NA GENES, MRC-NIMR.................. 66
ANNEX 10: PHYLOGENETIC COMPARISON OF INFLUENZA A/H3N2, HA GENES, MRC-NIMR ........................... 67
ANNEX 11: PHYLOGENETIC COMPARISON OF INFLUENZA A/H3N2, NA GENES, MRC-NIMR ........................... 68
ANNEX 12: PHYLOGENETIC COMPARISON OF INFLUENZA B YAMAGATA, HA GENES, MRC-NIMR .................. 69
ANNEX 13: PHYLOGENETIC COMPARISON OF INFLUENZA B YAMAGATA, NA GENES, MRC-NIMR .................. 70
ANNEX 14: PHYLOGENETIC COMPARISON OF INFLUENZA B VICTORIA, NA GENES, MRC-NIMR ...................... 71
ANNEX 15: ANTIVIRAL SENSITIVITY TESTING ON INFLUENZA A VIRUSES, MRC-NIMR ..................................... 72
ANNEX 16: SEQUENCING PRIMERS USED DURING THE 2014-2015 SEASON .................................................... 73
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Abbreviations
CDC: Centers for Disease Control and Prevention
CPE: cytopathic effect
CT: cycle threshold
ECDC: European Centre for Disease Control and Prevention
FOPH: Swiss Federal Office of Public Health
GISRS: Global Influenza Surveillance and Response System
HA: hemagglutinin
HI: hemagglutination inhibition test
HPAI: highly pathogenic avian influenza virus
HRI: highly reduced inhibition
HUG: University of Geneva Hospitals
ILI: influenza-like illness
LPAI: low pathogenic avian influenza virus
MC-ILI: medical consultations for influenza-like illness
MDCK: Madin-Darby canine kidney cells
MDCK-SIAT1: sialic acid-enriched MDCK cells
MP: matrix protein
MRC-NIMR: Medical Research Centre-National Institute of Medical Research
NA: neuraminidase
NAI: neuraminidase enzyme-inhibitor
NRCI: National Reference Centre of Influenza
NSP: non-structural protein
QIV: quadrivalent Influenza vaccine
RBC: red blood cells
RNP: ribonucleoprotein
rRT-PCR: real-time reverse-transcription polymerase chain reaction
TIV: trivalent Influenza vaccine
USDA: United States Department of Agriculture
VetVir: National Veterinarian Institute, Zurich, Switzerland
WHO: World Health Organization
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Acknowledgements
We would like to take this opportunity to extend our grateful thanks to :
- The Sentinel network, collaborating practitioners, and the persons who
accepted to participate in the study.
- Rita Born, Diana Guido, Francisca Morán Cadenas, Claudia Schmutz,
Andreas Birrer, Sabine Walser, and Daniel Koch, Swiss Federal Office of
Public Health (FOPH), Bern, Switzerland.
- John McCauley, Rod Daniels, and Vicky Gregory, World Health
Organization (WHO) Reference Laboratory, Medical Research Centre and
National Institute of Medical Research (MRC-NIMR), London, UK, for their
constant support and help during the epidemic.
- Maja Lièvre, Christian Fuster, and Wenghing Zhang, WHO Headquarters,
Geneva, Switzerland. Caroline S. Brown, Programme Manager, Influenza
and Other Respiratory Pathogens, Communicable Diseases, Health
Security and Environment, WHO Regional Office for Europe, Copenhagen,
Denmark, for her many efforts to promote European influenza surveillance
in non-European Union member countries.
- Christiane Monnet-Biston and Danielle Massimino, Laboratory of Virology,
University of Geneva Hospitals, Geneva, Switzerland, for their valuable
ongoing administrative support.
- Colette Nicolier University of Geneva Hospitals, Geneva, Switzerland, for
her major contribution to the NRCI website.
- Werner Wunderli, Zurich, Switzerland, for his contribution to this report.
- Members of the Swiss National Reference Centre for Emerging Viruses,
University of Geneva Hospitals, Geneva, Switzerland, who collaborate
regularly with the NRCI through fruitful and instructive discussion.
- All members of the Laboratory of Virology who have collaborated with the
NRCI.
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RESUME – ZUSAMMENFASSUNG – SUMMARY
Résumé
La surveillance Sentinelle de la grippe a débuté le 29 septembre 2014 pour se
terminer le 17 avril 2015. Le taux de consultations médicales pour symptômes de
grippe a dépassé le seuil épidémique de 70 cas pour 100'000 habitants pendant 12
semaines (S2/2015 à S13/2015). Les premiers virus Influenza de la saison ont été
détectés dès le début du mois de novembre (S45/2014) pour ensuite culminer au
cours de la semaine 6/2015 avec un taux de prélèvements positifs s’élevant à 74.4%.
Le taux maximal de consultations médicales pour symptômes de grippe de 445 cas
pour 100'000 habitants (52.5‰) a également été atteint en semaine 6/2015. Les
virus de l’Influenza A/H3N2 ont prédominé cette année avec 55.9% de l’ensemble
des virus grippaux détectés cette saison, surtout chez les plus de 65 ans (75%). Les
virus de l’Influenza B ont co-circulé (29.6%) avec les A/H3N2, devenant majoritaires
en fin de saison. Ces derniers étaient légèrement plus nombreux chez les 30-64
(36.4%). Quelques A/H1N1pdm09 ont également été détectés mais dans des
proportions plus faibles (14 %). La proportion de virus H1N1pdm09 était plus élevée
chez les 0-4 ans (22.2%) que pour les autres classes d’âge.
Les souches Influenza A/H3N2 ayant circulé cette saison étaient, en général,
faiblement reconnues par l’antiserum contre la souche vaccinale A/Texas/50/2012.
L’analyse phylogénétique effectuée sur le gène de l’hémagglutinine de ces virus a
confirmé cette observation. Ces résultats suggèrent que la couverture vaccinale des
souches A/H3N2 était sous-optimale. Les virus Influenza B appartenaient
majoritairement à la lignée Yamagata et seuls 7 B Victoria ont été observés. Ces
virus étaient antigéniquement proches de leur souche vaccinale respective. Les
résultats antigéniques contrastaient avec le résultats phylogénétiques qui groupaient
la majorité des virus Influenza B Yamagata avec la souche récente
B/Phuket/3073/13. Cette dernière étant la prochaine souche vaccinale d’Influenza B.
En résumé, les virus de l’Influenza B ayant circulé cette saison, n’ont été que
partiellement couverts par le vaccin 2014-15. La plupart des virus de l’Influenza
A/H1N1pdm09 étaient proches de la souche influenza A/St Petersburg/27/010, elle-
même très proche de la souche vaccinale A/California/7/2009 ; suggérant une bonne
couverture vaccinale contre les virus A/H1N1pdm09 présents en Suisse cet hiver.
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Aucun des virus de l’Influenza A et B analysés cette saison ne présentaient de
mutation connue pour leur conférer une résistance aux inhibiteurs de la
neuraminidase.
Plusieurs virus grippaux d’origine aviaire ont marqué l’actualité cette saison.
Notamment le A/H7N9 et le A/H5N1 qui ont régulièrement traversé la « barrière des
espèces » pour infecter des êtres humains en Asie (principalement en Chine) et en
Egypte, respectivement.
Depuis décembre 2014, les virus de l’Influenza A/H5N2 aviaire sont à l’origine d’une
épidémie de grippe conséquente chez le oiseaux d’élevage (poulets, dindes, poules
pondeuses…) actuellement en cours aux USA. Des virus A/H5N8 ont, quant à eux,
plus modestement touché des fermes avicoles en Europe cette saison.
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Zusammenfassung
Die Überwachung der Grippe durch das Sentinellasystem hat am 29. September
2014 begonnen, und wurde am 17. April 2015 beendet. Die Konsultationen aufgrund
grippeähnlicher Erkrankungen haben die epidemische Schwelle von 70 Fällen pro
100‘000 Einwohner während 12 Wochen überschritten (W2/2015 bis W13/2015). Die
ersten Influenza Viren der Saison sind Anfang November (W45/2014) festgestellt
worden, und der Anteil positiver Proben war im Laufe der Woche 6/2015 am
höchsten (74.4%). Der Höchstsatz von Konsultationen aufgrund grippeähnlicher
Erkrankungen von 445 Fällen pro 100‘000 Einwohner (52.5 ‰), ist ebenfalls in
Woche 6/2015 erreicht worden. Influenza A/H3N2 hat dieses Jahr mit 55.9% von der
Gesamtheit der Grippeviren überwogen, vor allem bei den über 65 Jährigen (75%).
Die Influenza B Viren (29.6%) haben parallel mit A/H3N2 zirkuliert , und wurden am
Ende der Saison dominant. Diese waren bei den 30-64 Jährigen leicht häufiger
(36.4%). Einige A/H1N1pdm09 sind ebenfalls festgestellt worden, aber zu einem
geringeren Anteil (14%). Der Anteil von H1N1pdm09 Viren war bei den 0-4 Jährigen
(22.2%) höher als für die anderen Altersklassen.
Die Influenza A/H3N2 Stämme, die in dieser Saison zirkulierten, waren im
Allgemeinen schwach vom Antiserum gegen den Impfstamm A/Texas/50/2012
erkennbar. Die phylogenetische Analyse, die auf dem Hämagglutinin Gen basierte,
hat diese Beobachtung bestätigt. Diese Ergebnisse weisen darauf hin, dass die
Abdeckung der Stämme A/H3N2 durch den Impfstoff suboptimal war. Die Influenza B
Viren gehörten mehrheitlich zur Yamagata Linie, und einzig 7 Fälle von B Victoria
wurden beobachtet. Diese Viren waren ihrem jeweiligen Impfstamm antigenisch
verwandt. Die antigenischen Ergebnisse kontrastierten mit den phylogenetischen
Ergebnissen, die die Mehrheit der Influenza B Yamagata Viren mit dem neuen
B/Phuket/3073/13 Stamm gruppierten. Der B/Phuket/3073/13 Stamm wird der
nächste Impfstamm von Influenza B sein. Zusammenfassend sind die Influenza B
Viren, die diese Saison zirkulierten, nur zum Teil durch den Impfstoff 2014-2015
abgedeckt worden. Die Mehrzahl der Influenza A/H1N1pdm09 Viren waren dem
Influenza A/St. Petersburg/27/010 nah, der selbst mit dem A/California/7/2009
Impfstamm verwandt ist. Dies zeigt, dass die Abdeckung durch den Impfstoff gegen
die A/H1N1pdm09 Viren die in diesem Winter in der Schweiz zirkulierten, gut war.
Keine der Influenza A und B Viren, die in dieser Saison analysiert wurden, enthielten
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eine Mutation, die für eine Resistenz gegen Neuraminidase Inhibitoren
verantwortlich ist.
Verschiedene Vogelgrippe Viren haben Schlagzeilen in dieser Saison gemacht.
Insbesondere A/H7N9 und A/H5N1, die regelmäßig die Speziesbarriere
übersprungen haben, und Menschen in Asien (besonders in China) und Ägypten
infizierten.
Seit Dezember 2014 gibt es in der USA eine A/H5N2 Vogelgrippe Epidemie bei
Zuchtvögeln (Hähnchen, Truthähne, Leghühner etc.). Ausserdem haben in dieser
Saison A/H5N8 Viren, in geringerem Masse, Geflügelzuchten in Europa betroffen.
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Summary
The Sentinel Network surveillance of influenza began on September 29, 2014, and
ended on April 17, 2015. The rate of medical consultations for an influenza-like
illness exceeded the epidemic threshold of 70 cases for 100‘000 inhabitants during
12 weeks (W2/2015 to W13/2015). The first seasonal influenza viruses were
detected in early November (W45/2014) and, culminated at week 6/2015. The
maximum rate of positive samples (74.4%) and the maximum rate of medical
consultations for influenza-like illness, i.e., 445 cases for 100'000 inhabitants (52.5‰)
were reached during week 6/2015. Influenza A/H3N2 viruses were prevalent this
year and represented 55.9% of the entire flu viruses detected during this season,
especially in the elderly (≥65 years, 75%). Influenza B viruses co-circulated (29.6%)
with the A/H3N2 viruses and even constituted the majority at the end of the season.
Influenza B strains were slightly more present in the 30-64 years old group (36.4%).
Some A/H1N1pdm09 were also detected but in lower proportions (14%). The ratio of
H1N1pdm09 virus was higher in the 0-4 years old group (22.2%) than in the other
age groups.
In general, influenza A/H3N2 strains that circulated this season were poorly
recognized by the antiserum against the vaccine strain A/Texas/50/2012. The
phylogenetic analysis carried out on the hemagglutinin (HA) genes confirmed this
observation. These results suggest that the A/H3N2 circulating strains were sub-
optimally covered by the vaccine. Influenza B viruses mainly belonged to the
Yamagata lineage and only 7 belonging to the B/Victoria lineage were observed.
These viruses were antigenically close to their respective vaccine stock. The
antigenic results contrasted with the phylogenic results which grouped most of the
influenza B Yamagata viruses with the recent influenza B/Phuket/3073/13, the latter
being the next influenza B vaccine strain. In conclusion, influenza B viruses
circulating this season were only partially covered by the 2014-2015 vaccine. Most of
the A/H1N1pdm09 influenza viruses were antigenically closely related to the
influenza A/St Petersburg/27/010 strain, itself being close to the vaccine strain
A/California/7/2009, which suggests a good vaccine coverage against
A/H1N1pdm09 viruses present in Switzerland in this season.
None of the influenza A and B viruses analyzed this season carried known mutations
providing resistance to neuraminidase inhibitors.
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Several influenza flu viruses of avian origin were prominent in the lay press this
season. In particular the A/H7N9 and the A/H5N1 viruses regularly crossed the
“species barrier” and infected humans in Asia (especially in China) and Egypt,
respectively.
Since December 2014, the avian influenza A/H5N2 viruses caused a consequent flu
epidemic in poultry, currently in progress in the USA. A/H5N8 influenza viruses were
responsible for more modest avian flu outbreaks in poultry farms in Europe.
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1 Introduction
The common cold and flu/influenza are both respiratory diseases, which frequently
circulate during winter in the Northern Hemisphere. As both share similar
flu/influenza-like symptoms, individuals have the tendency to mistake the common
cold for flu/influenza [1], and to underestimate flu/influenza severity and its public
heath impact. Indeed, colds are usually milder than flu and rarely require
hospitalization. In contrast, flu/influenza results generally in more acute symptoms
and has a preponderance to trigger complications in persons at risk, such as
children, the elderly >65 years old, pregnant women, and individuals suffering from
chronic disease(s) [2, 3]. The common cold can be caused by a wide variety of
viruses, whereas flu results from only an infection with influenza type A or B viruses
[4].
In Switzerland, it is estimated that 112’000 to 275’000 persons seek a medical
consultation for an Influenza-like illness (MC-ILI) suspicion per flu/influenza season.
In addition, several thousand hospitalizations and over 1000 deaths per year are
considered to be due to flu/influenza infections [5].
Due to flu/influenza virus pandemic potential and its major public health burden,
flu/Influenza virus infections are systematically reported to the Swiss Federal office of
Public Health (FOPH) by medical practitioners, hospitals and private diagnostic
laboratories. Since 1986, in addition to this obligatory notification, a network of
voluntary medical practitioners contribute to flu/influenza epidemiological surveillance
as part of the Swiss Sentinel surveillance system by the weekly reporting of MC-ILI
[5-7]. Flu/influenza virological surveillance is also achieved by the random collection
of nasopharyngeal swabs from community patients, which are then investigated at
the National Reference Centre (NRCI) in Geneva for the presence of influenza type A
and B, sub-typing, antigenic characterization, and antiviral resistance. Other
influenza A subtypes such as H5N1 (Egypt), H7N9 (Asia), including avian and swine
influenza strains could, occasionally be imported, and fall therefore under the
flu/influenza surveillance scope.
In this report, the NRCI present the details of the Swiss flu/influenza virological
surveillance data for 2014-2015 flu season.
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2 The flu/influenza virus
As mentioned previously flu/influenza disease is caused by the influenza virus, an
enveloped negative single-stranded ribonucleic acid (RNA) orthomyxiviridae virus
(Figure 1). There are three Influenza types, A, B and C, the latter being mainly
asymptomatic in humans [3]. Influenza A viruses have a wide host tropism, while
Influenza B viruses are exclusively human. [8]
Figure 1. The structure of an influenza viral particle. The hemagglutinin (HA1 and HA2), neuraminidase (NA) and ion channel M2 proteins are present at the surface of the virion. Their respective roles are virus attachment (HA1) to sialic acids and fusion (HA2), virion detachment from the cellular surface by cleaving the HA on the virus surface, and virion acidification required for fusion. The matrix protein (MP) is illustrated by a violet inner layer. The ribonucleoprotein (RNP) is present inside the viral capsid and surrounds the viral RNA. Image source [9].
Type A and B influenza viruses are responsible for the annual flu/influenza
epidemics. Since 2009, the major influenza subtypes circulating in Switzerland during
flu/Influenza seasons are H1N1pdm09 and H3N2 viruses for influenza A, and
Yamagata and Victoria lineages for influenza B.
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3 Methodology
3.1 Clinical identification of influenza cases
During the 2014-2015 flu/influenza season, 160 practitioners of the primary health
care system participated in the national flu/influenza surveillance system (Sentinel
Network). The geographic distribution of Sentinel participants is related to population
density in Switzerland as shown in Figure 2 (panels a and b). Medical practitioners
notify MC-ILI on a weekly basis. ILI is defined by: fever >38°C with or without a
feeling of sickness, myalgia, or an alteration of general status. In addition to fever,
acute respiratory symptoms, such as cough and/or sore throat, must be present [10].
This specific case definition was established in 2009 and has not changed since
then. A subgroup of 84 Sentinel practitioners (52.5%) also collected nasopharyngeal
swabs from patients with ILI. These clinical samples were then sent by regular mail
to the NRCI in Geneva for subsequent viral detection and characterization. The
sampling procedure of specimens is adapted to the flu/influenza epidemic phases as
follows:
1) Pre- and post-epidemic phase: the number of MC-ILI by Sentinel
practitioners remains below the pre-defined threshold level of 70 suspected
influenza cases per 100’000 inhabitants. The threshold value is defined by
the FOPH and based on data of the last 10 years (excluding pandemic
season 2009-10). During this phase, the screening for influenza viruses is
performed in all cases that fulfill the case definition.
2) Epidemic phase: the number of MC-ILI cases is ≥70 suspected Influenza
cases per 100’000 inhabitants. During this phase, the screening is only
performed in a subgroup of cases. Every fifth ILI case perpractitioner are
sent to the NRCI and screened for the presence of Influenza.
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Figure 2. Geographic distribution of the Swiss Sentinel Network practitioners (2014-2015) and population density in Switzerland (2013). a. Sentinel practitioners distribution. Yellow circles: location of participants (160) conducting clinical surveillance. Red circles: participants conducting both clinical surveillance and specimen collection (84). Circle size: participants per community. b. Population density in Switzerland per “commune”. Blue squares: absolute number of inhabitants per “commune”. Colored rectangles: number of inhabitants per km
2 of the total area.
3.2 Virological detection of influenza viruses
Nasopharyngeal swabs received at the NRCI were submitted to virus screening and
subtyping steps. The screening step, a one-step, real-time, reverse transcription
polymerase chain reaction (rRT-PCR, was adapted from the 2009 United States
Centers for Disease Control and Prevention (CDC) protocol. The CDC human
a.
b.
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influenza virus real-time RT-PCR diagnostic panel influenza A/B yping kit) has the
capacity to detect the presence of influenza A and/or B viral genomes in clinical
samples. The rRT-PCR targets are the matrix protein (MP) and the non-structural
protein (NSP) genes for influenza A and B viruses, respectively. For influenza A
or/and B specimens, a real-time PCR targeting the HA genes is performed in order to
discriminate between influenza A H1 and H3 subtypes, and B Yamagata (Yam) and
Victoria (Vic) lineages, respectively.
During the pre- and post-epidemic phases, a random selection of rRT-PCR-negative
specimens are inoculated on cells for viral culture (Figure 3). This strategy allows to
detect potential influenza strains that have “escaped” rRT-PCR detection. This could
be the case in the presence of drifted mutants carrying mutations in the genomic
regions targeted by the rRT-PCR screening.
3.3 Antigenic and genetic characterization of influenza viruses
During the surveillance period, a selection of influenza viruses detected each week
(see Figure 3 for details) are submitted to phenotypic and genotypic analysis.
Phenotypic analysis consists of an hemagglutination inhibition (HI) reaction
performed on viral cell culture supernatants. This assay evaluates the antigenic
similarity between the reference and the circulating influenza strains. The results of
this analysis provides a rapid estimation of the efficacy of the current influenza
vaccine-induced antibodies. Reference antisera have been kindly provided by the
World Health Organization (WHO) Collaborating Centre (WHO) Reference
Laboratory at the Medical Research Centre and National Institute of Medical
Research (MRC-NIMR), London, UK. HI reactions are performed with fixed guinea
pig red blood cells (RBC, Charles River, Lyon, France).
Subtyping of influenza strains by genotyping is achieved by sequencing selected viral
segments. At the NRCI we mainly focus on the sequencing of HA and neuraminidase
(NA) genes, and to a lesser extent on the MP genes. HA gene analysis allows to
determine the phylogeny (nucleotide level) of the circulating strains and how
genetically close they are to vaccine strains. Determining the NA gene sequence
allows to detect key mutations previously described as conferring antiviral resistance
to influenza viruses. Finally, MP gene sequencing allows to adapt the influenza A
screening rRT-PCR when necessary. In order to achieve a similar goal, a non-
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structural protein (NSP) sequencing protocol for influenza B will be developed at the
NRCI in the near future.
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84 practitioners collect
nasopharyngeal specimens from
persons with ILI
- -
rRT-PCR screening for Influenza A
rRT-PCR screening for Influenza B
n=937
Positive n=487 Negative n=450
Random sampling
n=57
Culture on cells
n= 145
Selection of specimens
with <30 Ct by rRT-PCR
≈ 5 first viruses/week≈
n=88
Phenotypic
characterization
HI
n=74
Genotypic
Characterization
HA gene sequencing
n=139
rRT-PCR
Subtyping/lineage
160 practitioners do clinical monitoring :
cases with ILI
Antiviral resistance
assessment
NA gene sequencing
n=139
Selection of specimens with Ct ≤30
≈ every fifth virus/subtype (pre and post-epidemic
phase) or every tenth virus/subtype (epidemic phase)≈
n= 139
Figure 3. Flow chart of Sentinel sample collection and processing. Numbers (n) represent the number of samples submitted to the described step during the 2014-2015 season.
20/73
3.3.1 Hemagglutination inhibition assay
This assay is based on the ability of the viral HA to bind to sialic acid present at the
surface of RBC. As HI analysis require a high concentration of influenza virus, a viral
amplification step is performed by inoculating the infected clinical samples on Madin-
Darby canine kidney (MDCK) cells and MDCK-sialic acid-enriched (MDCK-SIAT1)
cells, in parallel. According to our predefined selection criteria, a subgroup of five
specimens per week detected positive by rRT-PCR and with a cycle threshold (Ct)
value lower than 30 were inoculated on cells. In brief, 0.4 ml of transport medium
containing nasopharyngeal swab was incubated for 7 days under 5% CO2 at 33°C on
MDCK cells and 37°C on MDCK-SIAT1. The presence of virus was confirmed by the
presence of a cytopathic effect (CPE) under visible light (Leica®) and/or by an
immunofluorescence test using monoclonal influenza A and B antibodies combined
with mouse FITC-conjugate (Chemicon®, Temecula, CA, USA). Positive samples
were then submitted to an hemagglutination test in order to determine the virus titer.
A two-fold serial dilution was performed using 50 µl of viral suspension buffer in
SALK solution (5%). 25 µl of glutaraldehyde-fixed guinea pig RBC (1.5%) were
added for a 1 h incubation at 4°C. Hemagglutination titer is defined as the last dilution
in which a complete hemagglutination is still observed.
After titer determination, HI was performed as follows. 25 µl of reference antisera
were added in the first two wells of a 96-well plate. Two-fold dilutions were prepared
by adding 25 µl of SALK solution (5%) in the second well. 25 µl were then collected
from the same well, and the procedure repeated to the end of each line. 25 µl of viral
suspension containing 4 hemagglutination units were added to the antisera dilution
and incubated for 1 h at room temperature. 25 µl of guinea pig RBC were then added
to each well. The plates were incubated for 1 at 4°C. The HI titer corresponds to the
last antiserum dilution for which hemagglutination is still inhibited. This titer is
compared to the homologous titer obtained with reference strains submitted to their
corresponding antisera (antigenic table). The antigenic tables are flu/influenza
season and strain specific (Table 1) and, new ones are built each year.
A strain is considered as being antigenically related to a reference strain when the
obtained HI titer is no more than 4-fold below or above the one obtained with the
reference strain. Otherwise the strain is considered as being antigenically different
from the reference strain.
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Table 1. HI titers of reference influenza strains tested with the 2014-2015 reference antisera. HI reaction is performed as described in methodology section. HI titers mentioned in tables correspond to the highest dilution where an inhibition is still observed. In red: 2014-2015 flu vaccine strains.
H1N1pdm09 Anti-sera
Virus strain A/Brisbane/
59/07 A/California/
07/09 A/St Petersburg/
27/11 A/South Africa/
3626/13 A/Christchurch/
16/10
A/Brisbane/59/07
1024 <8 <8 <8 <8
A/California/07/09
<8 1024 64 32 32
A/St Petersburg/27/11
<8 512 512 64 32
A/South Africa/3626/13 16 512 512 256 128
A/Christchurch/16/10 <8 256 512 256 1024
H3N2 Anti-sera
Virus strain A/Victoria/ 361/11 egg
A/Texas/ 50/12
A/South Africa/ 4655/13
A/Switzerland/ 9715293/13
A/Hong Kong/146/13
A/Samara/ 73/13
A/HK/ 5738/14
A/Victoria/361/11
egg 2048 128 16 64 64 64 64
A/Texas/50/12
512 512 32 256 256 128 64
A/South Africa/4655/13
32 64 128 32 256 64 32
A/Switzerland/ 9715293/13 egg
128 128 32 512 128 128 512
A/Switzerland/ 9715293/13 cell
64 128 32 256 256 - -
A/Hong Kong/146/13
256 1024 32 256 1024 256 256
A/Samara/73/ 13
128 128 32 64 256 64 128
A/HK/5738/14 32 32 16 32 64 16 64
IB Anti-sera
Virus strain B/Brisbane/
60/08 B/Odessa/
3886/10 B/Johannesburg/
3964/12 B/Wisconsin/
01/10 B/Novosibirsk/
1/12 B/Massachusetts/
02/12 egg B/Phuket/ 3073/13
B/Brisbane/60/08 512 125 512 <8 <8 <8 <8
B/Odessa/3886/10 64 128 64 <8 <8 <8 <8
B/Johannesburg/3964/12 512 125 1024 <8 <8 <8 <8
B/Wisconsin/01/10 <8 <8 <8 512 128 256 64
B/Novosibirsk/1/12 <8 <8 <8 256 1024 256 32
B/Massachusetts/02/12 egg
<8 <8 <8 32 128 512 64
B/Phuket/3073/13 32 <8 <8 1024 256 1024 128
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3.3.2 Genetic characterization
At the NRCI, influenza strains are characterized by sequencing the HA 1 part of the
HA genes. As HA genes tend to evolve rapidly, comparing HA sequences of the
circulating strains with reference sequences, including those from the vaccine strains,
allows to evaluate viral diversity. A list of primers used for sequencing analysis are
presented in Annex 16.
3.3.3 Antiviral resistance
Antivirals against influenza viruses are not used routinely. However, hospitalized
patients, often immunocompromised cases, are increasingly treated with NA
inhibitors such as oseltamivir and more rarely zanamivir. This treatment can lead to
the selection of antiviral resistant strains [11] that could then be transmitted to the
community and potentially circulate in the population. Influenza viruses
spontaneously resistant to antivirals could also naturally emerge, as observed in the
past with seasonal influenza A/H1N1 [12, 13] and A/H3N2 viruses [14]. The resistant
variants can then either become the dominant species as for adamantane resistance
(M2 inhibitors), be replaced by fitter ones and/or sporadically reemerge.
Known mutations conferring antiviral resistance to a given Influenza subtype can be
monitored by sequencing the NA genes for NA inhibitor resistance, and M genes for
the M2 inhibitors. New antiviral resistances to NA inhibitors (NI) can be identified by
combining phenotypic NA enzyme-inhibitor (NAI) assays and NA genotyping
/sequencing. The latter technique is currently used at the NRCI and a phenotypic NAI
assay is currently under validation.
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4 Results of the 2014-2015 flu/influenza season
4.1 Detection of influenza viruses in nasopharyngeal samples
This winter influenza surveillance period was 29 weeks long from 29 September,
2014 (week 40/2014) to 17, April 2015 (week 16/2015). Of the 160 practitioners
participating in clinical surveillance in the six distinct regions, 84 collected a total of
937 nasopharyngeal swabs that were sent to the NRCI for screening. Overall,
487/937 samples (52%) were positive for influenza A and/or B by rRT-PCR (Figure
4a and Annex 1). Among these positive cases,343/487 were of type A (70.4%) and
144/487 (29.6 %) of type B (Annex 1). 272/487 (55.9%) of influenza A were A/H3N2,
68/487 (14%) A/H1N1pdm09, and 3/487 (0.6%) could not be further subtyped due to
a low viral load (Figure 4b). Concerning the influenza B viruses, 131/487 (26.9%)
were B Yamagata, 7/487 (1.4%) B Victoria, and 6/487 (1.2%) could not be
determined (Figure 4b).
Figure 4. Distribution of influenza viruses detected in nasopharyngeal specimens collected during the 2014-2015 season. a. Number and % of rRT-PCR-positive versus-negative specimens. (n=937). b. Distribution of the different types and subtypes of Influenza viruses (%). (n=487).
487 rRT-PCR positive (52%)
450 rRT-PCR negative (48%)
A undet.0.6%
A/H1N1 200914.0%
A/H3N255.9%
B undet.1.2%
B yam26.9%
B vic1.4%
a.
b.
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MC-ILI abruptly increased from week 52, reached the peak value at week 6, and
dropped from week 7 onwards. MC-ILI maintained above the epidemic threshold for
12 weeks. (data provided by FOPH, not shown).
From weeks 45/2014 to 9/2015, a majority of Influenza A/H3N2 and B/Yamagata
viruses were detected, with A/H3N2 being the dominant type. From week 10/2015 to
the end of the flu/influenza season the opposite observation was made.
A/H1N1pdm09 were regularly detected from weeks 1/2015 to 14/2015. Few sporadic
cases of B/Victoria were observed at the end of the season (weeks 10-16/2015).
(Figure 5)
As expected, a decrease in the number of samples collected was systematically
observed during weeks close to the holiday periods (weeks 52/2014-1/2015, 9/2015
and 13/2015). The maximal number of positive samples (n=58: 74.%) was reached at
week 6 and coincided with the peak of ILI suspected cases ‰ inhabitants (52.5‰).
(Figure 5)
Figure 5. Schematic illustration of 2014-2015 flu/influenza season. A undet.: influenza A, but the type could not be determined; A/H1N1 2009: influenza A/H1N1pdm09; A/H3N2 seasonal : influenza A/H3N2 viruses; B undet.: influenza B, but the type could not be determined; B-Yam: influenza B of the Yamagata lineage; B-Vic: influenza B of Victoria lineage; ILI 14/15 and 13/14: ILI suspected cases registered during the 2014-2015 and 2013-2014 season (‰); sampling: green arrow indicates the weeks when Sentinel practitioners sent a 1:5 samples for influenza screening (weeks 5 to 11).
0
10
20
30
40
50
60
70
80
90
100
40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Nu
mb
er
of
sa
mp
les
(n
)
Oct Nov Dec Jan Fev Mar Avr
A undet.
A H1N1 2009
A H3N2
B undet.
B-Yam
B-Vic
Total # of samples
‰ ILI CH 14/15
‰ ILI 13/14
Total # of positive samples
25/73
4.2 Characteristics of influenza viruses detected by the Sentinel Network
4.2.1 Stratification by age
In this section, the analyzed samples were classified according to the age of the
“source” individuals. Age groups were defined by the FOPH as follows: 0-4 years, 5-
14 years, 15-29, 30-64 years, and ≥ 65 years.
Among the 937 samples sent to the NRCI, 46.7% belonged to the 30-64 years old
group, 19.3% to the 15-29 years old, 15.5% to the 5-14 years old, 10.2% to the 0-4
years old, and finally 8% to the ≥ 65 years old group. The birth date was missing for
two individuals (0.2%). The proportion of Influenza positive versus negative samples
was similar for each age group (Figure 6a). Within the 486 Influenza positive samples
considered, 50% originated from the 30-64 years old , 17% from the 15-29 years old,
16% from the 5-14 years old, 9% from the 0-4 years old and 8% from the ≥65 years
old groups (Figure 6b).
The observed proportions of influenza subtypes were comparable between the five
age groups. B Victoria viruses were absent from the 15-29 years old group.
Nevertheless, only 7 B Vic were identified during the current flu/influenza season.
Of note, the rate of Influenza B Yamagata samples seemed to be slightly higher in
the 30-64 years old group when compared to the other four age groups. Influenza
A/H3N2 was the predominant subtype for all age groups, with the highest rate
observed in the ≥ 65 years old group (75%). (Figure 6c).
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Figure 6. Distribution of viruses detected and rate of positive samples detected (%) by age groups. a. Pie charts represent the proportion of positive versus negative samples per age group (n=936/937). b. Proportion of positive samples per age group (n=486/487). c. Histograms illustrate the distribution of the different Influenza viruses subtypes per age group. B Vic = B Victoria; B Yam = B Yamagata. Undet.= not able to be subtyped. Yo = years old.
4.2.2 Stratification by flu/influenza vaccination status
The vaccination status of individuals with a sample collected by Sentinel practitioners
was recorded according to the results of their viral analysis (Table 2).
Of the 937 persons with ILI, which were swabbed, at least 77 received the 2014-2015
flu/influenza vaccine before the beginning of the epidemic wave. Thirty-five of the 77
swabs were positive for influenza (45.5%), i.e. two influenza A/H1N1pdm09, 27
influenza A/H3N2, and six influenza B Yamagata. Among the 860 non-vaccinated
individuals, 452 had been detected positive for influenza (52.6%), as the following
distribution: 67 influenza A/H1N1pdm09, 244 A/H3N2, three influenza A not able to
be subtyped (undet), 125 influenza B Yamagata, 7 B Victoria and 6 B undetermined
viruses. Influenza A/H1N1pdm09 viruses were less prevalent in vaccinated
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0-4 5-14 15-29 30-64 ≥65
B-Vic
B-Yam
B undet.
H1N1 2009
A undet.
A H3N2
0-49% 5-14
16%
15-2917%
30-6450%
≥658%
POS NEG
47%
POS NEG
54%
POS NEG
POS
NEG
POS
NEG
56%
44%
55%
47%
53%
0-4
POS
NEG
n=486
0-4 yo
yo
5-14 yo
15-29 yo
30-64 yo ≥ 65 yo
55.6
22.2
15.6
4.4
2.2
60.8
12.7
22.8
2.5
67.5
1.3
22.5
1.3
8.8
47.1
15.7
33.9
0.8
0.8
1.7
75
7.5
15
2.5
a.
c.
b.
27/73
individuals than the 2013-2014 season and represented 2.9% and 7.6%,
respectively. For A/H3N2 viruses, similar vaccination rates were observed for both
the 2013-2014 and 2014-2015 seasons (10.3% and 10%, respectively). (Table 2).
Table 2. Samples distributed by analysis results and vaccination status of individuals. Neg: negative; undet.: Influenza virus not further analyzed (subtype and lineage) due to low viral dose. Yam: Yamagata-like virus, Vic: Victoria-like virus
4.3 Antigenic and genetic characterization of influenza viruses
A subset of influenza-positive samples were submitted to HI assays and genotyping
of HA, NA and M genes.
HI tested samples were chosen as follows: each week the first five rRT-PCR positive
samples (Ct value < 30) were cultured on MDCK and MDCK-SIAT cells. (Figure 3).
Viruses with sufficient hemagglutination titers were further characterized. Of the 88
rRT-PCR positive samples cultured, 76 grew on MDCK and/or MDCK-SIAT cells.
74/76 were successfully subtyped by HI. (Table 3 and Annexes 2-4)
Table 3. Antigenic characterization of influenza viruses season 2014-15
H3N2 H1N1 pdm 09 B-Yam B-Vic
A/South Africa/4655/13 24
A/Texas/50/12 4
A/St Petersburg/27/11 10
A/California/7/09 1
A/South Africa/3626/13 5
B/Massachusetts/02/12 19
B/Phuket/3073/13 1
B/Novosibirsk/1/12 5
B/Wisconsin/1/10 2
B/Odessa/3886/10 3
937 nasopharyngeal swabs
Neg
Influenza A Influenza B Total
Pos. H1N1 pdm09
H3N2 A
undet. Yam Vic
B undet.
Vaccinated 42 2 27 - 6 - - 35
Non vaccinated 408 67 244 3 125 7 6 452
Proportion vaccinated (%) 2.9 10 0 4.6 0 0
28/73
Samples selected for sequencing were chosen as follows: every fifth virus (rRT-PCR
Ct ≤30) per subtype during the pre and post-epidemic phases, and every tenth virus
(rRT-PCR Ct ≤30) per subtype during the epidemic phase.
HA and NA sequencing were performed for 139 samples; and 34 M genes were also
sequenced. 131/139 HA sequences were recovered, among which 63/69 belonged to
A/H3N2, 29/30 to A/H1N1, 35/36 to B Yamagata and 4/4 to B Victoria subtypes.
125/139 NA were successfully sequenced.:63/69 were A/H3N2, 29/30 A/H1N1, 29/36
B Yamagata and 4/4 B Victoria. All of the 18/18 A/H1N1 and 16/16 A/H3N2 M
sequences were recovered.
Of note, no significant changes were observed in the sequenced M genes suggesting
that our rRT-PCR screening was still sufficiently sensitive to detect the circulating
viruses.
A selection of 10 influenza viruses (4 A/H3N2, 3 A/H1N1pdm09 and 3 B) were also
sent to the WHO Collaborating Centre for Reference and Research on influenza
(MRC-NIMR) for HI analysis, HA and NA gene sequencing, and antiviral activity
assessment ( phenotypic analysis: see Annexes 5-7; phylogenic analysis: see
Annexes 8-14; and antiviral resistance: see Annex 15).
4.3.1 Characterization of influenza A/H3N2
As described in the last periodic reports of the MRC-NIMR, the antigenic
characterization of A/H3N2 viruses has become more and more difficult by HI assay.
Indeed, most of the current A/H3N2 strains, including ours, exhibited low reactivity
with the chosen reference antisera. According to the MRC-NIMR, this phenomenon
could be explained by a variable agglutination of RBC guinea pigs and a NA-
mediated agglutination of RBC.
28/74 HI characterized strains were A/H3N2, 24 were identified as A/South
Africa/4655/13-like strains and, as expected, had very low reactivity with the
reference antisera. The last 4 A/H3N2 (****8336, ****1338,****6955,****9293;
Annexes 2a and 2b) with much better HI titers were identified as A/Texas/50/12-like
strains. In contrast to the 24 A/South Africa/4655/13-like strains, the 4
A/Texas/50/12-like strains were antigenically similar to the 2014-2015 vaccine strain,
which was also a A/Texas/50/12-like (Annexes 2a and 2b).
29/73
4/4 of the A/H3N2 viruses sent to the MRC-NIMR were recovered. Of these 3/4 had
no HA activity but exhibited CPE and NA activity. Thus only 1/4 viruses could be
analyzed by HI. This was A/Switzerland/656/2014. According to the MRC-NIMR, it
was recognized well by antisera raised against A/Victoria/361/2011 Cell (genetic
group 3C.1), A/Samara/73/2013 (genetic group 3C.3), A/Stockholm/6/2014 (group
3C.3a), and A/Hong Kong/5738/2014 (group 3C.2a) but less well with the antiserum
raised against A/Switzerland/9715293/2013 (group 3C.3a). The MRC-NIMR noted
that the latter antiserum had a very low homologous titer in the test (Annex 5). As
expected for A/H3N2 viruses, A/Switzerland/656/2014 was poorly recognized by
antisera raised against the egg-propagated viruses (in genetic groups, 3C.1, 3C.2,
3C.2a, 3C.3a), except for the antiserum raised against the egg-propagated
A/Stockholm/6/2014, which recognized the test virus within 2-fold of the titers of the
antiserum for the homologous virus. (Annex 5). The A/Switzerland/656/2014 tested at
the NRCI was identified as an A/South Africa/4655/13-like virus (3C.3) (Annex
2a).[15].
At the genetic level, A/H3N2 viruses are divided into seven distinct genetic groups.
Two derived from A/Perth/16/2009 and five from A/Victoria/208/2009. The majority of
the current A/H3N2 viruses HA genes fall into subgroups 3B and 3C of group 3 of the
latter genetic clade. 3C subgroup is further subdivided into 3 groups: 3C.1, 3C.2 and
3C.3. [15]. The 2014/2015 vaccine virus, A/Texas/50/2012, belongs to genetic
subgroup 3C.1. Subgroups 3C.2 and 3C.3 can be defined by different mutations in
the HA sequence as follows:
3C.2: N145S in HA1 and D160N in HA2 (e.g. A/Hong Kong/146/2013).
3C.2a: also carries N144S, K160T, N225D, Q311H in HA1 and some also
carry F159Y in HA1 (e.g. A/Hong kong/5738/2014).
3C.3: T128A , R142G and N145S in HA1 (e.g.A/Samara/73/2013).
3C3a: also carries A138S, F159S and N225D in HA1. Some also carry K326R
in HA1 (e.g. A/Switzerland/9715293/2013). [15].
139/487 of the 2014-2015 season viruses isolated at the NRCI were genetically
characterized by HA sequencing. 131/139 HA sequences were recovered, among
which 63 belonged to A/H3N2 strains. 33/63 sequences clustered with the A/Hong
Kong/5738/2014 and contained the typical mutations K144S, N225D, K160T, and
Q311H as well as F159Y of the 3C.2a genetic group. The sequence from virus
30/73
A/Switzerland****9293/2015 was genetically divergent from group 3C.2a, but part of
group 3C.2 with the mutation N145S. 17/63 sequences were part of the 3C.3b group.
They clustered with the MRC-NIMR isolated strain A/Newcastle/22/2014 and almost
all contained mutations: K83R, K261Q and N122D. 10/63 sequences were part of the
3C.3 group. Finally, only 2/63 sequences clustered within the 3C.3a genetic group,
with the 2015-2016 vaccine strain A/Switzerland/9715293/2013. Both had mutations
A138S, F159S and N225D. (Figure 7).
The HA genes of 2 of the isolates that could not be analyzed by the MRC-NIMR by
HI, were further sequenced. As expected, both belonged to genetic group 3C.2a
(Annexes 5 and 10).
Of note, viruses of the 3C.2a group are known to have a glycosylation motif at 158-
160 in HA1. This particular motif seems to be responsible for a loss of binding to
RBC, thus explaining the poor or abolished reactivity of those viruses in HI. [16].
From the MRC-NIMR point of view, the titers obtained with viruses from the genetic
group 3C.2a could be explained by NA agglutination or/and reverted polymorphism
158-160 in HA1.
In summary, strains from groups 3C.2a and 3C.3a increased in frequency during the
2014-2015 flu/influenza season and those in 3C.2a became predominant worldwide
[17]. These two groups are considered antigenically distinct from the vaccine group
3C.1. This observations led to the recommendation of A/Switzerland/9715293/2013
as A/H3N2 strain for the 2015 Southern Hemisphere and the 2015/2016 Northern
Hemisphere vaccines. [18].
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Figure 7. Phylogenetic analysis of the HA gene of A/H3N2 viruses. In black: some of the influenza virus detected in the Sentinel Network during the 2014-2015 season. Red: 2014-2015 vaccine strain. Turquoise: 2015-2016 vaccine strain. Green: MRC-NIMR reference strains. Violet: some typical mutations described by the MRC-NIMR. Pink star corresponds to the expected location of A/Newcastle/22/2014. Blue: A/H3N2 genetic group 3C. Sequences were aligned using Geneious 6.1.8 MAFFT alignment (v7.017) with default settings. A consensus tree was built from 1000 original trees in ML (70% support threshold) constructed using Geneious 6.1.8 PHYML default settings.
4.3.2 Characterization of influenza A/H1N1pdm09 viruses
Among the 76 influenza viruses, which grew in cell culture, 17 were identified as
influenza A/H1N1pdm09. 16/17 gave sufficient HA titers to be further characterized.
One of these influenza viruses was well recognized by the vaccine antiserum
directed against influenza A/California/7/2009, and 10 gave better HI titers to the
influenza A/Saint-Petersburg/27/2011 that is considered to be antigenically close to
the A/California/7/2009. The five last strains were identified as A/South
Africa/3626/13-like. (Table 3 and Annex 3)
A198S
V293I
N312S
Q33R
N278K
K144N
N145S
A138S
F159S
L157S
K83R
K261Q
N122D
F159Y
Q311H
N225D
K160T
K144S
L7I
R142G
T128AN225D
3C.3a
3C.2a
3C.2
3C.2
3C.3
3C.3
3C.1
3C.3b
*
32/73
The three A/H1N1pdm09 sent to the MRC-NIMR were successfully recovered. All
were recognized well by the antiserum raised against the current vaccine virus
A/California/7/2009 at titers within two-fold of its homologous titer, as well as by
antisera against almost all of the other reference viruses. Results of these analyses
confirmed that these strains were well recognized by antiserum raised against the
vaccine virus A/California/7/2009. (Annex 6).
Influenza A/H1N1pdm09 HA1 sequences can be divided into up to 10 different
groups. Influenza A/California/7/2009, the 2015-2016 vaccine strain is a
representative of group 1 [15]. Most of the viruses isolated recently match into the
genetic group 6. This group is further subdivided into subgroups 6A, 6B and 6C.
6B: carry substitutions K163Q, and A256T in HA1 (e.g. A/South
Africa/3626/2013).
6C: V234I in HA1; some carry also the substitutions V30A and A186T in HA1.
Genetic subgroups 6B and C differ from A/California/7/2009 but are antigenically
similar enough to cross-react as observed in the HI assays.
All HA1 sequences of influenza A/H1N1pdm09 detected in Switzerland displayed the
D97N and S203T mutations described in the genetic group 6 and the K163Q and
A256T mutations specific to the subgroup 6B strains represented by the influenza
A/South-Africa/3626/2013. (Figure 8 and Annex 8)
The HA genes from two of the three isolates sent to the MRC-NIMR were analyzed;
both HA sequences clustered into genetic group 6B, similar to all the other A/H1N1
HA sequences isolated at the CNRI (Annexes 6 and 8). Taken together, these results
show that influenza A/H1N1/pdm09 viruses circulating in Switzerland this season
belonged to the subgroup 6B. This was the case for most of the A/H1N1/pdm09
viruses isolated throughout Europe [17].
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Figure 8. Phylogenetic analysis of the HA1 gene of A/H1N1pdm09-like viruses. In red, 2014-2015 vaccine strain. In green, selected reference sequences. In blue, A/H1N1pdm09 genetic groups. Violet: some typical mutations described by the MRC-NIMR. Sequences were aligned using Geneious 6.1.8 MAFFT alignment (v7.017) with default settings. A consensus tree was built from 1000 original trees in ML (70% support threshold) constructed using Geneious 6.1.8 PHYML default settings
4.3.3 Characterization of influenza B viruses
Thirty influenza B viruses grown in cell culture were characterize by HI. Twenty-
seven belonged to the Yamagata lineage and three were part of the Victoria lineage.
Among the 27 influenza B Yamagata, 19 reacted with the antisera raised against
B/Massachusetts/02/2012 egg, one against B/Phuket/3073/2013, five against
B/Novosibirsk/1/12-like, and two against B/Wisconsin/1/2010 strains. The overall HI
titers obtained for almost all the tested strains remained low. The Three influenza B
Victoria analyzed by HI were recognized by the antiserum against B/Odessa/3886/10
within two-fold of the antiserum for the homologous strain titer. (Annexes 4a and 4b).
6B
K163Q
A256T
D97N
S203T
P83S
6
6A
7
3
5
4
34/73
Two of the three influenza B viruses sent to the MRC-NIMR were successfully grown
in cell culture. They were poorly recognized by antisera raised against cell culture-
propagated viruses in clade 2 (B/Estonia/55669/2011 and B/Massachusetts/02/
2012). In addition, recognition by the antiserum raised against the cell culture-
propagated B/Phuket/3073/2013 (clade 3) was also very low with titers eight-fold or
16-fold reduced over the titer of the antiserum for the homologous virus. The
analyzed viruses were recognized at titers at or within two-fold or four-fold of the
homologous titers by antisera raised against the egg-propagated clade 3 viruses
(B/Wisconsin/1/2010, B/Phuket/3073/2013 and B/Hong Kong/3417/2014). These
viruses were recognized very poorly by the antiserum raised against the clade 2 virus
propagated in eggs (B/Massachusetts/02/2012). (Annex 7).
Forty Influenza B viruses HA genes were submitted to sequencing. 40/40 were
recovered successfully. Among these, 36/40 were B Yamagata-like viruses all
belonging to the clade 3 (Figure 9). The last four sequences corresponded to B
Victoria like viruses from clade 1A (Figure 10). From the two samples sent to the
MRC-NIMR, both had HA genes clustering in clade 3, the B/Wisconsin/1/2010 and
B/Phuket/3073/2013 clade. Clade 3 viruses are currently the predominant clade of
influenza B viruses in circulation. (Annexes 7 and 12)
Of note, B/Switzerland/****9016/2014, B/Switzerland/*****1773/2015, and
B/Switzerland/*****7033/2015 had NA genes from the B Victoria lineage. Thus, three
B/Yamagata-Victoria reassortants were isolated during the 2014-2015 season
(Figure 11).
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Figure 9. Phylogenetic analysis of the HA1 gene of B Yamagata-like viruses. Red, 2014-2015 vaccine strain. Blue, 2015-2016 vaccine strain. Green, selected reference sequences used by the WHO. Pink star: viruses that have a NA of the Victoria lineage. Sequences were aligned using Geneious 6.1.8 MAFFT alignment (v7.017) with default settings. A consensus tree was built from 1000 original trees in ML (70% support threshold) constructed using Geneious 6.1.8 PHYML default settings.
* *
*
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Figure 10. Phylogenetic analysis of the HA1 gene of B Victoria-like viruses. R, 2014-2015 vaccine strain. Green, selected reference sequences used by the WHO. Violet: some typical mutations described by the MRC-NIMR. Sequences were aligned using Geneious 6.1.8 MAFFT alignment (v7.017) with default settings. A consensus tree was built from 1000 original trees in ML (70% support threshold) constructed using Geneious 6.1.8 PHYML default settings.
1B
1A
N75K
N165K
S172P
L58P
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Figure 11. Phylogenetic analysis of the NA gene of B Yamagata and Victoria-like viruses. Red, 2014-2015 vaccine strains. Green, selected reference sequences used by the WHO. Blue and pink: viruses from the Yamagata and Victoria lineages, respectively. Sequences were aligned using Geneious 6.1.8 MAFFT alignment (v7.017) with default settings. A consensus tree was built from 1000 original trees in ML (70% support threshold) constructed using Geneious 6.1.8 PHYML default settings.
Of note, NA gene sequencing by the MRC-NIMR confirmed the phylogenetic
classification previously determined on the HA1 gene basis. (Annexes 9, 11and 13).
2
3
1B
1A
Yamagata
Victoria
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4.4 Antiviral resistance
One hundred thirty-nine influenza viruses were submitted to NA gene sequencing
analysis to assess the antiviral resistance of circulating strains. Among the 63/69
A/H3N2, 29/30 A/H1N1, 29/36 B Yamagata and 4/4 B Victoria NA sequences
successfully recovered, none harbored the common strain-specific mutations
associated with resistance to neuraminidase inhibitors (Table 4).
This means that influenza viruses circulating in the community did not acquire known
mutations providing antiviral resistance.
The 10 Sentinel influenza viruses (7 H1N1pdm09 and 3 H3N2) sent to MRC-NIMR
had sufficient activity for resistance to the inhibitors oseltamivir and zanamivir in NA
inhibition assays to be assessed. All were sensitive to the two NA inhibitors,
oseltamivir and zanamivir (Annex 15).
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Table 4. Key mutations conferring antiviral resistance to influenza viruses according to type or subtype. Only mutations identified in clinical and surveillance samples are listed here. a.a.= amino acid. NI = normal inhibition:<10-fold above normal inhibition for influenza A and <5-fold normal inhibition for influenza B. RI = reduced inhibition: :10-100-fold above normal inhibition for influenza A and 5 to 50-fold normal inhibition for Iinfluenza B. :HRI = highly reduced inhibition >100-fold above normal inhibition for influenza A and >50-fold normal inhibition for influenza B. In bold red: common amino acid substitutions associated with HRI by oseltamivir and peramivir that have been associated with clinical resistance. Del: deletion. Adapted from the WHO's Global Influenza Surveillance and Response System (GISRS) Information Centre. http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/nai_overview/en/.
a.a. substitution Virus
Type/subtype Oseltamivir Zanamivir Peramivir References
E119V H3N2 RI/HRI NI NI [19-26]
E119I H3N2 HRI RI NI [22]
D151V/D H3N2 NI HRI Unk [27]
Del 245–248d H3N2 HRI NI Unk [28]
R292K H3N2 HRI NI/RI/HRI RI/HRI [20, 21, 23, 27, 29-
32]
N294S H3N2 HRI NI NI [20, 32]
E119V+T148I H3N2 HRI HRI HRI [26]
E119V+I222V H3N2 HRI NI NI [25, 33]
I223R H1N1pdm RI RI Unk [34-36]
I223K H1N1pdm RI NI NI [37]
H275Y H1N1pdm HRI NI RI/HRI [38-47]
Q313K+I427T H1N1pdm RI NI/RI NI [39, 48]
D199N+H275Y H1N1pdm HRI NI HRI [45, 49]
I223K+H275Y H1N1pdm HRI RI HRI [41]
I223R+H275Y H1N1pdm HRI RI HRI [36, 37, 41]
I223V+H275Y H1N1pdm HRI NI HRI [2, 50]
S247N+H275Y H1N1pdm HRI NI HRI [51]
E105K B NI RI HRI [52]
E117A B RI/HRI RI/HRI HRI [53, 54]
Q138R B NI RI RI [55]
P139S B RI RI HRI [55]
G140R B RI RI HRI [55]
R150K B HRI NI/RI/HRI HRI [19, 31, 56-58]
D197E B RI NI RI [54, 59, 60]
D197N B NI/RI NI/RI NI [19, 24, 27, 61]
D197Y B RI/HRI RI HRI [54, 62]
A200A/T B RI RI Unk [47]
I221L B HRI RI Unk [63]
I221T B NI/RI NI RI [43, 54, 59, 61, 64]
A245T B RI RI RI [43]
H273Y B NI/RI NI RI/HRI [27, 45, 54]
N294S B RI NI RI [54, 65]
K360E B NI NI HRI [43]
R374K B HRI RI/HRI HRI [27, 54]
A395E B RI NI RI [43]
D432G B NI NI RI [43]
G140R+N144K B NI RI HRI [45]
Y142H+G145R B RI NI HRI [43]
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5 Using the FluSurver tool for rapid identification of known antiviral resistance mutations
At the NRCI, known influenza antiviral resistances to NA inhibitors are detected by
reading “manually” NA sequences and looking for mutated positions. This is a heavy
and time-consuming process. Since 2009, Dr Sebastian Maurer-Stroh and his group,
at the A*STAR Bioinformatics Institute (Singapore), in collaboration with, the
Genome Institute of Singapore, the Instituto Nacional de Medicina Genómica (Mexico
City, Mexico), the National Public Health Laboratory of the Ministry of Health
Singapore, the Instituto Adolfo Lutz (Sao Paulo, Brazil), the WHO Collaborating
Centre for Reference and Research on Influenza and the Global Initiative for Sharing
All Influenza Data (GISAID) have been developing a web-based bioinformatics tool
for the screening of influenza A and B sequences. This tool, named FluSurver
(http://flusurver.bii.a-star.edu.sg/), is intended to allow the rapid recognition of known
mutations, as well as the identification of new phenotypically or epidemiologically
interesting candidate mutations. A detailed description of FluSurver functionalities
may be found at the following link: http://flusurver.bii.a-
star.edu.sg/help/Poster_FluSurver_GISAID_2013_v3.pdf. Note that candidate
mutations will require further experimental testing with, for example, the NA-Fluor
influenza NAI resistance detection assay, in order to confirm the resistant phenotype.
In order to evaluate the usefulness and validity of the FluSurver tool in the context of
the NRCI Sentinel surveillance of flu/influenza in Switzerland, we submitted all our
2014-2015 HA and NA sequences to FluSurver screening. The sequences of
resistant influenza stains included in the WHO NI susceptibility reference virus panel
were used as positive controls (Table 5).
Table 5. CDC NI susceptibility reference virus panel v2.0 (WHO)
Strain Mutation Sensitive Resistant FluSurver scanning result
A/H1N1 pdm09
H274Y* A/California/12/2012 A/Texas/23/2012 H275Y° identified as related to
drug resistance
A/H3N2 E119V* A/Washington/01/2007 A/Texas/12/2007 E119V* identified as potentially
related to drug resistance
B Yam D198N* B/Rochester/02/2001 B/Rochester/02/2001 D197N° identified as potentially
related to drug resistance
B Vic R152K* B/Memphis/20/96 B/Memphis/20/96 R150K° identified as potentially
related to drug resistance
*universal N2 numbering, ° equivalent strain specific N position.
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FluSurver allowed the rapid and correct scanning of all our influenza A HA and NA,
as well as our influenza B NA sequences. We could also easily identify the predicted
mutations associated with different genetic clades and double check our phylogenic
trees.
The FluSurver scan correctly identified the well-known H274Y and E119V mutations
on the resistant strains. It also recognized the D198N and R152K (N2 numbering)
mutations as interesting mutations, which could potentially be associated with drug
resistance. Nevertheless, and in contrast to the H274Y and E119V mutations, the
D198N and R152K polymorphic positions were not directly described as associated
with drug resistance in the influenza B background, thus suggesting that an update of
the FluSurver mutations’ list and associated literature is required. This last point will
be crucial to ensure the usefulness and pertinence of use of this smart and powerful
tool.
Of concern, we identified a scanning problem for the influenza B HA sequences.
Indeed, the program does not use the “standard” amino acid numbering, thus leading
to an incorrect attribution of the amino acid positions in the background of the
different strains. This information was shared with the FluSurver staff and is under
correction.
FluSurver was recently integrated in the GISAID platform [66]. In this context, it
allows the direct screening of chosen sequences within the database.
6 WHO recommendation for the composition of influenza virus vaccines for the 2015-2016 flu/influenza season
Twice a year, on February for the northern hemisphere and in September for the
Southern Hemisphere, the WHO organizes a consultation meeting during which,
flu/influenza experts determine the recommended composition of the next season
influenza vaccine. 2015-2016 flu/influenza vaccine recommended strains are
described in table 6.
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7 Human infection with animal influenza viruses
Transmission of influenza viruses of animal origin to humans, notably avian and
porcine, can potentially lead to severe epidemics and, in worst cases, to pandemics.
To allow the early identification and rapid containment of new potential animal-to-
human transmission events, several countries, including Switzerland, have
introduced the regular screening of animals (mainly poultry/wild birds and farm pigs)
for the presence of the respective influenza strains.
7.1 Surveillance of swine-to-human flu/influenza viruses transmission in
Switzerland
Respiratory specimens from farm pigs exhibiting respiratory symptoms are collected
by veterinarians in Switzerland. These samples are then analyzed at the National
Veterinarian Institute, (Vetvir, Zurich, Switzerland). In parallel, samples from
consenting employees from screened pig farms who have been in contact with
influenza-infected animals and who present respiratory symptoms are also submitted
to influenza detection at the NRCI. To detect influenza A viruses of swine origin, we
use a rRT-PCR specially designed by the United States CDC [67] to detect influenza
A virus of human and animal origin, both avian and porcine.
During the 2014-2015 flu/influenza season, two pig farm employees’ samples were
sent to the NRCI for testing. Both samples were negative for influenza. (Table 7).
Table 7. Pig breeders influenza rRT-PCR results
Vaccine strains 2015-2016
A (H1N1)pdm09 A/California/7/2009-like virus
A (H3N2) A/Switzerland/9715293/2013-like virus
B B/Phuket/3073/2013-like virus*
Table 6. Recommended influenza vaccine composition for the 2015-2016
season. *B/Brisbane/60/2008-like virus is advised for quadrivalent vaccines.
Sample ID Birth date Sexe Result Origin Sender Sample date
*****286 29.07.1976 M NEGATIVE 3012 Bern Suis AG, Bern 25.02.2015
*****321 10.12.1974 M NEGATIVE 3012 Bern Suis AG, Bern 25.02.2015
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7.2 Other influenza A subtypes (non-Sentinel data)
In addition to A/H1N1 and A/H3N2, two other influenza strains, A/H5N1 and A/H7N9,
are of major concern for public health. Even if these avian strains do not (or not
efficiently) transmit from human-to-human yet, they are responsible for local
outbreaks in Egypt for A/H5N1 and China for A/7H7N9 (Figures 12 and 13).
Moreover, as they co-circulate with seasonal influenza strains, recombination events
leading to human adaptation are to be feared.
The avian A/H5N1 strain was identified for the first time in humans in 1997 in
southern China and Hong Kong. Due to its high mortality rate in poultry, A/H5N1 is
classified as a highly pathogenic avian influenza virus (HPAI). It sporadically infects
humans in contact with infected birds, particularly poultry, causing a severe
respiratory disease.[68].
A total of 784 confirmed human cases of A/H5N1 have been reported worldwide by
WHO since 2003 (last update 29.05.15). 37% were identified in Egypt. Since January
2015, 89 new cases have been reported almost exclusively in Egypt (Figure 12a),
thus exceeding the total number of cases (85) monitored during the four previous
years (2011-2014) (Figure 12b). From January to March 2015, 116 new human
cases, including 36 deaths, have been reported to WHO from Egypt. [68].
In 2013, the avian influenza A H7N9 strain was isolated for the first time in humans in
eastern China. Despite being low pathogenic in birds, A/H7N9 causes severe
respiratory disease in humans, with a possible fatal outcome. Most infected
individuals have reported previous contacts with either poultry or wild birds No
human-to-human transmission has been clearly reported so far. [69].
More than 600 persons have been infected with A/ H7N9 strain since the beginning
of the outbreak with 227 deaths reported as of February 23, 2015. All cases
originated from China. To date only 3 cases were exported outside China, 2 in
Canada and 1 in Malaysia. [69].
Similar to A/H5N1, A/H7N9 can infect individuals of all ages. But in contrast to
A/H5N1, which is predominant in children and young adults, A/H7N9 is more
prevalent in adults and elderly, particularly in men. [70]
Of note, laboratory-confirmed cases of both a/H5N1 and A/H7N9 are being reported
regularly.
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Figure 12. Influenza A/H5N1. a. Worldwide distribution of A/H5N1 cases through 2014 [68]. b. Cumulative number of laboratory-confirmed H5N1 cases and deaths from 2003 to 2015, year of onset. http://www.who.int/influenza/human_animal_interface/EN_GIP_20150303cumulativeNumberH5N1cases.pdf?ua=1).
Apart from A/H5N1 and A/H7N9, other influenza A viruses routinely circulating in wild
and domestic animals, can also infect humans (Figure 13 and Table 8). A detailed
description of these cases can be found in the Eurosurveillance volume 19, issue 18,
May 2014. A couple of A/H1N1v (1), A/H3N2v (1) and A/H1N2v (2) cases were
detected this season in USA and Sweden respectively [71].
a.
b.Country 2003-2010 2011-2012 2013-2014 2015 Total
cases deaths cases deaths cases deaths cases deaths cases deaths
Cambodia 10 8 11 11 35 18 0 0 56 37
China 40 26 3 2 4 2 1 0 48 30
Egypt 119 40 50 20 35 13 88 26 292 99
Indonesia 171 141 21 19 5 5 0 0 197 165
Thailand 25 17 0 0 0 0 0 0 25 17
Viet Nam 119 59 4 2 4 3 0 0 127 64
Other 32 15 5 0 2 2 0 0 40 17
Total 516 306 94 54 85 43 89 26 784 429
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Figure 13. A/H7N9, A/H5N1, A/H10N8 and A/H5N6 cases distribution in China, 2003-2014. A/H5N1 and A/H7N9 are mainly present in eastern China. Note that some cases have also been identified in the autonomous region of Xinjiang Uygur in western China.[69].
Strain origin Number of cases (1959-2014)
HP H7N7 Avian 89
LP H7N7 Avian 4
LP H6N1 Avian 1
H10N7 Avian 4
H9N2 Avian 15
LP H7N3 Avian 2
HP H7N3 Avian 3
H7N2 Avian 4
H5N6 Avian 2
H10N8 Avian 3
H1N1 Swine 47
H1N2 Swine 2
H3N2 Swine 7
H3N2v Swine 340
Table 8. Natural infections with avian and swine influenza viruses other that H5N1 and H7N9. H3N2v is a human-swine-avian reassortant virus. H1N1 also includes trH1N1, a human-swine-avian reassortant.
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8 Avian influenza A in animals (Current update)
HPAI and low PAI (LPAI) co-circulate in the avian population and sporadically lead
to outbreaks in both poultry and wild birds.
HPAI A/H5N8 viruses have been monitored in Asia since 2010. In 2014, they
emerged in Europe causing outbreaks in Germany, the Netherlands, United
Kingdom, and Italy. A/H5N8 outbreaks were also reported in the USA in 2015. [72].
Since December 2014, a major LPAI A/H5N2 epidemic in poultry is spreading in
North America (USA and Canada). The virus is present in over 17 states, affecting
more than 29 million birds, including chickens, ducks, pheasants, and turkeys, with
chickens particularly affected. Details about the current A/H5N2 outbreaks can be
found at the United States Department of Agriculture (USDA), Animal and Plant
Health Inspection Service (http://www.aphis.usda.gov/wps/portal/aphis/home). As the
outbreaks affected large commercial farms, a major economic impact from this threat
may be expected on the poultry market.
Of note, neither A/H5N8 nor A/H5N2 human infections have been reported.
According to the European ECDC and the United States CDC, the expected risk of
human infection from these avian influenza subtypes is low.
Since the beginning of 2015, several A/H5N1 avian influenza outbreaks have also
been reported, notably in Burkina Faso, Israel, Turkey, Egypt, and Nigeria [72].
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9 Discussion
The 2014-2015 flu/influenza season was comparable to previous seasons, notably
2012-2013, 2008-2009, and 2009-2010 in terms of maximum MC-ILI rates (Table 9).
The epidemic phase was longer than the previous season with a 12-week duration,
but the 2012-2013 season was unexpectedly weak. As expected, the maximum
proportion of MC-ILI reached during this season was much higher than last year, but
similar to several previous flu/influenza seasons (Table 9). Nine hundred eighty-
seven samples were sent to the NRCI during the 29 weeks of the Sentinel
surveillance of influenza.
As observed last year, a high variety of influenza viruses were detected in the
community. Influenza A/H3N2 and, in contrast to last year, B Yamagata strains were
co-circulating. A/H1N1pdm09 In (H1N1)pdm09 viruses were much less abundant
than last year, and only 4 B Victoria could be isolated at the end of the season.
HI analysis of this year’s A/H3N2 viruses suggests a high antigenic variation in the
community. Most of the circulating influenza A/H3N2 viruses were antigenically
divergent from the A/Texas/50/2012 vaccine strain [15]. Genetic analysis of HA3
genes confirmed the diversity of A/H3N2 strains among influenza viruses detected in
Switzerland. This strain divergence was observed also throughout Europe [15], and
Season Duration
(n = weeks) Maximum value
of MC-ILI (‰) Peak
week/year Influenza viruses
circulating
2001/02 8 42 6 / 02 B, H3N2
2002/03 8 43 9 / 03 H3N2, B
2003/04 7 69 1 / 04 H3N2
2004/05 8 60 6 / 05 H3N2, B, H1N1
2005/06 5 21 12 / 06 B, H3N2, H1N1
2006/07 5 41 6 / 07 H3N2, H1N1
2007/08 8 31.2 3 / 08 B, H1N1
2008/09 7 54.4 4 / 09 H3N2, B
2009/10 9 54.1 49 / 09 H1N1pdm09
2010/11 10 35.8 5 / 11 H1N1pdm09, H3N2
2011/12 5 26.7 9 / 12 H3N2
2012/13 12 57.3 6 / 13 B, H1N1pdm09, H3N2
2013/14 7 17.9 8 / 14 H3N2, H1N1pdm09, B
2014/15 12 52.9 6 / 15 H3N2, B, H1N1pdm09
Mean 8 ± 2 43 ± 15
Table 9. Overview the last 14 seasons. Duration: number of weeks when MC-ILI values were equal or above the epidemic threshold. Bold: influenza viruses with an annual detection rate above 50 specimens.
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in the USA [17]. However, all Swiss variants were of the same 3C genetic group.
From these results, one may expect a lower recognition of the circulating A/H3N2
viruses by the antibodies induced by the 2014-2015 vaccine, thus a weaker and/or
only partial vaccine-induced protection. Due to the high antigenic and genetic
discrepancy of the circulating A/H3N2 strains in comparison to the vaccine strain, the
latter will be replaced by the A/Switzerland/9715293/2013 strain in the 2015-2016
flu/influenza vaccine. Of note A/Switzerland/9715293/2013 has already been
incorporated in the 2015 Southern Hemisphere vaccine. As observed during previous
seasons, A/H3N2 was more prevalent in elderly (≥65 years) (75%) [73, 74].
No major antigenic changes were detected for A/H1N1pfm09 strains in HI compared
to last year. They remained antigenically close to the A/California/7/2009 vaccine,
despite the fact that both fit in two distinct genetic groups. Indeed, all circulating
influenza A/H1N1pdm09 belonged to the 6B genetic group, whereas
A/California/7/2009 is part of the genetic group 1. This results certainly accounted for
the conservation of A/California/7/2009 strain in the 2015-2016 flu vaccine.
Nevertheless, increasing amount of evidence suggest that conventional HI assays
using primary ferret antisera may not be representative of the immune responses
raised against influenza in humans. Indeed, except for infants, humans are rarely
naïve for influenza; either due to previous vaccination or natural infection, or both.
Previous studies, support the fact that previous exposure to A/H1N1 or
A/H1N1pdm09 strains shapes the individuals immune responses, therefore impacting
their responses to future influenza infections [75-77]. Recently Linderman et al.
identified a particular mutation, K163Q/E, in the HA of viruses from the A/H1N1 6B
group, which is responsible for a reduced serologic reactivity in-between sera from
individuals (42%) who were born in-between 1965-1979 and the circulating 2013-
2014 influenza strains [77]. Furthermore, Huang et al. shown that the serum
antibodies from a A/California/7/2009 vaccinated individual born before 1977 manly
recognized epitopes localized in an HA region containing residue K163 [78]. These
antibodies recognized the A/USSR/90/1977 stain (K163) that was close to
A/California/7/2009 and was circulating during the donor’s childhood, but did not
react with strains containing the K163Q/E mutations. This suggests that
A/California/7/2009 vaccination may have manly recalled B cell responses to
A/USSR/90/1977 previous exposure. This observation contrasted with the fact that
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primary ferret antisera reacted with A/California/7/2009 as well as with viruses from
group 6B.
In contrast to last year, influenza B viruses were quite abundant and were even the
dominant influenza type at the end of the season. Almost all detected influenza B
virus belonged to the Yamagata lineage, and only 7 were from the influenza B
Victoria lineage. Interestingly, among the 40 B viruses analyzed by sequencing, three
were reassortants from the Yamagata (HA) and Victoria (NA) lineages. As
mentioned, influenza B viruses are divided into two antigenicaly distinct lineages,
which started to co-circulate worldwide since 1985 [79, 80]. Reassortants of both
lineages, are regularly observed worldwide [81-83]. As these two lineages are
antigenically distinct, no or little cross-reaction is expected between Victoria-like
antiserum and Yamagata-like strains. This suggests that the 2014-2015 trivalent flu
vaccine, containing only a Yamagata-like strain, would not (or only poorly) cover the
few circulating B Victoria strains [79, 84]. Interestingly, even if experimental studies
on the trivalent (contains only one B strain) versus the quadrivalent (contains both B
Yamagata and Victoria strains) influenza vaccines (TIV and QIV respectively) have
shown that the latter produces superior antibody titers against the B strain absent
from the TIV, similar estimates of vaccine efficacy were reported for both vaccines.
The the clinical benefit of the enhanced antibody titers against the additional B strain
in response to the QIV has not been established yet. (Discussed in [85]).
Apart from amantadine, to which the majority of influenza A/H3N2 and
A/H1N1pdm09 are resistant since 2008 and 2009, respectively, no spontaneous
antiviral resistance could be detected in community samples collected by the Swiss
Sentinel Network.
In Europe, more than 1500 A/H3N2 samples were tested and four appeared to be
oseltamivir-resistant (3 E119V and 1 R292K): among these, one was also resistant to
zanamivir (R292K). Two of 500 influenza A/H1N1pdm09 viruses tested were
resistant to oseltamivir (H275Y). All tested influenza B were sensitive to NAI. As
expected all influenza A samples were resistant to amantadine, the M2 inhibitor. [17].
In the USA, 1.6% of the 64 A/H1N1pdm09 viruses tested were resistant to
oseltamivir but not to zanamivir. None of the 3232 A/H3N2 and of the 896 influenza B
viruses investigated for NAI resistance were resistant. [86].
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Geneva, 22 June 2015
Dr. Samuel Cordey
Dr Ana Rita Gonçalves Cabecinhas
Mme Patricia Suter-Boquete
Prof. Laurent Kaiser
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Annex 1: Weekly report of influenza virus detection and virus characteristics
Undet.A (H1N1)
pdm09
A (H3N2)
seasonalTotal Undet. Bvic Byam Total
40 28-Sep-14 4-Oct-14 0.6 0 - - - 0 - - - 0 0 0.00
41 5-Oct-14 11-Oct-14 1.3 4 - - - 0 - - - 0 0 0.00
42 12-Oct-14 18-Oct-14 1 12 - - - 0 - - - 0 0 0.00
43 19-Oct-14 25-Oct-14 1.6 8 - - - 0 - - - 0 0 0.00
44 26-Oct-14 1-Nov-14 1.3 8 - - - 0 - - - 0 0 0.00
45 2-Nov-14 8-Nov-14 1.5 13 - - - 0 - - 1 1 1 7.69
46 9-Nov-14 15-Nov-14 2.3 19 - - 1 1 - - - 0 1 5.26
47 16-Nov-14 22-Nov-14 1.4 14 - - - 0 - - - 0 0 0.00
48 23-Nov-14 29-Nov-14 2.5 12 - - 1 1 - - - 0 1 8.33
49 30-Nov-14 6-Dec-14 2.5 14 - - - 0 - - - 0 0 0.00
50 7-Dec-14 13-Dec-14 3.5 13 - - 2 2 - - - 0 2 15.38
51 14-Dec-14 20-Dec-14 4.3 20 - - 4 4 - - 2 2 6 30.00
52 21-Dec-14 27-Dec-14 5.6 6 - - - 0 - - - 0 0 0.00
1 28-Dec-14 3-Jan-15 13.7 12 - 2 3 5 - - - 0 5 41.67
2 4-Jan-15 10-Jan-15 12.9 54 - 6 19 25 - - 2 2 27 50.00
3 11-Jan-15 17-Jan-15 18.3 77 - 11 23 34 1 - 6 7 41 53.25
4 18-Jan-15 24-Jan-15 32.3 90 - 6 34 40 - - 4 4 44 48.89
5 25-Jan-15 31-Jan-15 43.8 82 - 15 35 50 - - 3 3 53 64.63
6 1-Feb-15 7-Feb-15 52.5 78 1 7 41 49 - - 9 9 58 74.36
7 8-Feb-15 14-Feb-15 52.9 83 - 6 35 41 - - 14 14 55 66.27
8 15-Feb-15 21-Feb-15 47.9 62 1 5 29 35 - - 8 8 43 69.35
9 22-Feb-15 28-Feb-15 39.9 41 - 3 16 19 - - 5 5 24 58.54
10 1-Mar-15 7-Mar-15 27.1 42 - 3 10 13 - 1 16 17 30 71.43
11 8-Mar-15 14-Mar-15 16.9 47 - 1 12 13 2 1 16 19 32 68.09
12 15-Mar-15 21-Mar-15 12.4 51 1 2 5 8 - 1 26 27 35 68.63
13 22-Mar-15 28-Mar-15 8.8 38 - - - 0 1 1 12 14 14 36.84
14 29-Mar-15 4-Apr-15 5.5 20 - 1 2 3 1 2 2 5 8 40.00
15 5-Apr-15 11-Apr-15 6.3 11 - - - 0 1 - 4 5 5 45.45
16 12-Apr-15 18-Apr-15 3.5 6 - - - 0 - 1 1 2 2 33.33
3 68 272 6 7 131
Weeks Dates ‰ ILISamples
received
Sentinel Surveillance, Winter 2014-15
937 487
Influenza BTotal
virus (n)
Influenza A
343 144
% pos
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Annex 2a: Hemagglutination inhibition of influenza A/H3N2 viruses
Antisera
Virus strains WHO
A/Victoria/361
/11 EGG
A/Texas/50
/12
A/South Africa/
4655/13
A/Switzerland/
9715293/13 EGG
A/Hong-Kong/
146/13
A/Samara/73/1
3
A/Hong-
Kong/5738/14
A/Victoria/361/11 EGG 2048 128 16 64 64 64 64
A/Texas/50/12 512 512 32 256 256 128 64
A/South Africa/4655/13 32 64 128 32 256 64 32
A/Switzerland/9715293/13 EGG 128 128 16 512 128 128 512
A/Hong Kong/146/13 256 256 32 256 1024 256 256
A/Samara/73/13 128 128 32 64 256 128 128
A/Hong-Kong/5738/14 32 32 16 32 64 16 64
# Patient ID HA titer Virus strains NRCI
1 ****0656 16 A/South Africa/4655/13 32 32 64 32 64
2 ****8372 32* A/South Africa/4655/13 32 32 32 32 16
3 ****8236 16 A/Texas/50/12 512 256 256 128 512
4 ****1338 16 A/Texas/50/12 512 128 128 128 128
5 ****9053 16 A/South Africa/4655/13 128 64 64 64 64
6 ****5329 16 A/South Africa/4655/13 128 64 64 64 64
7 ****9158 32 A/South Africa/4655/13 64 32 128 64 64 64 128
8 ****9293 8* A/Texas/50/12 256 512 128 256 64
9 ****4674 32 A/South Africa/4655/13 64 64 64 64 128
10 ****2716 64 A/South Africa/4655/13 64 64 32 32 64
11 ****6955 32 A/Texas/50/12 256 256 128 256 512 16 64
12 ****2151 32* A/South Africa/4655/13 64 64 128 64 64
13 ****1942 32 A/South Africa/4655/13 128 32 64 32 32
14 ****6082 32 A/South Africa/4655/13 128 32 64 32 32
15 ****2015 64 A/South Africa/4655/13 32 16 32 32 32
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Annex 2b: Hemagglutination inhibition of influenza A/H3N2 viruses
Antisera
Virus strains WHO
A/Victoria/361
/11 EGG
A/Texas/50
/12
A/South Africa/
4655/13
A/Switzerland/
9715293/13 EGG
A/Hong-Kong/
146/13
A/Samara/73/
13
A/Hong-
Kong/5738/14
A/Victoria/361/11 EGG 2048 128 16 64 64 64 64
A/Texas/50/12 512 512 32 256 256 128 64
A/South Africa/4655/13 32 64 128 32 256 64 32
A/Switzerland/9715293/13 EGG 128 128 16 512 128 128 512
A/Hong Kong/146/13 256 256 32 256 1024 256 256
A/Samara/73/13 128 128 32 64 256 128 128
A/Hong-Kong/5738/14 32 32 16 32 64 16 64
# Patient ID HA titer Virus strains NRCI
16 ****7481 32* A/South Africa/4655/13 32 32 32 32 16 64 16
17 ****6448 32* A/South Africa/4655/13 64 32 32 32 32
18 ****3887 32 A/South Africa/4655/13 32 32 32 32 32
19 ****9601 16* A/South Africa/4655/13 16 16 32 16 32
20 ****6172 16 A/South Africa/4655/13 32 32 32 16 16 16 64
21 ****3880 16 A/South Africa/4655/13 32 32 32 64 32
22 ****3900 32 A/South Africa/4655/13 128 64 32 32 64 64 64
23 ****7251 16* A/South Africa/4655/13 64 64 64 32 32 32 64
24 ****0346 16 A/South Africa/4655/13 64 64 64 32 32 16 16
25 ****8508 8 A/South Africa/4655/13 64 32 32 32 NF
26 ****8519 8 A/South Africa/4655/13 128 32 32 32 NF
27 ****9143 8 A/South Africa/4655/13 64 64 64 64 NF
28 ****2015 16 A/South Africa/4655/13 64 64 32 32
Titres were established in MDCK cells. * corresponds to MDCK-SIAT.
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Annex 3: Hemagglutination inhibition of influenza A/H1N1 pdm09 viruses
Antisera
Virus strain WHO A/California/7/09
A/St Petersburg/
27/11
A/South Africa/
3626/13
A/Christchurch/
16/10
A/California/7/09 1024 64 32 32
A/St Petersburg/27/11 512 512 64 32
A/South Africa/3626/13 512 512 512 256
A/Christchurch/16/10 256 512 256 1024
# Patient ID HA titer Virus strain NRCI
1 ****8955 8 A/St Petersburg/27/11 256 512 256 128
2 ****9485 64 A/St Petersburg/27/11 64 64 64 32
3 ****2870 32 A/ST Petersburg/27/11 32 64 32 64
4 ****5163 32 A/St Petersburg/27/11 64 128 64 64
5 ****7153 32 A/St Petersburg/27/11 32 64 32 32
6 ****6177 16* A/California/7/09 128 64 128 <16
7 ****2101 32 A/St Petersburg/27/11 128 128 128 128
8 ****9614 16* A/St Petersburg/27/11 128 128 64 64
9 ****6265 32 A/South Africa/3626/13 256 256 256 128
10 ****3894 32 A/St Petersburg/27/11 32 64 32 32
11 ****3903 32 A/St Petersburg/27/11 256 512 64 128
12 ****6204 16 A/St Petersburg/27/11 128 128 64 64
13 ****8489 16 A/South Africa/3626/13 256 512 256 128
14 ****4449 8 A/South Africa/3626/13 128 256 256 ND
15 ****2104 8 A/South Africa/3626/13 1024 2048 1024 256
16 ****3783 16* A/South Africa/3626/13 512 512 256 256
Titers were established in MDCK cells. * corresponds to MDCK-SIAT.
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Annex 4a: Hemagglutination inhibition of influenza B viruses
Antisera
Virus strains WHO
B/Brisbane/60
/08
B/Odessa/3886
/2010
B/Johannesburg/
3964/12
B/Wisconsin/01
/10
B/Novosibirsk/1/
12
B/Massachusetts/
02/12 Egg
B/Phuket/
3073/13
B/Brisbane/60/08 512 128 512 <8 <8 <8 <8
B/Odessa/3886/2010 64 128 64 <8 <8 <8 <8
B/Johannesburg/3964/12 512 128 1024 <8 <8 <8 <8
B/Wisconsin/01/10 <8 <8 <8 512 128 256 64
B/Novosibirsk/1/12 <8 16 <8 256 1024 256 32
B/Massachusetts/1/12 Egg <8 16 <8 32 128 512 64
B/Phuket/3073/13 32 <8 <8 1024 256 1024 128
# Patient ID HA titer Virus strains NRCI
1 ****0771 16 B/Massachusetts/02/12
64 128 128 64
2 ****7457 128 B/Massachusetts/02/12
64 128 64 16
3 ****4241 128 B/Massachusetts/02/12
32 128 64 16
4 ****5878 128 B/Massachusetts/02/12
64 128 64 32
5 ****7033 64 B/Massachusetts/02/12
128 128 64 32
6 ****6261 128 B/Massachusetts/02/12
64 128 64 32
7 ****8094 64 B/Massachusetts/02/12
64 64 64 32
8 ****9566 16* B/Massachusetts/02/12
128 256 128 64
9 ****3422 128 B/Massachusetts/0212
64 128 64 16
10 ****9663 16* A/Massachusetts/02/12
128 64 128 128
11 ****0427 128 B/Massachusetts/02/12
64 128 128 32
12 ****9266 256 B/Massachusetts/02/12
64 128 128 32
13 ****0046 32 B/Massachusetts/02/12
64 256 128 32
14 ****9903 16* B/Massachusetts/02/12
128 128 256 32
15 ****1721 64 B/Phuket/3073/13
64 128 64 32
16 ****5225 128 B/Odessa/3886/10 128 256 256
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Annex 4b: Hemagglutination inhibition of influenza B viruses
Antisera
Virus strains WHO
B/Brisbane/60
/08
B/Odessa/3886
/2010
B/Johannesburg/
3964/12
B/Wisconsin/01
/10
B/Novosibirsk/1/
12
B/Massachssetts/
02/12 Egg
B/Phuket/
3073/13
B/Brisbane/60/08 512 128 512 <8 <8 <8 <8
B/Odessa/3886/2010 64 128 64 <8 <8 <8 <8
B/Johannesburg/3964/12 512 128 1024 <8 <8 <8 <8
B/Wisconsin/01/10 <8 <8 <8 512 128 256 64
B/Novosibirsk/1/12 <8 16 <8 256 1024 256 32
B/Massachssetts/1/12 Egg <8 16 <8 32 128 512 64
B/Phulket/3073/13 32 <8 <8 1024 256 1024 128
# Patient ID HA titer Virus strains NRCI
17 ****9985 64 B/Massachusetts/02/12
128 256 128 32
18 ****1682 64 B/Massachusetts/02/12
64 256 128 32
19 ****1738 128 B/Massachusetts/02/12
64 128 128 16
20 ****2869 64* B/Novosibirsk/02/12
128 512 256 32
21 ****3085 64 B/Massachusetts/02/12
128 256 128 32
22 ****2956 16* B/Novosibirsk/02/12
128 512 256 64
23 ****9017 64 B/Massachusetts/02/12
32 128 256 32
24 ****8905 128 B/Novosibirsk/02/12
64 512 128 32
25 ****8877 64 B/Odessa/3886/10 64 256 128
26 ****3678 128 B/Odessa/3886/10 64 256 128
27 ****7032 128 B/Novosibirsk/02/12
128 256 128 32
28 ****6942 128 B/Wisconsin/1/10
128 128 64 32
29 ****7038 128 B/Massachusetts/02/12
32 64 64 <16
30 ****6924 128 B/Wisconsin/1/10
128 128 64 16
Titers were established in MDCK cells. * corresponds to MDCK-SIAT.
62/73
Annex 5: Antigenic analyses of influenza A/H3N2 viruses (with 20nM oseltamivir) 2015-02-24, MRC-NIMR
Hamagglutination inhibition titre
1
Post-infection ferret antisera
Viruses
Collection Passage A/Perth A/Vic A/Texas A/Samara A/HK A/Stock A/Stock A/Switz A/Switz A/HK A/HK
date history 16/09 361/11 50/12 73/13 146/13 6/14 6/14 9715293/13 9715293/13 5738/14 5738/14
F18/11
T/C F09/12
Egg F42/13
F24/13 F40/13 T/C
F14/14 Egg
F20/14 T/C NIBSC
F13/14 Egg F32/14
T/C F30/14
NIB F53/14
Genetic group
3C.1 3C.1 3C.3 3C.2 3C.3a 3C.3a isolate
2 3C.3a
3C.3a cl123
3C.2a 3C.2a cl 121
REFERENCE VIRUSES
A/Perth/16/2009
2009-07-04 E3/E2 320 80 80 80 160 40 80 < 40 < <
A/Victoria/361/2011 3C.1 2011-10-24 MDCK2/SIAT5 160 320 320 640 320 640 160 80 160 160 40
A/Texas/50/2012 3C.1 2012-04-15 E5/E2 320 640 1280 640 640 160 640 40 640 80 40
A/Samara/73/2013 3C.3 2013-03-12 C1/SIAT4 160 320 320 640 640 640 320 80 320 320 40
A/Hong Kong/146/2013 3C.2 2013-01-11 E3/E3 320 320 640 640 2560 160 640 40 640 320 40
A/Stockholm/6/2014 3C.3a 2014-02-06 SIAT1/SIAT2 < 80 40 160 80 320 80 80 80 80 40
A/Stockholm/6/2014 3C.3a 2014-02-06 E4/E1 isolate 2 80 160 160 80 320 160 320 80 640 160 40
A/Switzerland/9715293/2013 3C.3a 2013-12-06 SIAT1/SIAT3 < 40 40 160 80 640 160 80 80 80 <
A/Switzerland/9715293/2013 3C.3a 2013-12-06 E4/E1 clone 123 40 160 160 160 320 320 320 80 640 160 <
A/Hong Kong/5738/2014 3C.2a 2014-04-30 MDCK1/MDCK2/SIAT1 < 80 80 160 160 320 80 40 80 160 40
A/Hong Kong/5738/2014 3C.2a 2014-04-30 E5/E2 clone121 40 40 80 160 160 160 80 40 40 320 640
TEST VIRUSES
A/Switzerland/656/2014
2014-11-14 SIAT1 160 160 160 320 160 320 160 < 80 80 <
1<=<40 Vaccine
63/73
Annex 6: Antigenic analyses of influenza A/H1N1pdm09 viruses (2015 01 28), MRC-NIMR
Haemagglutination inhibition titer
Post-infection ferret antisera
Viruses
Collection Passage A/Cal A/Bayern A/Lviv A/Chch A/HK A/Astrak A/St. P A/St. P A/HK A/Sth Afr
date history 7/09 69/09 N6/09 16/10 3934/11 1/11 27/11 100/11 5659/12 3626/13
F30/11 F11/11 F14/13 F30/10 F21/11 F22/13 F23/11 F24/11 F30/12 F3/14
Genetic group
4 3 5 6 7 6A 6B
REFERENCE VIRUSES
A/California/7/2009
2009-04-09 EP1/E3 640 1280 1280 160 160 320 320 320 160 320
A/Bayern/69/2009
2009-07-01 MDCK5/MDCK2 160 320 320 40 40 80 80 80 80 80 G155E
A/Lviv/N6/2009
2009-10-27 MDCK4/S1/MDCK3 640 1280 1280 160 80 160 320 160 320 160 G155E>G, D222G
A/Christchurch/16/2010 4 2010-07-12 E1/E3 1280 2560 2560 5120 2560 2560 2560 5120 2560 2560
A/Hong Kong/3934/2011 3 2011-03-29 MDCK2/MDCK3 320 160 320 320 640 1280 640 1280 1280 640
A/Astrakhan/1/2011 5 2011-02-28 MDCK1/MDCK5 1280 640 640 640 1280 2560 1280 2560 2560 1280
A/St. Petersburg/27/2011 6 2011-02-14 E1/E3 1280 1280 1280 640 1280 2560 2560 5120 2560 2560
A/St. Petersburg/100/2011 7 2011-03-14 E1/E3 1280 1280 1280 1280 1280 2560 2560 5120 5120 2560
A/Hong Kong/5659/2012 6A 2012-05-21 MDCK4/MDCK2 320 160 160 320 1280 1280 640 2560 1280 1280
A/South Africa/3626/2013 6B 2013-06-06 E1/E2 640 640 640 320 640 1280 640 2560 1280 1280
TEST VIRUSES
A/Switzerland/485/2015 6B 2015-01-07 MDCK1 2560 1280 640 1280 2560 5120 5120 5120 5120 5120
A/Switzerland/955/2014
2014-12-30 MDCK1 640 320 320 640 1280 1280 1280 5120 2560 1280
A/Switzerland/988/2014 6B 2014-12-30 MDCK1 1280 640 640 1280 2560 2560 2560 5120 5120 2560
Vaccine
64/73
Annex 7: Antigenic analyses of influenza B viruses (Yamagata lineage) 2015-01-28, MRC-NIMR
Haemagglutination inhibition titer
Post-infection ferret sera
Viruses
Collection Passage B/Fl1,3
B/Fl1 B/Bris
2 B/Wis
2 B/Stock
2 B/Estonia
2 B/Mass
2 B/Mass
2 B/Phuket
2 B/Phuke
t2
B/HK4
date history 4/06 4/06 3/07 1/10 12/11 55669/11 02/12 02/12 3073/13 3073/13 3417/14
SH479 F1/10 F21/12 F10/13 F12/12 F26/11
Egg
F28/13
T/C
F15/13
Egg
F36/14
T/C
F35/14
St Judes
F715/14
Genetic group
1 1 2 3 3 2 2 2 3 3 3
REFERENCE VIRUSES
B/Florida/4/2006 1 2006-12-15 E7/E1 5120 640 1280 320 640 160 1280 320 160 < 320
B/Brisbane/3/2007 2 2007-09-03 E2/E2 5120 1280 1280 640 640 320 1280 320 320 < 640
B/Wisconsin/1/2010 3 2010-02-20 E3/E3 1280 320 320 640 320 < 160 80 160 20 320
B/Stockholm/12/2011 3 2011-03-28 E4/E1 2560 160 160 160 320 < 160 80 80 < 160
B/Estonia/55669/2011 2 2011-03-14 MDCK1/MDCK1 1280 160 160 80 80 1280 40 640 80 40 320
B/Massachusetts/02/2012 2 2012-03-13 E3/E4 5120 640 1280 320 320 160 1280 160 160 < 320
B/Massachusetts/02/2012 2 2012-03-13 MDCK1/C2/MDCK3 5120 640 640 640 320 640 640 640 320 40 640
B/Phuket/3073/2013 3 2013-11-21 E4/E3 1280 320 320 640 640 80 320 80 160 20 320
B/Phuket/3073/2013 3 2013-11-21 M2/M2 2560 320 320 640 640 160 160 320 320 640 320
B/Hong Kong/3417/2014 3 2014-06-04 E5 320 80 80 160 80 < 40 40 40 < 320
TEST VIRUSES
B/Switzerland/457/2014 3 2014-12-17 MDCK1 640 80 160 160 80 20 40 80 80 80 320
B/Switzerland/016/2014 3 2014-12-16 MDCK1 640 160 160 320 160 20 80 80 80 40 640
1. < = <40; 2. < = <10; 3. hyperimmune sheep serum; 4. RDE serum pre-absorbed with TRBC Vaccine
65/73
Annex 8: Phylogenetic comparison of influenza A/H1N1pdm09, HA genes, MRC-NIMR
66/73
Annex 9: Phylogenetic comparison of influenza A/H1N1pdm09, NA genes, MRC-NIMR
67/73
Annex 10: Phylogenetic comparison of influenza A/H3N2, HA genes, MRC-NIMR
68/73
Annex 11: Phylogenetic comparison of influenza A/H3N2, NA genes, MRC-NIMR
69/73
Annex 12: Phylogenetic comparison of influenza B Yamagata, HA genes, MRC-NIMR
70/73
Annex 13: Phylogenetic comparison of influenza B Yamagata, NA genes, MRC-NIMR
71/73
Annex 14: Phylogenetic comparison of influenza B Victoria, NA genes, MRC-NIMR
72/73
Annex 15: Antiviral sensitivity testing on influenza A viruses, MRC-NIMR
The IC50 values are given for oseltamivir and zanamivir
Collection date Virus name Type/Subtype OS IC50 OS sensitivity Zan IC50 Zan sensitivity HI result 1 Centre ID
17.12.2014 B/Switzerland/457/2014 BY 23.89 Normal inhibition 1.28 Normal inhibition CHE
16.12.2014 B/Switzerland/016/2014 BY 41.76 Normal inhibition 5.4 Normal inhibition CHE
07.11.2014 B/Switzerland/771/2014 BY no HA titre CHE
07.01.2015 A/Switzerland/485/2015 H1pdm 0.95 Normal inhibition 0.46 Normal inhibition CHE
30.12.2014 A/Switzerland/955/2014 H1pdm 0.53 Normal inhibition 0.28 Normal inhibition CHE
30.12.2014 A/Switzerland/988/2014 H1pdm 0.53 Normal inhibition 0.26 Normal inhibition CHE
05.01.2015 A/Switzerland/785/2015 H3 0.45 Normal inhibition 0.2 Normal inhibition CHE
17.12.2014 A/Switzerland/236/2014 H3 0.25 Normal inhibition 0.28 Normal inhibition CHE
28.11.2014 A/Switzerland/373/2014 H3 0.46 Normal inhibition 0.41 Normal inhibition CHE
14.11.2014 A/Switzerland/656/2014 H3 2.22 Normal inhibition 1.17 Normal inhibition CHE
73/73
Annex 16: Sequencing primers used during the 2014-2015 season
Primers used for classical RT-PCR detection of influenza viruses
Influenza virus Target gene Primer or probe Origin and reference
A/H1N1pdm 2009
Hemaggutinin (H1)
Forward cswHAF1 R.Daniel, MRC-NIMR London Forward cswHAF31 Feb 2011
Forward cswHAF451
Forward cswHAF848
Reverse cswHAR475
Reverse cswHAR873
Reverse cswHAR1263
Reverse cswHAR1313
Neuraminidase (N1)
Forward cswN1F1 R.Daniel, MRC-NIMR London Forward cswN1F401
Reverse cswN1R424
Forward cswN1F1076
Reverse cswN1R1099
Reverse cswN1R1424
Reverse cswN1R1440
Matrix (M1)
Forward M93c Y. Thomas, CNRI, Geneva
Reverse MF821Y Aug 2009
A/H3N2 seasonal
Hemagglutinin (H3)
Forward AH3G J. Ellis London
Reverse AH3H Jan 2006
Forward AH3B
Reverse AH3CII
Reverse AH3I
Forward H3HAF567
Reverse H3HAR650
Neuraminidase (N2)
Forward H3N2F1 V. Gregory , MRC-NIH London Reverse N2R410 Modified by Y. Thomas,
CNRI Geneva Forward N2F387 Mar 2011
Reverse N2R778
Forward N2F1083
Reverse N2R1447
Matrix
Forward M93c Y.Thomas, CNRI, Geneva
Reverse MF820R Feb 2007
B seasonal
Hemagglutinin
Forward BHA1F1 V.Gregory, MRC-NIMR London Reverse BHA1R1 Jan 2006
Forward BHAF
Forward BHA25
Forward BHAF458
Reverse BHAR652
Neuraminidase
Forward BNAF1 V. Gregory , MRC-NIMR London Forward BNAF336 Modified by Y.Thomas,
CNRI Geneva Forward BNAF725 2011
Forward BNAF1096
Reverse BNAR1487
Reverse BNAR1119
Reverse BNAR748
