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Genetic diversity of Influenza B virus in 2009–2010 and 2010–2011 in Tunisia
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Original article
Genetic diversity of Influenza B virus in 2009–2010 and 2010–2011 in Tunisia Diversité génétique du virus de la grippe B en Tunisie en 2009–2010 et 2010–2011 A. El Moussi a,∗ , M.A. Ben Hadj Kacem a , J. Ledesma b , F. Pozo b , M. Teresa Cuevas b , I. Casas b , A. Slim a b
a National Influenza Centre-Tunis, Unit Virology, Microbiology Laboratory, Charles Nicolle’s Hospital, boulevard 9-Avril, 1006 Tunis, Tunisia National Influenza Centre-Madrid, Influenza and Respiratory Viruses Laboratory, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km 2 Majadahonda, 28220 Madrid, Spain
Received 20 October 2012; received in revised form 26 March 2013; accepted 17 June 2013 Available online 19 July 2013
Abstract Objective. – The authors had for aim to characterize influenza B strains having circulated in Tunisia to identify new mutations and compare them with reference strains. Methods. – The epidemiological surveillance of influenza allowed identifying 19 patients with symptoms related to respiratory infection, who had been infected by influenza B strains isolated in several regions of Tunisia in 2009–2010 and in 2010–2011. Laboratory identification and detection of mutations in the segment encoding hemagglutinin of influenza viruses was performed by real time PCR and sequencing. Results. – The two influenza B Tunisian strains of the 2009–2010 season belonged to the Victoria lineage, whereas 2010–2011 season strains belonged to B/Victoria/2/87 and B/Yamagata/16/88 lineages with a dominance of the Yamagata lineage (76%). This study allowed identifying amino acid substitutions: T121A, S150I, N165Y, T181A, G183R, D196N, S229D, M251V and K253R in the B/Yamagata lineage; L58P, N75K, K109N, N165K, S172P and K257R into the B/Victoria lineage. These mutations were specific of Tunisian groups of variants. Most influenza B-Yamagata lineage viruses (69%) were associated with severe cases. Conclusion. – Molecular analysis of the various influenza B strains circulating in Tunisia is useful to detect new mutations that can modify the phenotype of influenza strains. © 2013 Elsevier Masson SAS. All rights reserved. Keywords: Influenza B; Mutation; Phylogeny; Sequencing; B/Yamagata lineage; B/Victoria lineage
Résumé Objectif. – Caractériser les souches de la grippe B ayant récemment circulées en Tunisie afin de déceler les nouvelles mutations en comparaison avec les souches de référence. Méthodes. – Dans le cadre de la surveillance épidémiologique de la grippe, 19 patients présentant des symptômes liés à l’infection des voies respiratoires se sont révélés infectés par le virus de la grippe B durant les deux saisons 2009–2010 et 2010–2011. L’identification ainsi que la détection des mutations dans le segment codant l’hémagglutinine des virus grippaux ont été réalisées au laboratoire par la technique de RT-PCR en temps réel et le séquenc¸age. Résultats. – Il s’est avéré que les deux souches Tunisiennes de la grippe B de la saison 2009–2010 appartiennent à la lignée Victoria, alors que les souches de la saison 2010–2011 sont des deux lignées B/Victoria/2/87 et B/Yamagata/16/88 avec une dominance de la lignée Yamagata (76 %). Cette étude a permis d’identifier des mutations à l’origine de substitutions d’acide aminé : T121A, S150I, N165Y, T181A, G183R, D196N, S229D, M251V et K253R dans la lignée B-Yamagata ; L58P, N75K, K109N, N165K, S172P et K257R dans la lignée B-Victoria. Ces mutations sont spécifiques de groupes de variants Tunisiens. La majorité des virus de la lignée Yamagata étudiés (69 %) sont associés à des cas graves.
∗
Corresponding author. E-mail address: [email protected] (A. El Moussi).
0399-077X/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.medmal.2013.06.005
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Conclusions. – L’analyse moléculaire régulière des différentes souches de la grippe B circulant en Tunisie s’avère utile pour détecter de nouvelles mutations capables de modifier le phénotype des souches grippales. © 2013 Elsevier Masson SAS. Tous droits réservés. Mots clés : Grippe B ; Mutation ; Phylogénie ; Séquenc¸age ; Lignée B-Yamagata ; Lignée B-Victoria
1. Introduction Influenza is a very contagious infectious disease due to a genetically instable virus that spreads rapidly worldwide during seasonal outbreaks. Among viruses responsible for this disease, the influenza B virus seems to affect humans only even though it was isolated in seals [1]. These type B viruses mutate more slowly and have weaker pandemic potential than type A viruses. Nevertheless, during the last 20 years, influenza B has caused seasonal outbreaks in humans, as well as influenza A sub-type H1 and H3 strains [2]. A great influenza B activity was reported in several countries of the Northern hemisphere, in March 2012 [3]. Surveillance of influenza viruses is implemented in developed countries, but also in some African countries [4] and in the World Health Organization Regional Office for the Eastern Mediterranean (EMRO) region. This surveillance is essential to determine and compare the nucleotide sequences of viral genes, especially for the gene encoding hemagglutinin, HA. This allows characterizing viral isolates and assessing the yearly evolution of influenza strains. 2. Material and methods 2.1. Studied population We included patients consulting in the institutions of the Basic Healthcare network (French acronym DSSB) or admitted to the hospital for symptoms suggesting a flu syndrome with or without signs of severity. 3865 samples were positive for influenza viruses among the 7350 collected in the community during the 2009 pandemic. Hundred and eighty-one patients were found to be positive for influenza viruses among the 894 examined, during flu season 2010–2011. 2.2. Samples Respiratory samples were collected from all patients included (naso-pharyngeal, nose, throat, rhino-pharyngeal aspiration, protected tracheal sampling [PTS], BAL, etc.). These samples were put in an adapted virological medium for transportation (Vircell, Spain® ) then sent at +4 ◦ C to the National Influenza Centre at the Virology Unit of the Charles Nicolle hospital, Microbiology Laboratory in Tunis. 2.3. Molecular analysis The extraction of viral RNA was performed with the QIAamp Viral RNA mini kit (QIAGEN). The detection was made with a molecular technique relying on real time RT-PCR according to the recommended protocol, with primers and probes
from the CDC in Atlanta [5]. This technique is used routinely by the National Influenza Centre Laboratory for the surveillance of influenza viruses type A (A(H1N1)pdm09, A/H1N1, A/H3N2), and influenza B viruses. A partial sequencing was then performed targeting the segment encoding hemagglutinin (HA) (864 bp) for samples positive for influenza B, according to the protocol of National Influenza Centre Madrid [6]. This protocol is based on Sanger’s technique and performed on the machine “3130 Genetic Analyzer”. The sequences obtained were assembled using the Macaw software and compared to the reference sequences for influenza B, available on GenBank (http://www.ncbi.nih.gov/genomes/flu/swineflu.html) and Global initiative on Sharing Avian Influenza Data database (http://www.platform .gisiad.org), with the specialized software Clustal W included in MEGA version 4 [7]. 3. Results The analysis of influenza viruses by real time RT-PCR allowed detecting two influenza B viruses during season 2009–2010 and 17 influenza viruses during season 2010–2011. They were subdivided in two groups according to the severity of symptoms: 13 cases were classified as severe and six as mild. The mean age of cases was 29 years (range: 10 months to 51 years) (Table 1). The molecular analysis was performed on samples collected in various regions of Tunisia. It revealed the circulation of the B-Victoria lineage during season 2009–2010 and the co-circulation of B/Victoria lineage (4 strains) and B-Yamagata lineage (13 strains) in 2010–2011 season. The comparison of Tunisian Victoria strains with the reference strain revealed the following mutations: L58P, N75K, K109N, N165K, S172P, and K257R (Fig. 1). Furthermore, these strains presented sporadic substitutions in various positions of hemagglutinin such as: P31Q, K56E, H122R, V146I, G256W, I267N, and I290M (Table 2). Only two strains (B/Tunisia/8340/2009 and B/Tunisia/8348/2009) present a substitution of amino acid valine by isoleucine in position 146. This substitution is found in Nglycosylation site of the hemagglutinin domain 1. The mutations found in all Yamagata-like Tunisian strains compared with the reference B/Florida/4/2006 strain were: K88R, S150I, I165N, D196N, S229D, and M251V (Table 3). Other substitutions defining the two Yam/88 Tunisian groups were also identified: T121A, T181A, G183R, and K253R (Fig. 2). Sporadic mutations were also found in the Yamagata strains: S148N, L268W, I282L, and G248R. Furthermore, all Yamagata-like Tunisian strains had a new potential N-glycosylation site N196 with the substitution of amino acid aspartic acid (D) by asparagine (N). The phylogenetic analysis of Victoria strains (B/Tunisia/ 6/2011, B/Tunisia/756/2011, B/Tunisia/485/2011, and
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Table 1 Characteristics of patients and Tunisian influenza B strains studied. Caractéristiques des patients et des souches de la grippe B tunisiennes étudiés. Tunisian Influenza B strains
GenBank access number
Region
Date of sampling
Age
Sex
Clinical status
B/Tunisia/8348/2009 B/Tunisia/8340/2009 B/Tunisia/6/2010 B/Tunisia/7/2011 B/Tunisia/478/2011 B/Tunisia/485/2011 B/Tunisia/477/2011 B/Tunisia/484/2011 B/Tunisia/756/2011 B/Tunisia/1193/2011 B/Tunisia/1195/2011 B/Tunisia/1570/2011 B/Tunisia/1428/2011 B/Tunisia/1869/2011 B/Tunisia/1914/2011 B/Tunisia/2127/2011 B/Tunisia/2128/2011 B/Tunisia/2130/2011 B/Tunisia/2211/2011
JN037698 JN037697 JN037700 JN037706 JN037710 JN037705 JN037714 JN037713 JN037715 JN037701 JN037702 JN037703 JN037699 JN037704 JN037707 JN037708 JN037709 JN037711 JN037712
Tunis Nabeul Jendouba Tunis Sousse Sousse Tunis Sousse Bizerte Monastir Monastir Mahdia Sousse Mahdia Gabes Tunis Tunis Tunis Gabes
11 June 2009 11 June 2009 30 Dec. 2010 03 Jan. 2011 08 Jan. 2011 08 Jan. 2011 11 Jan. 2011 08 Jan. 2011 20 Jan. 2011 02 Feb. 2011 02 Feb. 2011 06 Feb. 2011 07 Feb. 2011 10 Feb. 2011 12 Feb. 2011 20 Feb. 2011 20 Feb. 2011 20 Feb. 2011 20 Feb. 2011
10 months 23 years 24 years 13 years 5 years 1 years 50 years 30 years 20 years 51 years 23 years 16 years 9 years 42 years 46 years 35 years 50 years 50 years 34 years
M F F M M M F F M F M M F M M M F F M
Mild case Mild case Severe case Mild case Mild case Mild case Mild case Mild case Severe case Severe case Severe case Severe case Severe case Severe case Severe case Severe case Severe case (ICU) Severe case Severe case
Fig. 1. Comparison of amino acid sequences of HA protein domain 1 gene of Victoria lineage Tunisian influenza B strains with reference strain B/Malaysia/2506/2004, showing the new and specific substitutions. Comparaison de séquences d’acides aminés déduites du domaine 1 de gène de la protéine HA représentatifs des souches de la grippe B de la lignée Victoria tunisiennes avec celle de la souche de référence B/Malaysia/2506/2004 montrant les nouvelles substitutions spécifiques du virus de la grippe B Tunisien. Table 2 Amino acid substitution of the HA protein (HA1 domain) of influenza B Victoria lineage. Variation des acides aminés de la protéine HA (de domaine HA1) de la grippe B de lignée Victoria. Position of the amino acid (without precursor protein) Strains B/Victoria
31
56
58
75
109
122
146
165
172
256
257
267
290
B/Malaysia/2506/2004 8340/2009 8348/2009 1428/2011 6/2011 485/2011 756/2011
P – – – Q Q –
K – – E – – –
L – – P P P P
N K K K K K K
K N N N N N N
H – – – R R R
V I I – – – –
N K K K K K K
S P P P P P P
G – – W – – –
K – – R R R R
I – – – – N –
I – – M – – –
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Table 3 Amino acids substitution of the HA protein (HA1 domain) of influenza B Yamagata lineage. Variation des acides aminés de la protéine HA (de domaine HA1) de la grippe B de lignée Yamagata. Position of the amino acid (without precursor protein) Strains B/Yamagata
88
121
148
150
165
181
183
196
229
251
253
268
282
284
B/Florida/4/2006 1193/2011 1195/2011 478/2011 B/7/2011 477/2011 1570/2011 1869/2011 2127/2011 2128/2011 2130/2011 2211/2011 484/2011 1914/2011
K R R R R R R R R R R R R R
T A A A A A – – – – – – – –
S – – – – – – – – – – – – N
S I I I I I I I I I I I I I
Y N N N N N N N N N N N N N
T – – – – – A A A A A A A A
G R R R R R – – – – – – – –
D N N N N N N N N N N N N N
S D D D D D D D D D D D D D
M V V V V V V V V V V V V V
K – – – – – R R R R R R R R
L – – W – – – – – – – – – –
I – – L – – – – – – – – – –
G – – – – – – – – – – – R –
Fig. 2. Comparison of amino acid sequences of HA protein domain 1 gene of Victoria lineage Tunisian influenza B strains with reference strain B/Florida/4/2006, showing the new and specific substitutions. Comparaison de séquences d’acides aminés déduites du domaine 1 de gène de la protéine HA représentatifs des souches de la grippe B de la lignée Yamagata tunisiennes avec celle de la souche de référence B/Florida/4/2006 montrant les nouvelles substitutions spécifiques du virus de la grippe B tunisien.
B/Tunisia/1428/2011) indicated that they belonged to the Brisbane/60 group characterized by specific mutations of Tunisian strains H122R, K255R, and P31Q (Fig. 3). Furthermore, the Tunisian strains were genetically related to those of neighbor countries Morocco and Algeria. The studied Yamagata-like strains (13 strains) belonged to the Bangladesh/3333 group (Fig. 4). These strains
were subdivided in two groups: eight belonged to the subgroup B/Serbia/1894/2011 and with the following mutations: T181A and K253N; the five remaining strains (B/Tunisia/7/2011, B/Tunisia/477/2011, B/Tunisia/478/2011, B/Tunisia/1193/2011, and B/Tunisia/1195/2011), with the mutations T121A and G183R, were not included in any of the four subgroups of the Bangladesh/3333 group described in the
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Fig. 3. Phylogenetic tree of HA sequences Tunisian influenza B strains Victoria lineage isolated during the 2009–2010 and 2010–2011 seasons. Arbre phylogénétique de la séquence HA des souches de la grippe B de la lignée Victoria isolées pendant les deux saisons 2009–2010 et 2010–2011.
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Fig. 4. Phylogenetic tree of HA sequences Tunisian influenza B strains Yamagata lineage isolated during the 2009–2010 and 2010–2011 seasons. Arbre phylogénétique de la séquence HA des souches de la grippe B de la lignée Yamagata isolées pendant les deux saisons 2009–2010 et 2010–2011.
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European Centre for Disease Prevention and Control (ECDC) report [8]. 4. Discussion The surveillance of influenza viruses circulating in the whole world is crucial for several reasons. It relies on the detection, typing, sub-typing, and sequencing the genome of strains in order to detect mutations able to bring new characteristics to the virus. This allows the WHO, each year, to indicate the most appropriate vaccinal composition for influenza, according to surveillance data collected worldwide thanks to the surveillance network. In Tunisia, the surveillance of influenza strains is managed by the National Influenza Centre at the Charles Nicolle hospital Virology Unit Microbiology Laboratory in Tunis. There are three circulating strains transmissible from human to human; among these influenza viruses the influenza B virus is important because it is responsible for sporadic cases and epidemics. But it remains less pathogenic than the influenza A virus [9]. The segments that encode hemagglutinin (HA) and neuraminidase (NA) (segments 4 and 6) are the most frequent targets of genetic characterization used for this surveillance [10,11]. Indeed, the mutations in these sequences are the most likely to induce a phenotypic change and especially antigenic, allowing the virus to escape the immune system. Helix 190 (194–196), which is part of the receptor binding site of HA, is undoubtedly one of the most important epitopes of influenza B. This study revealed mutations in this helix, belonging to hemagglutinin domain 1 (HA1), inducing the addition of a potential N-glycosylation site (NKT) in Tunisian B/Yamagata strains, in position 196. As reported, the influenza B virus similar to the influenza A virus [12–15], uses the suppression or addition of a glycosylation site to conduct an antigenic drift [16–18]. Most Tunisian Yamagata strains were detected in severe cases (9/13 or 69%). This supports the hypothesis that the gain of a glycosylation may cause an increased virulence of the mutated virus. Recently, Shen et al. confirmed that this mutation allowed a positive selection of the virus and escaping the immune system [19]. Our study allowed us to have an idea of the virus’s virulence, but more strains need to be sequenced so as to obtain statistically robust calculations. Indeed, even though influenza B viruses are less pathogenic than influenza A viruses, and even if they are responsible for sporadic diseases, they can be associated with severe complications and death [20,21]. The sequencing of influenza virus genes could constitute a sufficiently large database to obtain information on their virulence. The pathogenicity of influenza B is still not explained molecularly. Complementary studies are needed. The phylogenetic analysis of HA nucleotide sequences revealed a great genetic diversity of the studied viruses. We detected only two type B viruses (0.1%) during the 2009 pandemic. The comparison of strain sequences (B/Tunisia/8340/2011 and B/Tunisia/8348/2011) with those of reference strains revealed that it was Victoria lineage influenza B, similar to older Victoria strain: B/Victoria/2/87. The comparison of these strains with the Victoria strains detected during season 2010–2011 (n = 4), revealed new mutations of influenza
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B expressed by two amino acid substitutions: K257R and L58P already mentioned in the ECDC report. During 2010–2011 season, four out of 17 viruses B were localized in the Victoria lineage and belonged to the B/Brisbane/60/2008 group. The 13 other strains (after week 40 of season 2010–2011) were clustered in the Yamagata lineage and belonged to the B/Bangladesh/3333/2007 group. The HA sequences of Tunisian B/Yamagata strains were classified in two groups. The first specific group of eight viruses was characterized by two mutations (T121A and G183R). The second group of five viruses was defined by two substitutions: T181A, K253R, mentioned in the ECDC report [22]. All the segments of the hemagglutinin gene had evolved into two distinct major lineages (B/Victoria and B/Yamagata) and their amino acid substitution profiles were specific and maintained in these two lineages. During 2010–2011 season, most Tunisian strains belonged to the Yamagata lineage (76%) contrary to other countries (4%) where the dominant circulating strain was Victoria lineage [23]. The B/Yamagata virus was not included in the 2011–2012 trivalent vaccine. The co-circulation of two lineages in Tunisia, B/Victoria/2/87 (Vic87) and B/Yamagata/16/88 (Yam88), have been identified elsewhere since 1983 [24]. The variants belonging to these two lineages had been identified in 2002 [25,26]. The dynamic evolution of influenza B virus, with the passage of a dominant position between the Vic87 and the Yam88 lineages, is due to immune selection. In fact, the dominant circulation of Yama88 lineage in the 1990s was associated to a very important antigenic drift of this lineage [27]. Likewise, influenza A and influenza B are clearly in competition to infect humans. It is probable that the infection by one type of virus inhibits infection as well as the replication of the other [28,29]. 5. Conclusion The molecular characterization of influenza B virus has been greatly contributive. It allows genotyping, which is essential for the traceability of circulating strains. It also allows detecting mutations that could explain specific phenotypes. We demonstrated that, during the influenza A virus (H1N1)pdm09 pandemic, the Victoria lineage influenza B viruses had been very weakly active. During the next season, the influenza B activity was more important and characterized by a co-circulation of two lineages, Victoria and Yamagata, with the predominance of the latter. Specific mutations of Tunisian strains belonging to the Yamagata lineage B/Bangladesh/3333 group have been identified and could constitute a new subgroup of these viruses. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We would like to thank warmly Ines Laaribi, Mónica Gónzalez-Esguevillas, Nieves Cruz, Ana Calderón, Noelia
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Reyes, Manuela Lopez-Valero, Mar Molinero, and Silvia Moreno for their excellent technical contribution. We would also like to thank all people involved in the various surveillance networks for their implication monitoring influenza viruses in Tunisia. References [1] Osterhaus AD, Rimmelzwaan GF, Martina BE, Bestebroer TM, Fouchier RA. Influenza B virus in seals. Science 2000;288:1051–3. [2] Nakagawa T, Higashi N, Nakagawa N. Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen. J Virol Methods 2008;148:296–9. [3] World Health Organization. Recommended composition of influenza vaccines for use in the 2012 southern hemisphere influenza season. Wkly Epidemiol Rec 2011;86:457–68. [4] Centers for Disease Control Prevention (CDC). Update: influenza activity–United States and worldwide, 2000–01 season, and composition of the 2001–02 influenza vaccine. MMWR Morb Mortal Wkly Rep 2001;50:466–79. [5] The WHO Collaborating Centre for influenza at CDC Atlanta, United States of America. CDC protocol of realtime RTPCR for swine influenza A(H1N1) 2009. http://www.who.int/csr/resources/publications/swineflu/ CDCrealtimeRTPCRprotocol 20090428.pdf [6] Ruiz-Carrascoso G, Casas I, Pozo F, Pérez-González C, Reina J, PérezBre˜na P. Development and implementation of influenza A virus subtyping and detection of genotypic resistance to neuraminidase inhibitors. J Med Virol 2010;82:843–53. [7] Tamura K, Dudley J, Nei M, et al. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [8] Influenza virus characterization surveillance report. Summary for Europe. Community Network of Reference Laboratories (CNRL) for Human Influenza in Europe; March 2012. Available from: http://www.ecdc.europa. eu/en/publications/Publications/1204-TED-CNRL-report.pdf [9] Kim YH, Kim HS, Cho SH, Seo SH. Influenza B virus causes milder pathogenesis and weaker inflammatory responses in ferrets than influenza A virus. Viral Immunol 2009;22:423–30. [10] Nerome R, Hiromoto Y, Sugita S, et al. Evolutionary characteristics of influenza B virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism. Arch Virol 1998;143:1569–83. [11] McCullers JA, Wang GC, He S, Webster RG. Reassortment and insertiondeletion are strategies for the evolution of influenza B viruses in nature. J Virol 1999;73:7343–8. [12] Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 1982;3:417–27.
[13] Daniels RS, Douglas AR, Skehel JJ, Wiley DC. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J Gen Virol 1983;64:1657–62. [14] Schulze IT. Effects of glycosylation on the properties and functions of influenza virus hemagglutinin. J Infect Dis 1997;76:S24–8. [15] Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000;69:531–69. [16] Schild GC, Oxford JS, de Jong JC, Webster RG. Evidence for host-cell selection of influenza virus antigenic variants. Nature 1983;303:706–9. [17] Ikonen N, Pyhala R, Axelin T, Kleemola M, Korpela H. Reappearance of influenza B/Victoria/2/87-lineage viruses: epidemic activity, genetic diversity and vaccination efficacy in the Finnish Defence Forces. Epidemiol Infect 2005;133:263–71. [18] Wang Q, Cheng F, Lu M, Tian X, Ma J. Crystal structure of unliganded influenza B virus hemagglutinin. J Virol 2008;82:3011–20. [19] Shen J, Kirk BD, Ma J, Wang Q. Diversifying selective pressure on influenza B virus hemagglutinin. J Med Virol 2009;81:114–24. [20] Sleeman K, Sheu TG, Moore Z, Kilpatrick S, Garg S, Fry AM, et al. Influenza B viruses with mutation in the neuraminidase active site, North Carolina, USA, 2010–11. Emerg Infect Dis 2011;17:2043–6. [21] Barclay WS, Palese P. Influenza B viruses with site-specific mutations introduced into the HA gene. J Virol 1995;69:1275–9. [22] Influenza virus characterization surveillance report. Summary for Europe. Community Network of Reference Laboratories (CNRL) for human influenza in Europe; March 2012. Available from: http://www.ecdc.europa. eu/en/publications/Publications/1204-TED-CNRL-report.pdf [23] European Surveillance Centre for Disease Prevention and Control. Surveillance Report. Weekly influenza surveillance overview. Main surveillance developments in week 19/2011 (09–15 May 2011). http://www.ecdc. europa.eu/en/publications/Publications/110520 SUR Weekly Influenza Surveillance Overview%20pdf. pdf [24] Rota PA, Wallis TR, Hamon MW, Rota JS, Kendal AP, Nerome K. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 1990;175:59–68. [25] Rota PA, Hemphill ML, Whistler T, Regnery HL, Kendal AP. Antigenic and genetic characterization of the haelagglutinins of recent cocirculating strains of influenza B virus. J Gen Virol 1992;73:2737–42. [26] Chen JM, Guo YJ, Wu KY, et al. Exploration of the emergence of the Victoria lineage of influenza B virus. Arch Virol 2007;152: 415–22. [27] Chen R, Holmes EC. The evolutionary dynamics of human influenza B virus. J Mol Evol 2008;66:655–63. [28] Anestad G. Surveillance of respiratory viral infections by rapid immunofluorescence diagnosis, with emphasis on virus interference. Epidemiol Infect 1987;99:523–31. [29] Mikheeva A, Ghendon YZ. Intrinsic interference between influenza A and B viruses. Arch Virol 1982;73:287–94.
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Original article
Genetic diversity of Influenza B virus in 2009–2010 and 2010–2011 in Tunisia Diversité génétique du virus de la grippe B en Tunisie en 2009–2010 et 2010–2011 A. El Moussi a,∗ , M.A. Ben Hadj Kacem a , J. Ledesma b , F. Pozo b , M. Teresa Cuevas b , I. Casas b , A. Slim a b
a National Influenza Centre-Tunis, Unit Virology, Microbiology Laboratory, Charles Nicolle’s Hospital, boulevard 9-Avril, 1006 Tunis, Tunisia National Influenza Centre-Madrid, Influenza and Respiratory Viruses Laboratory, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km 2 Majadahonda, 28220 Madrid, Spain
Received 20 October 2012; received in revised form 26 March 2013; accepted 17 June 2013 Available online 19 July 2013
Abstract Objective. – The authors had for aim to characterize influenza B strains having circulated in Tunisia to identify new mutations and compare them with reference strains. Methods. – The epidemiological surveillance of influenza allowed identifying 19 patients with symptoms related to respiratory infection, who had been infected by influenza B strains isolated in several regions of Tunisia in 2009–2010 and in 2010–2011. Laboratory identification and detection of mutations in the segment encoding hemagglutinin of influenza viruses was performed by real time PCR and sequencing. Results. – The two influenza B Tunisian strains of the 2009–2010 season belonged to the Victoria lineage, whereas 2010–2011 season strains belonged to B/Victoria/2/87 and B/Yamagata/16/88 lineages with a dominance of the Yamagata lineage (76%). This study allowed identifying amino acid substitutions: T121A, S150I, N165Y, T181A, G183R, D196N, S229D, M251V and K253R in the B/Yamagata lineage; L58P, N75K, K109N, N165K, S172P and K257R into the B/Victoria lineage. These mutations were specific of Tunisian groups of variants. Most influenza B-Yamagata lineage viruses (69%) were associated with severe cases. Conclusion. – Molecular analysis of the various influenza B strains circulating in Tunisia is useful to detect new mutations that can modify the phenotype of influenza strains. © 2013 Elsevier Masson SAS. All rights reserved. Keywords: Influenza B; Mutation; Phylogeny; Sequencing; B/Yamagata lineage; B/Victoria lineage
Résumé Objectif. – Caractériser les souches de la grippe B ayant récemment circulées en Tunisie afin de déceler les nouvelles mutations en comparaison avec les souches de référence. Méthodes. – Dans le cadre de la surveillance épidémiologique de la grippe, 19 patients présentant des symptômes liés à l’infection des voies respiratoires se sont révélés infectés par le virus de la grippe B durant les deux saisons 2009–2010 et 2010–2011. L’identification ainsi que la détection des mutations dans le segment codant l’hémagglutinine des virus grippaux ont été réalisées au laboratoire par la technique de RT-PCR en temps réel et le séquenc¸age. Résultats. – Il s’est avéré que les deux souches Tunisiennes de la grippe B de la saison 2009–2010 appartiennent à la lignée Victoria, alors que les souches de la saison 2010–2011 sont des deux lignées B/Victoria/2/87 et B/Yamagata/16/88 avec une dominance de la lignée Yamagata (76 %). Cette étude a permis d’identifier des mutations à l’origine de substitutions d’acide aminé : T121A, S150I, N165Y, T181A, G183R, D196N, S229D, M251V et K253R dans la lignée B-Yamagata ; L58P, N75K, K109N, N165K, S172P et K257R dans la lignée B-Victoria. Ces mutations sont spécifiques de groupes de variants Tunisiens. La majorité des virus de la lignée Yamagata étudiés (69 %) sont associés à des cas graves.
∗
Corresponding author. E-mail address: [email protected] (A. El Moussi).
0399-077X/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.medmal.2013.06.005
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Conclusions. – L’analyse moléculaire régulière des différentes souches de la grippe B circulant en Tunisie s’avère utile pour détecter de nouvelles mutations capables de modifier le phénotype des souches grippales. © 2013 Elsevier Masson SAS. Tous droits réservés. Mots clés : Grippe B ; Mutation ; Phylogénie ; Séquenc¸age ; Lignée B-Yamagata ; Lignée B-Victoria
1. Introduction Influenza is a very contagious infectious disease due to a genetically instable virus that spreads rapidly worldwide during seasonal outbreaks. Among viruses responsible for this disease, the influenza B virus seems to affect humans only even though it was isolated in seals [1]. These type B viruses mutate more slowly and have weaker pandemic potential than type A viruses. Nevertheless, during the last 20 years, influenza B has caused seasonal outbreaks in humans, as well as influenza A sub-type H1 and H3 strains [2]. A great influenza B activity was reported in several countries of the Northern hemisphere, in March 2012 [3]. Surveillance of influenza viruses is implemented in developed countries, but also in some African countries [4] and in the World Health Organization Regional Office for the Eastern Mediterranean (EMRO) region. This surveillance is essential to determine and compare the nucleotide sequences of viral genes, especially for the gene encoding hemagglutinin, HA. This allows characterizing viral isolates and assessing the yearly evolution of influenza strains. 2. Material and methods 2.1. Studied population We included patients consulting in the institutions of the Basic Healthcare network (French acronym DSSB) or admitted to the hospital for symptoms suggesting a flu syndrome with or without signs of severity. 3865 samples were positive for influenza viruses among the 7350 collected in the community during the 2009 pandemic. Hundred and eighty-one patients were found to be positive for influenza viruses among the 894 examined, during flu season 2010–2011. 2.2. Samples Respiratory samples were collected from all patients included (naso-pharyngeal, nose, throat, rhino-pharyngeal aspiration, protected tracheal sampling [PTS], BAL, etc.). These samples were put in an adapted virological medium for transportation (Vircell, Spain® ) then sent at +4 ◦ C to the National Influenza Centre at the Virology Unit of the Charles Nicolle hospital, Microbiology Laboratory in Tunis. 2.3. Molecular analysis The extraction of viral RNA was performed with the QIAamp Viral RNA mini kit (QIAGEN). The detection was made with a molecular technique relying on real time RT-PCR according to the recommended protocol, with primers and probes
from the CDC in Atlanta [5]. This technique is used routinely by the National Influenza Centre Laboratory for the surveillance of influenza viruses type A (A(H1N1)pdm09, A/H1N1, A/H3N2), and influenza B viruses. A partial sequencing was then performed targeting the segment encoding hemagglutinin (HA) (864 bp) for samples positive for influenza B, according to the protocol of National Influenza Centre Madrid [6]. This protocol is based on Sanger’s technique and performed on the machine “3130 Genetic Analyzer”. The sequences obtained were assembled using the Macaw software and compared to the reference sequences for influenza B, available on GenBank (http://www.ncbi.nih.gov/genomes/flu/swineflu.html) and Global initiative on Sharing Avian Influenza Data database (http://www.platform .gisiad.org), with the specialized software Clustal W included in MEGA version 4 [7]. 3. Results The analysis of influenza viruses by real time RT-PCR allowed detecting two influenza B viruses during season 2009–2010 and 17 influenza viruses during season 2010–2011. They were subdivided in two groups according to the severity of symptoms: 13 cases were classified as severe and six as mild. The mean age of cases was 29 years (range: 10 months to 51 years) (Table 1). The molecular analysis was performed on samples collected in various regions of Tunisia. It revealed the circulation of the B-Victoria lineage during season 2009–2010 and the co-circulation of B/Victoria lineage (4 strains) and B-Yamagata lineage (13 strains) in 2010–2011 season. The comparison of Tunisian Victoria strains with the reference strain revealed the following mutations: L58P, N75K, K109N, N165K, S172P, and K257R (Fig. 1). Furthermore, these strains presented sporadic substitutions in various positions of hemagglutinin such as: P31Q, K56E, H122R, V146I, G256W, I267N, and I290M (Table 2). Only two strains (B/Tunisia/8340/2009 and B/Tunisia/8348/2009) present a substitution of amino acid valine by isoleucine in position 146. This substitution is found in Nglycosylation site of the hemagglutinin domain 1. The mutations found in all Yamagata-like Tunisian strains compared with the reference B/Florida/4/2006 strain were: K88R, S150I, I165N, D196N, S229D, and M251V (Table 3). Other substitutions defining the two Yam/88 Tunisian groups were also identified: T121A, T181A, G183R, and K253R (Fig. 2). Sporadic mutations were also found in the Yamagata strains: S148N, L268W, I282L, and G248R. Furthermore, all Yamagata-like Tunisian strains had a new potential N-glycosylation site N196 with the substitution of amino acid aspartic acid (D) by asparagine (N). The phylogenetic analysis of Victoria strains (B/Tunisia/ 6/2011, B/Tunisia/756/2011, B/Tunisia/485/2011, and
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Table 1 Characteristics of patients and Tunisian influenza B strains studied. Caractéristiques des patients et des souches de la grippe B tunisiennes étudiés. Tunisian Influenza B strains
GenBank access number
Region
Date of sampling
Age
Sex
Clinical status
B/Tunisia/8348/2009 B/Tunisia/8340/2009 B/Tunisia/6/2010 B/Tunisia/7/2011 B/Tunisia/478/2011 B/Tunisia/485/2011 B/Tunisia/477/2011 B/Tunisia/484/2011 B/Tunisia/756/2011 B/Tunisia/1193/2011 B/Tunisia/1195/2011 B/Tunisia/1570/2011 B/Tunisia/1428/2011 B/Tunisia/1869/2011 B/Tunisia/1914/2011 B/Tunisia/2127/2011 B/Tunisia/2128/2011 B/Tunisia/2130/2011 B/Tunisia/2211/2011
JN037698 JN037697 JN037700 JN037706 JN037710 JN037705 JN037714 JN037713 JN037715 JN037701 JN037702 JN037703 JN037699 JN037704 JN037707 JN037708 JN037709 JN037711 JN037712
Tunis Nabeul Jendouba Tunis Sousse Sousse Tunis Sousse Bizerte Monastir Monastir Mahdia Sousse Mahdia Gabes Tunis Tunis Tunis Gabes
11 June 2009 11 June 2009 30 Dec. 2010 03 Jan. 2011 08 Jan. 2011 08 Jan. 2011 11 Jan. 2011 08 Jan. 2011 20 Jan. 2011 02 Feb. 2011 02 Feb. 2011 06 Feb. 2011 07 Feb. 2011 10 Feb. 2011 12 Feb. 2011 20 Feb. 2011 20 Feb. 2011 20 Feb. 2011 20 Feb. 2011
10 months 23 years 24 years 13 years 5 years 1 years 50 years 30 years 20 years 51 years 23 years 16 years 9 years 42 years 46 years 35 years 50 years 50 years 34 years
M F F M M M F F M F M M F M M M F F M
Mild case Mild case Severe case Mild case Mild case Mild case Mild case Mild case Severe case Severe case Severe case Severe case Severe case Severe case Severe case Severe case Severe case (ICU) Severe case Severe case
Fig. 1. Comparison of amino acid sequences of HA protein domain 1 gene of Victoria lineage Tunisian influenza B strains with reference strain B/Malaysia/2506/2004, showing the new and specific substitutions. Comparaison de séquences d’acides aminés déduites du domaine 1 de gène de la protéine HA représentatifs des souches de la grippe B de la lignée Victoria tunisiennes avec celle de la souche de référence B/Malaysia/2506/2004 montrant les nouvelles substitutions spécifiques du virus de la grippe B Tunisien. Table 2 Amino acid substitution of the HA protein (HA1 domain) of influenza B Victoria lineage. Variation des acides aminés de la protéine HA (de domaine HA1) de la grippe B de lignée Victoria. Position of the amino acid (without precursor protein) Strains B/Victoria
31
56
58
75
109
122
146
165
172
256
257
267
290
B/Malaysia/2506/2004 8340/2009 8348/2009 1428/2011 6/2011 485/2011 756/2011
P – – – Q Q –
K – – E – – –
L – – P P P P
N K K K K K K
K N N N N N N
H – – – R R R
V I I – – – –
N K K K K K K
S P P P P P P
G – – W – – –
K – – R R R R
I – – – – N –
I – – M – – –
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Table 3 Amino acids substitution of the HA protein (HA1 domain) of influenza B Yamagata lineage. Variation des acides aminés de la protéine HA (de domaine HA1) de la grippe B de lignée Yamagata. Position of the amino acid (without precursor protein) Strains B/Yamagata
88
121
148
150
165
181
183
196
229
251
253
268
282
284
B/Florida/4/2006 1193/2011 1195/2011 478/2011 B/7/2011 477/2011 1570/2011 1869/2011 2127/2011 2128/2011 2130/2011 2211/2011 484/2011 1914/2011
K R R R R R R R R R R R R R
T A A A A A – – – – – – – –
S – – – – – – – – – – – – N
S I I I I I I I I I I I I I
Y N N N N N N N N N N N N N
T – – – – – A A A A A A A A
G R R R R R – – – – – – – –
D N N N N N N N N N N N N N
S D D D D D D D D D D D D D
M V V V V V V V V V V V V V
K – – – – – R R R R R R R R
L – – W – – – – – – – – – –
I – – L – – – – – – – – – –
G – – – – – – – – – – – R –
Fig. 2. Comparison of amino acid sequences of HA protein domain 1 gene of Victoria lineage Tunisian influenza B strains with reference strain B/Florida/4/2006, showing the new and specific substitutions. Comparaison de séquences d’acides aminés déduites du domaine 1 de gène de la protéine HA représentatifs des souches de la grippe B de la lignée Yamagata tunisiennes avec celle de la souche de référence B/Florida/4/2006 montrant les nouvelles substitutions spécifiques du virus de la grippe B tunisien.
B/Tunisia/1428/2011) indicated that they belonged to the Brisbane/60 group characterized by specific mutations of Tunisian strains H122R, K255R, and P31Q (Fig. 3). Furthermore, the Tunisian strains were genetically related to those of neighbor countries Morocco and Algeria. The studied Yamagata-like strains (13 strains) belonged to the Bangladesh/3333 group (Fig. 4). These strains
were subdivided in two groups: eight belonged to the subgroup B/Serbia/1894/2011 and with the following mutations: T181A and K253N; the five remaining strains (B/Tunisia/7/2011, B/Tunisia/477/2011, B/Tunisia/478/2011, B/Tunisia/1193/2011, and B/Tunisia/1195/2011), with the mutations T121A and G183R, were not included in any of the four subgroups of the Bangladesh/3333 group described in the
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Fig. 3. Phylogenetic tree of HA sequences Tunisian influenza B strains Victoria lineage isolated during the 2009–2010 and 2010–2011 seasons. Arbre phylogénétique de la séquence HA des souches de la grippe B de la lignée Victoria isolées pendant les deux saisons 2009–2010 et 2010–2011.
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Fig. 4. Phylogenetic tree of HA sequences Tunisian influenza B strains Yamagata lineage isolated during the 2009–2010 and 2010–2011 seasons. Arbre phylogénétique de la séquence HA des souches de la grippe B de la lignée Yamagata isolées pendant les deux saisons 2009–2010 et 2010–2011.
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European Centre for Disease Prevention and Control (ECDC) report [8]. 4. Discussion The surveillance of influenza viruses circulating in the whole world is crucial for several reasons. It relies on the detection, typing, sub-typing, and sequencing the genome of strains in order to detect mutations able to bring new characteristics to the virus. This allows the WHO, each year, to indicate the most appropriate vaccinal composition for influenza, according to surveillance data collected worldwide thanks to the surveillance network. In Tunisia, the surveillance of influenza strains is managed by the National Influenza Centre at the Charles Nicolle hospital Virology Unit Microbiology Laboratory in Tunis. There are three circulating strains transmissible from human to human; among these influenza viruses the influenza B virus is important because it is responsible for sporadic cases and epidemics. But it remains less pathogenic than the influenza A virus [9]. The segments that encode hemagglutinin (HA) and neuraminidase (NA) (segments 4 and 6) are the most frequent targets of genetic characterization used for this surveillance [10,11]. Indeed, the mutations in these sequences are the most likely to induce a phenotypic change and especially antigenic, allowing the virus to escape the immune system. Helix 190 (194–196), which is part of the receptor binding site of HA, is undoubtedly one of the most important epitopes of influenza B. This study revealed mutations in this helix, belonging to hemagglutinin domain 1 (HA1), inducing the addition of a potential N-glycosylation site (NKT) in Tunisian B/Yamagata strains, in position 196. As reported, the influenza B virus similar to the influenza A virus [12–15], uses the suppression or addition of a glycosylation site to conduct an antigenic drift [16–18]. Most Tunisian Yamagata strains were detected in severe cases (9/13 or 69%). This supports the hypothesis that the gain of a glycosylation may cause an increased virulence of the mutated virus. Recently, Shen et al. confirmed that this mutation allowed a positive selection of the virus and escaping the immune system [19]. Our study allowed us to have an idea of the virus’s virulence, but more strains need to be sequenced so as to obtain statistically robust calculations. Indeed, even though influenza B viruses are less pathogenic than influenza A viruses, and even if they are responsible for sporadic diseases, they can be associated with severe complications and death [20,21]. The sequencing of influenza virus genes could constitute a sufficiently large database to obtain information on their virulence. The pathogenicity of influenza B is still not explained molecularly. Complementary studies are needed. The phylogenetic analysis of HA nucleotide sequences revealed a great genetic diversity of the studied viruses. We detected only two type B viruses (0.1%) during the 2009 pandemic. The comparison of strain sequences (B/Tunisia/8340/2011 and B/Tunisia/8348/2011) with those of reference strains revealed that it was Victoria lineage influenza B, similar to older Victoria strain: B/Victoria/2/87. The comparison of these strains with the Victoria strains detected during season 2010–2011 (n = 4), revealed new mutations of influenza
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B expressed by two amino acid substitutions: K257R and L58P already mentioned in the ECDC report. During 2010–2011 season, four out of 17 viruses B were localized in the Victoria lineage and belonged to the B/Brisbane/60/2008 group. The 13 other strains (after week 40 of season 2010–2011) were clustered in the Yamagata lineage and belonged to the B/Bangladesh/3333/2007 group. The HA sequences of Tunisian B/Yamagata strains were classified in two groups. The first specific group of eight viruses was characterized by two mutations (T121A and G183R). The second group of five viruses was defined by two substitutions: T181A, K253R, mentioned in the ECDC report [22]. All the segments of the hemagglutinin gene had evolved into two distinct major lineages (B/Victoria and B/Yamagata) and their amino acid substitution profiles were specific and maintained in these two lineages. During 2010–2011 season, most Tunisian strains belonged to the Yamagata lineage (76%) contrary to other countries (4%) where the dominant circulating strain was Victoria lineage [23]. The B/Yamagata virus was not included in the 2011–2012 trivalent vaccine. The co-circulation of two lineages in Tunisia, B/Victoria/2/87 (Vic87) and B/Yamagata/16/88 (Yam88), have been identified elsewhere since 1983 [24]. The variants belonging to these two lineages had been identified in 2002 [25,26]. The dynamic evolution of influenza B virus, with the passage of a dominant position between the Vic87 and the Yam88 lineages, is due to immune selection. In fact, the dominant circulation of Yama88 lineage in the 1990s was associated to a very important antigenic drift of this lineage [27]. Likewise, influenza A and influenza B are clearly in competition to infect humans. It is probable that the infection by one type of virus inhibits infection as well as the replication of the other [28,29]. 5. Conclusion The molecular characterization of influenza B virus has been greatly contributive. It allows genotyping, which is essential for the traceability of circulating strains. It also allows detecting mutations that could explain specific phenotypes. We demonstrated that, during the influenza A virus (H1N1)pdm09 pandemic, the Victoria lineage influenza B viruses had been very weakly active. During the next season, the influenza B activity was more important and characterized by a co-circulation of two lineages, Victoria and Yamagata, with the predominance of the latter. Specific mutations of Tunisian strains belonging to the Yamagata lineage B/Bangladesh/3333 group have been identified and could constitute a new subgroup of these viruses. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We would like to thank warmly Ines Laaribi, Mónica Gónzalez-Esguevillas, Nieves Cruz, Ana Calderón, Noelia
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Reyes, Manuela Lopez-Valero, Mar Molinero, and Silvia Moreno for their excellent technical contribution. We would also like to thank all people involved in the various surveillance networks for their implication monitoring influenza viruses in Tunisia. References [1] Osterhaus AD, Rimmelzwaan GF, Martina BE, Bestebroer TM, Fouchier RA. Influenza B virus in seals. Science 2000;288:1051–3. [2] Nakagawa T, Higashi N, Nakagawa N. Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen. J Virol Methods 2008;148:296–9. [3] World Health Organization. Recommended composition of influenza vaccines for use in the 2012 southern hemisphere influenza season. Wkly Epidemiol Rec 2011;86:457–68. [4] Centers for Disease Control Prevention (CDC). Update: influenza activity–United States and worldwide, 2000–01 season, and composition of the 2001–02 influenza vaccine. MMWR Morb Mortal Wkly Rep 2001;50:466–79. [5] The WHO Collaborating Centre for influenza at CDC Atlanta, United States of America. CDC protocol of realtime RTPCR for swine influenza A(H1N1) 2009. http://www.who.int/csr/resources/publications/swineflu/ CDCrealtimeRTPCRprotocol 20090428.pdf [6] Ruiz-Carrascoso G, Casas I, Pozo F, Pérez-González C, Reina J, PérezBre˜na P. Development and implementation of influenza A virus subtyping and detection of genotypic resistance to neuraminidase inhibitors. J Med Virol 2010;82:843–53. [7] Tamura K, Dudley J, Nei M, et al. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [8] Influenza virus characterization surveillance report. Summary for Europe. Community Network of Reference Laboratories (CNRL) for Human Influenza in Europe; March 2012. Available from: http://www.ecdc.europa. eu/en/publications/Publications/1204-TED-CNRL-report.pdf [9] Kim YH, Kim HS, Cho SH, Seo SH. Influenza B virus causes milder pathogenesis and weaker inflammatory responses in ferrets than influenza A virus. Viral Immunol 2009;22:423–30. [10] Nerome R, Hiromoto Y, Sugita S, et al. Evolutionary characteristics of influenza B virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism. Arch Virol 1998;143:1569–83. [11] McCullers JA, Wang GC, He S, Webster RG. Reassortment and insertiondeletion are strategies for the evolution of influenza B viruses in nature. J Virol 1999;73:7343–8. [12] Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 1982;3:417–27.
[13] Daniels RS, Douglas AR, Skehel JJ, Wiley DC. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J Gen Virol 1983;64:1657–62. [14] Schulze IT. Effects of glycosylation on the properties and functions of influenza virus hemagglutinin. J Infect Dis 1997;76:S24–8. [15] Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000;69:531–69. [16] Schild GC, Oxford JS, de Jong JC, Webster RG. Evidence for host-cell selection of influenza virus antigenic variants. Nature 1983;303:706–9. [17] Ikonen N, Pyhala R, Axelin T, Kleemola M, Korpela H. Reappearance of influenza B/Victoria/2/87-lineage viruses: epidemic activity, genetic diversity and vaccination efficacy in the Finnish Defence Forces. Epidemiol Infect 2005;133:263–71. [18] Wang Q, Cheng F, Lu M, Tian X, Ma J. Crystal structure of unliganded influenza B virus hemagglutinin. J Virol 2008;82:3011–20. [19] Shen J, Kirk BD, Ma J, Wang Q. Diversifying selective pressure on influenza B virus hemagglutinin. J Med Virol 2009;81:114–24. [20] Sleeman K, Sheu TG, Moore Z, Kilpatrick S, Garg S, Fry AM, et al. Influenza B viruses with mutation in the neuraminidase active site, North Carolina, USA, 2010–11. Emerg Infect Dis 2011;17:2043–6. [21] Barclay WS, Palese P. Influenza B viruses with site-specific mutations introduced into the HA gene. J Virol 1995;69:1275–9. [22] Influenza virus characterization surveillance report. Summary for Europe. Community Network of Reference Laboratories (CNRL) for human influenza in Europe; March 2012. Available from: http://www.ecdc.europa. eu/en/publications/Publications/1204-TED-CNRL-report.pdf [23] European Surveillance Centre for Disease Prevention and Control. Surveillance Report. Weekly influenza surveillance overview. Main surveillance developments in week 19/2011 (09–15 May 2011). http://www.ecdc. europa.eu/en/publications/Publications/110520 SUR Weekly Influenza Surveillance Overview%20pdf. pdf [24] Rota PA, Wallis TR, Hamon MW, Rota JS, Kendal AP, Nerome K. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 1990;175:59–68. [25] Rota PA, Hemphill ML, Whistler T, Regnery HL, Kendal AP. Antigenic and genetic characterization of the haelagglutinins of recent cocirculating strains of influenza B virus. J Gen Virol 1992;73:2737–42. [26] Chen JM, Guo YJ, Wu KY, et al. Exploration of the emergence of the Victoria lineage of influenza B virus. Arch Virol 2007;152: 415–22. [27] Chen R, Holmes EC. The evolutionary dynamics of human influenza B virus. J Mol Evol 2008;66:655–63. [28] Anestad G. Surveillance of respiratory viral infections by rapid immunofluorescence diagnosis, with emphasis on virus interference. Epidemiol Infect 1987;99:523–31. [29] Mikheeva A, Ghendon YZ. Intrinsic interference between influenza A and B viruses. Arch Virol 1982;73:287–94.