Original Paper Intervirology 2014;57:344–352 DOI: 10.1159/000365758
Received: April 3, 2014 Accepted after revision: July 5, 2014 Published online: October 3, 2014
Characterization of Human Influenza Viruses in Lebanon during 2010–2011 and 2011–2012 Post-Pandemic Seasons Hassan Zaraket a, c Clyde Dapat d Soha Ghanem b Zainab Ali b Mireille Lteif b Hiroki Kondo d Isolde C. Dapat d Kousuke Saito d Ghazi Kayali c Hiroshi Suzuki d, e Ghassan Dbaibo b Reiko Saito d a
Department of Experimental Pathology, Immunology and Microbiology, and b Division of Pediatric Infectious Diseases, Department of Pediatrics and Adolescent Medicine and the Center for Infectious Diseases Research, American University of Beirut, Beirut, Lebanon; c Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tenn., USA; d Division of International Health, Graduate School of Medical and Dental Sciences, Niigata University, and e School of Nursing, Niigata Seiryo University, Niigata, Japan
Abstract Objective: To genetically characterize human influenza viruses and their susceptibilities to antivirals during two post-pandemic seasons in Lebanon. Methods: Influenza virus was isolated from nasopharyngeal swabs that were obtained from patients with influenza-like illness during 2010–2012 and further analyzed both phenotypically and genotypically. Results: During the 2010–2011 season, both 2009 pandemic H1N1 (H1N1p) and B viruses co-circulated with equal prevalence, while the H3N2 virus predominated during the 2011–2012 season. All H3N2 and H1N1 viruses were resistant to amantadine. Importantly, all viruses of the influenza A and B types were susceptible to the neuraminidase (NA) inhibitors oseltamivir, zanamivir, peramivir, and laninamivir. Nonetheless, all 2011–2012 H1N1p isolates had three mutations (V241I, N369K, and N386S) in the NA gene that were suggested to be permissive of the H275Y mutation, which confers resistance to
© 2014 S. Karger AG, Basel 0300–5526/14/0576–0344$39.50/0 E-Mail [email protected] www.karger.com/int
oseltamivir. We also detected one H1N1p virus during the 2010–2011 season with a 4-fold decrease in susceptibility to oseltamivir due to an NA-S247N mutation. This isolate was phylogenetically distinct from other H1N1p viruses that were isolated in other regions. Conclusions: Influenza A viruses with reduced susceptibility to oseltamivir and mutations permissive for acquiring NA resistance-conferring mutation with minimal burden on their fitness were isolated in Lebanon. © 2014 S. Karger AG, Basel
Introduction
Annual influenza outbreaks affect on average 5–20% of the world’s population, resulting in a significant burden on human health [1]. These outbreaks are caused by seasonal influenza viruses that undergo continuous antigenic changes [2]. Occasionally, pandemics can occur due to antigenic shifts in the HA protein. Until the 2008– 2009 winter season, two influenza A subtypes, H3N2 and H1N1, and influenza B circulated in humans. However, in spring 2009, a novel influenza virus (pandemic H1N1 2009; H1N1p) emerged and quickly spread around the Hassan Zaraket Department of Experimental Pathology, Immunology and Microbiology American University of Beirut PO Box 113/6044/B21, Beirut (Lebanon) E-Mail hassan.zaraket @ aub.edu.lb
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Key Words Influenza virus · Pandemic H1N1 · Neuraminidase inhibitors · Antiviral drug susceptibility · Lebanon · Middle East
Flu B A/H1N1p A/H3N2 ILI
70 Number of cases
world [3]. In the following season, the seasonal H1N1 virus continued to circulate at a minimal level but eventually became extinct. However, a return and, in some cases, dominance of seasonal H3N2 and influenza B viruses was observed worldwide [4, 5]. In addition to their antigenic evolution, influenza viruses evolve under drug pressure or natural selection, and new virus populations that are resistant to treatment occasionally evolve and sometimes prevail if viral fitness is maintained [6, 7]. As a result, the use of antiviral drugs like amantadine, an M2-channel inhibitor, has been largely limited due to the surge of amantadine-resistant H3N2 influenza viruses [8]. Neuraminidase inhibitors (NAIs), including oseltamivir and zanamivir, became the drugs of choice for treatment of H3N2 influenza infections [9]. The utility of oseltamivir in treating influenza was jeopardized by the worldwide rapid emergence of oseltamivir-resistant seasonal H1N1 viruses during 2007 through 2009 [6, 7]. Nonetheless, this virus was soon replaced by the H1N1p virus, which is largely sensitive to oseltamivir but resistant to amantadine [10]. This has revived the utility of oseltamivir in treating influenza. Surveillance studies aimed at investigating the evolution and characteristics of influenza viruses worldwide are essential to early detection of new antigenically variant viruses as well as those with decreased antiviral drug susceptibility to allow informed recommendations for vaccination strategies and therapeutic interventions. Influenza surveillance efforts in the Middle East, including Lebanon, the site of this study, have been very limited. Characterization of influenza viruses in this region is critical to compliment the global effort to monitor and control influenza. The H1N1p influenza virus was first detected in Lebanon in May 2009. One study showed that a large outbreak caused by the pandemic virus occurred in October of the same year in Lebanon [11]. The prevalence of H1N1p viruses in the post-pandemic seasons and their susceptibilities to antiviral drugs is unknown in Lebanon. The goal of this study was to characterize influenza viruses that circulated in Lebanon during two post-pandemic influenza seasons.
60 50 40 30 20 10 0
A S ON D J F MAM J J A S ON D J F MA M J 2010 2011 2012
Fig. 1. Graphical representation of influenza cases in Lebanon during the 2010–2012 period. The dotted line represents all ILI cases that were identified during the study period. The letters on the xaxis represent months of the year.
Virus Isolation and Characterization 100 μl of the swab suspension was inoculated onto Madin-Darby canine kidney (MDCK) cells and incubated at 37° and 5% CO2. Influenza virus type and subtype were characterized using cyclingprobe real-time PCR [12–14]. RNA Extraction, PCR, and Sequencing Following RNA extraction and reverse transcription [15, 16], the HA (HA1 subunit) and neuraminidase (NA) genes were sequenced and aligned using BioEdit software. Phylogenetic trees were constructed using the Neighbor-Joining (MEGA program). Clusters were defined using WHO-designated reference strains [17]. Sequences generated in this study were deposited in the Japanese GenBank database (http://www.ddbj.nig.ac.jp/; accession No. KF000008-62). Antiviral Drug Susceptibility Testing Genotypic analysis of the single-nucleotide polymorphisms M2-S31N and NA-H275Y, which confer resistance to amantadine and oseltamivir, respectively, was performed using cycling probe method [12, 13]. In vitro antiviral drug susceptibilities of the isolated viruses were determined by measuring the 50% inhibitory concentrations (IC50) of oseltamivir (Sequoia Research, UK), zanamivir (Sequoia Research, UK), peramivir (Shionogi, Japan), and laninamivir (Daiichi Sankyo, Japan) using a fluorescencebased NA inhibition assay with methylumbelliferone N-acetylneuraminic acid (MUNANA) as the substrate at a final concentration of 0.025 mM [18].
Materials and Methods
Post-Pandemic Influenza in Lebanon
Results
A total of 202 samples were collected from patients with ILI during August 2010 through June 2012 (fig. 1). These included 73 samples from the 2010–2011 season and 129 samples from the 2011–2012 season. ILI activity Intervirology 2014;57:344–352 DOI: 10.1159/000365758
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Sample Collection Nasal swabs were collected from patients presenting with influenza-like illness (ILI) symptoms (i.e. fever of at least 37.5°, cough, rhinorrhea, myalgia, and headache) at a pediatric clinic in Beirut. One swab was immediately tested using a rapid influenza diagnostic test, Quick-Ex Flu kit (Denka Seiken, Japan), and a second swab was stored in virus transport media at –80° until further analysis.
2010 –2011 H1N1p
2011 –2012 H1N1p
1,000
1,000 n=3
n = 10 100
100
IC50 (nM)
S247N 1
1 1.23 ± 0.28
a
1.07 ± 0.13
0.63 ± 0.91
0.1
0.01
10
0.14 ± 0.02
0.10 ± 0.04 Peramivir
Oseltamivir
Zanamivir
Laninamivir
0.37 ± 0.13
0.1
0.31 ± 0.11
0.01
b
Peramivir
2011 –2012 H3N2
Oseltamivir
Zanamivir
1,000
100
100
10
10
1
c
n = 11
25.98 ± 3.94 29.90 ± 14.73
IC50 (nM)
IC50 (nM)
n = 25
0.01
Laninamivir
2010 –2011 influenza B
1,000
0.1
0.23 ± 0.01
1
0.24 ± 0.32
0.05 ± 0.10
0.39 ± 0.13
0.42 ± 0.12
Peramivir
Oseltamivir
Zanamivir
Laninamivir
11.51 ± 6.19 2.92 ± 0.90
0.1
d
0.01
Peramivir
Oseltamivir
Zanamivir
Laninamivir
Fig. 2. Box plots of IC50 values of NAIs against 2010–2011 H1N1p (a), 2011–2012 H1N1p (b), 2011–2012 H3N2 (c), and 2010–2011 influenza B (d) viruses collected in Lebanon. IC50 values of peramivir, oseltamivir, zanamivir, and laninamivir were determined using a fluorescence-based assay with MUNANA as a substrate.
The black bars indicate the 75th percentile + 3 × interquartile range (3 IQR). Values below the box plot represent mean ± SD. The mean value was calculated from viruses with IC50 values below the cutoff limit (75th percentile + 3 IQR). Samples with IC50 greater than the cutoff limit are considered outliers.
was highest during the months of January and February. In the 2010–2011 season, 12 (17%) samples tested positive for influenza A and 15 (21%) samples tested positive for influenza B using the rapid test kit. Among these, 10 H1N1p viruses and 11 influenza B viruses were isolated.
The influenza B cases were detected in the earlier part of the season, followed by H1N1p viruses. No H3N2 viruses were detected during the 2010–2011 season. In the 2011– 2012 season, 42 (33%) samples tested positive for influenza A by the rapid kit, and no influenza B viruses were
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IC50 (nM)
10
detected. Of these, 23 H3N2 and 3 H1N1p viruses were isolated. Cycling probe real-time PCR analysis revealed that all viruses from both subtypes had the M2-S31N mutation. None of the H1N1p viruses from either the 2010–2011 or 2011–2012 season had the NA-H275Y mutation. Using an in vitro fluorescence-based NA inhibition assay, all H1N1p viruses collected in this study were found susceptible to all the tested NAIs with IC50 values of 0.06–0.19 nM for peramivir, 0.6–5.11 nM for oseltamivir, 0.46–1.01 nM for zanamivir, and 0.19–0.57 nM for laninamivir (fig. 2a, b). One H1N1p isolate (A/Lebanon/10L-21/2011) had a 4-fold higher oseltamivir IC50 (IC50 = 5.11 nM) than the average IC50 (1.18 nM) for the rest of the H1N1p isolates from both seasons. No difference was observed in IC50values of the other three NAIs against the 10L-21 isolate. For the H3N2 viruses, IC50 values were 0.17–0.30 nM for peramivir, 0.94–1.2 nM for oseltamivir, 0.26–0.51 nM for zanamivir, and 0.23–0.73 nM for laninamivir (fig. 2c). Influenza B isolates had IC50 values that were higher than the influenza A viruses at 1.85–4.27 nM for peramivir, 19.58–32.05 nM for oseltamivir, 11.10–49.18 nM for zanamivir, and 3.41–21.82 nM for laninamivir (fig. 2d). In summary, the IC50 values suggest that all influenza A viruses isolated in the study are highly susceptible to NAIs. Influenza B isolates had up to 50-fold higher IC50 values than influenza A isolates. Phylogenetic analysis of the HA genes from H1N1p viruses revealed that the 2010–2011 season belonged to five distinct clusters: 1, 5, 6, 7 and 9 (fig. 3a). Cluster 1 included isolates from the 2009 pandemic outbreak with the exception of one isolate (A/Lebanon/10L-21/2011) from the 2010–2011 season, which had 2 additional mutations (S134T and R265K) in the HA gene. Two isolates (A/Lebanon/10L-34/2011 and A/Lebanon/10L-38/2011) from the 2010–2011 season formed a small cluster, designated cluster 9, characterized by 4 unique mutations (N44D, A267V, G270S, and I272L). None of these mutations belonged to any of the reported antigenic sites. In addition to the 2010–2011 season isolates, cluster 6 also accommodated the 2011–2012 isolates, which accumulated 3 additional HA mutations, including an H144R mutation in the Ca antigenic site and a V158I mutation adjacent to the Sa antigenic site [19]. Overall, the H1N1p viruses analyzed in this study had 6 amino acid mutations in the antigenic sites: 3 in Ca, 2 in Sb, and 1 in Sa, compared with A/California/07/2009. Three of these mutations were not detected in the earlier H1N1p viruses. To determine the genetic basis of the reduced susceptibility to oseltamivir of the 10L-21 (A/Lebanon/10L-21/
2011) isolate from the 2010–2011 season and its genetic relatedness to other isolates, we sequenced the NA genes of all H1N1p viruses isolated in this study. In general, the Lebanese H1N1p NA genes had a clustering pattern that corresponded with their counterpart HA clusters and had 5–7 mutations compared with the A/California/07/2009 vaccine strain (fig. 3). The 10L-21 isolate with reduced susceptibility to oseltamivir belonged to cluster 1 in the NA tree similar to its position in the HA tree. This isolate was characterized by 3 mutations: S247N, N385T, and N386K. The N385T and N386K mutations were located away from the enzyme-active site, suggesting no role in the reduced susceptibility to oseltamivir. The S247N mutation interacts with amino acid residues that in turn interact with oseltamivir and could interfere with stability of the inhibitor in the enzyme-active pocket [20]. The 10L-34 and 10L-38 strains formed a small isolated cluster in the NA tree just like in the HA tree and were characterized by 4 unique mutations: Q313E, T383R, I396T, and S442I. The HA genes of 8 Lebanese H3N2 influenza viruses collected during the 2011–2012 season were sequenced. All the sequenced viruses were A/Perth/16/2009-like and belonged to the Vic/208 lineage in the HA phylogeny (fig. 4). Two of the isolates belonged to clade 3A, and the rest belonged to clade 3B, which also contained strains from other Middle Eastern countries. These isolates accumulated 9–10 mutations, including 2 antigenic site A mutations (K144N/D and N145S). In case of influenza B, all of the 11 isolates collected during the 2010–2011 season were sequenced. All of these isolates were Victorialike and belonged to the Brisbane/60 clade (fig. 5). The Lebanese strains had 1–2 mutations compared with the B/Brisbane/60/2008 vaccine strain (2009–2012).
Post-Pandemic Influenza in Lebanon
Intervirology 2014;57:344–352 DOI: 10.1159/000365758
In this study, we found that in the first post-pandemic season (2010–2011), both influenza B and A/H1N1p cocirculated at a similar rate. In the 2011–2012 season, the A/H3N2 virus dominated and the H1N1p virus co-circulated with a lower prevalence. Similar circulation patterns have been reported elsewhere in the world [4, 21]. The extinction of seasonal H1N1 viruses has been suggested to be a consequence of the original antigenic sin, whereby infection with the H1N1p virus boosted the HA stalk antibodies against the seasonal H1N1 virus [22]. A similar phenomenon has been observed when H2N2 pandemic viruses, which emerged in 1957, replaced previously circulating H1N1 viruses. The H2N2 virus circulated for 11 347
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Discussion
A/Lebanon/11L043/2012 Jan A/Lebanon/11L008/2011 Dec A/Lebanon/11L045/2012 Jan A/Lebanon/10L-45/2011 Feb D103N A/St. Petersburg/27/2011 S191T S90I A/Lebanon/10L-40/2011 Jan (Sb) G161E (Sa) 98 A/Lebanon/10L-41/2011 Jan A/England/04860492/2010 Nov A/Singapore/GP4588/2010 Dec ¥ 98 A/Darwin/74/2011 Mar ¥ H144Q A/Perth/29/2011 Mar ¥ A/Astrakhan/1/2011 R211K (Ca) , A/Archangelsk/CRIE-SPA/2011 Jan I222V, V255L 95 A/Izmir/12/2011 Feb A/Lebanon/10L-13/2011 Jan N103D A/Ankara/03/2010 Dec A/Ghom/198/2010 Dec A/Lebanon/10L-30/2011 Jan A/Lebanon/10L-48/2011 Feb A192T A/Brazil/AVS11/2011 Jun (Sb) A/Ankara/02/2011 Jan A/Texas/JMS415/2010 Feb A/Lebanon/09L-04/2009 May N131D 4 A/Sydney/DD3-10/2010 Jul A/Christchurch/16/2010 S191T A/Matara/2132/2011 Feb (Sb) A/Brunei/1/2011 Jan ¥ A/Ghom/215/2010 Dec A/Karaj/5327/2010 Dec A/Tehran/5958/2010 Dec A/Athens/INS564/2011 Feb 7 S149G A/Karaj/5924/2010 Dec A203T 82 S209T A/Qatar/2311001414/2011 Jan 73 (Ca) A/Lebanon/10L-53/2011 Feb A/St. Petersburg/100/2011 A/Jeddah/3657/2010 Nov 89 A/Jeddah/3669/2010 Nov D228E (Ca) P303S A/Jeddah/3673/2010 Nov A/Lebanon/09L-30/2009 Nov 2 A/Bahrain/N11659/2009 Oct A140T A/Ancona/01/2010 Jan S189P A/New York/10/2009 A/Amman/WRAIR3448N/2010 Dec 3 A/Hong Kong/3934/2011 A/Ghana/FS-11-1803/2011 A/Boston/DOA08/2011 Jan 8 P89S A/Lebanon/09L-15/2009 Oct I327V A/Abu Dhabi/1134/2009 June A/Kuwait/4536/2009 Kuwait A/California/05/2009 A/Lebanon/09L-06/2009 Oct A/Dakar/WRAIR0019T/2010 Jan L38I A/Lebanon/09L-28/2009 Nov 93 A/Lebanon/10L-21/2011 Jan ¥ S134T, R265K N44D, A267V, A/Lebanon/10L-34/2011 Jan G270S, I272L 100 A/Lebanon/10L-38/2011 Jan
NA
H144R V158I, V255L 99
A/California/07/2009 (2009-2013)
6
A/Lebanon/11L045/2012 Jan N386S D451G 97 A/Lebanon/11L043/2012 Jan A/Lebanon/11L008/2011 Dec A/Lebanon/10L-40/2011 Jan V453M A/Lebanon/10L-45/2011 Feb A/England/04860492/2010 Nov N44S A/Lebanon/10L-53/2011 Feb A/St. Petersburg/100/2011 A/Athens/INS564/2011 Feb A/Ghom/215/2010 Dec A/Karaj/5327/2010 Dec 85 A/Tehran/5958/2010 Dec A/Karaj/5924/2010 Dec V81A A/Ghom/198/2010 Dec A/Archangelsk/CRIE-SPA/2011 Jan V241I 88 A/Lebanon/10L-13/2011 Jan N369K N141D A/Brazil/AVS11/2011 06/4 D416E A/Lebanon/10L-48/2011 Feb A/Lebanon/10L-30/2011 Jan L40I, I46T, S95I, S247N A/Perth/29/2011 Mar ¥ A/Brunei/1/2011 Jan ¥ S247N S299A 96 A/Singapore/GP4588/2010 Dec ¥ 85 A/Darwin/74/2011 Mar ¥ A/Sydney/DD3-10/2010 Jul 4 A/Amman/WRAIR3448N/2010 Dec 3 V106I Q313E, T383R A/Lebanon/10L-34/2011 Jan I396T, S442I 99 A/Lebanon/10L-38/2011 Jan 9 A/New York/10/2009 A/Jeddah/3673/2010 Nov A/Lebanon/09L-15/2009 Oct A/Jeddah/3669/2010 Nov N248D A/Lebanon/09L-30/2009 Nov 2 A/Lebanon/09L-06/2009 Oct 81 A/Ancona/01/2010 Jan A/Texas/JMS415/2010 Feb A/Jeddah/3657/2010 Nov Y381F A/Lebanon/09L-04/2009 May A/Boston/DOA08/2011 Jan V106I S247N A/Dakar/WRAIR0019T/2010 Jan 84 N385T, N386K A/Lebanon/10L-21/2011 Jan ¥ 1 A/Lebanon/09L-28/2009 Nov A/California/05/2009 N44S, V53I, V62I, N386S
5
1
6
Color version available online
HA
7
5
A/California/07/2009 (2009-2013) 0.001
9
Fig. 3. Phylogenetic analysis of the hemagglutinin (HA1) and NA genes of influenza A/H1N1 pandemic 2009 viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. A/California/07/09 was used as the root for the tree and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated on the branches. The HA protein antigenic site
to which a mutation belongs is in parentheses. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2009 (previously reported by Zaraket et al. [11]), 2010–2011, and 2011–2012 seasons are shown in red, green, and blue, respectively. ¥ Viruses with the S247N mutation. In the NA tree, only reference samples for which the NA gene sequence was available in the GenBank database are included.
years until it was replaced by H3N2 virus in 1968 due to antibodies specific to the retained N2 subtype [23]. The H1N1p viruses continued to evolve, accumulating mutations in antigenic sites, but remained antigenically similar to the A/California/07/2009 vaccine strain.
In the post-pandemic 2010–2011 season, H1N1p viruses collected in Lebanon were diverse, belonging to multiple clusters, suggesting multiple introductions of this virus into Lebanon. This was in contrast to mostly homologous H1N1p viruses that we detected in previous
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0.001
A198S N145S (A)
V223I
S45N T48I 92 A/Victoria/361/2011
(2012-2013)
A/Myanmar/M019/2011
S312Q A/Hong Kong/3969/2011 N144D (A) A/Stockholm/18/2011 N145S (A) 99 A/Lebanon/11L007/2011 Dec
3A
A/Virginia/03/2010 A/Kuwait/4554/2009 Dec A/Hong Kong/1736/2010 A/Serbia/71/2011 A/Hiroshima-C/27/2010 A/Myanmar/M049/2009 Q312 A/Iraq/7-FSS/2011 Jan A/Alabama/04/2011 A/Victoria/208/2009 A/Vietnam/BN420/2010 T212A D53N (C) A/Tehran/181/2012 Jan 92 Y94H I230N 93 A/Tehran/8331/2011 Nov A/Esfahan/8343/2011 Nov E280A A/Iowa/19/2010 A/Myanmar/M417/2010 A/Afghanistan/2950/2009 Oct A/Thailand/CU-B1697/2009 A/Norway/1330/2010 T212A A/Tianjin-Nankai/1687/2010 K260M 77 A/Taiwan/42/2010 I261Q 97 A/Iran/755/2009 June
Vic/208
A/Lebanon/11L018/2012 Jan A/Lebanon/11L033/2012 Feb K173 85 A/Lebanon/11L040/2012 Feb A/Bahrain/40/2011 Dec A/Lebanon/11L046/2012 Feb 3B A/Lebanon/11L013/2011 Dec A/Lebanon/11L023/2012 Jan A/England/259/2011 A/Hormozgan/7981/2011 Nov A/Jordan/20110030780/2011 Dec A/Lebanon/11L002/2011 Nov A/Oman/916/2011 July A/Alborz/96/2012 Jan A/Khorasan8032//2011 Nov A/Niigata/11F526/2012 Mar A/Ghom/9677/2011 Dec N278K A/Hong Kong/5047/2011 3C A/Gunma/11G001/2011 Dec
Color version available online
G5E, E62I A198P, N145S (A)
Q312S
K158N N189K E62K N144K (A)
82
A/Lebanon/L34/2009 Jan A/Lebanon/L47/2009 Jan A/Lebanon/L82/2009 Feb A/Tehran/93/2008 Nov A/Kuwait/WRAIR1561P/2009 Jan A/Wisconsin/67/2005 I179V
A/Brisbane/10/2007 (2008-2010)
Perth/16
A/Victoria/210/2009 (2011-2012) A/Perth/16/2009 (2010-2011)
G50E K173Q
Brisbane/10
0.002
work during the pandemic [11]. In the case of H3N2, all viruses from the 2011–2012 season sequenced in this study belonged to the A/Victoria/208/2011-like lineage, and most of them fell into clade 3B. The Lebanese isolates closely resembled WHO-designated reference strains A/Stockholm/18/2011 and A/England/259/2011 which exhibited 64- and 32-fold reduction, respectively, in their HA inhibition (HAI) titers of sera raised against the A/Victoria/210/2009 vaccine strain [17]. These viruses accumulated several mutations, including 3–4 in the antigenic sites compared with the vaccine strain
which explains the antigenic drift marked reduction in their HAI titers. The prevalence of oseltamivir-resistant H1N1p viruses remained low worldwide in the post-pandemic period, albeit at a slight increase from their level during the pandemic period [24–26]. Nonetheless, the rate of oseltamivir resistance is much higher among high-risk groups such as immunocompromised patients, and clusters of high community transmission rates have been reported [25, 27]. Hurt et al. [28] reported a significant increase in the frequency H275Y oseltamivir-resistant H1N1p virus-
Post-Pandemic Influenza in Lebanon
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Fig. 4. Phylogenetic analysis of the hemagglutinin (HA1) genes of influenza A/H3N2 viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. A/Brisbane/10/2007 was used as the root for the tree and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated on the branches. The HA antigenic site to which a mutation belongs is in parentheses. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2008–2009 and 2011–2012 seasons are shown in red and blue, respectively.
Color version available online
B/Niigata/11F374/2012 B/Ireland/12M17522/2012 B/Myanmar/12M444/2012 B/Latvia/3-39620p/2011 B/Tehran/5277/2010 Nov 75 B/Shanghai-Baoshan/193/2011 B/Qatar/2310012781/2010 Dec B/Lebanon/10L-39/2011 Jan T197L B/Lebanon/10L-20/2011 Jan 87 B/Lebanon/10L-25/2011 Jan A154T 98 B/Lebanon/10L-28/2011 Jan B/Lebanon/10L-32/2011 Jan R118K B/Kyoto/10K470/2011 Feb B/Lebanon/10L-11/2011 Jan B/Lebanon/10L-37/2011 Jan 89 B/Lebanon/10L-47/2011 Feb B/Lebanon/10L-43/2011 Jan B/BRUNEI/1/2010 Jan 98 B/Nagasaki/10N089/2011 Feb 89 B/Texas/08/2011 77
Brisbane/60
V146I N75K K109N N165K S172P
B/Brisbane/60/2008 (2009-2012)
L58P
B/Wisconsin/01/2010 (2012-2013)
R48K P108A S227G
es in a community in Australia. These resistant viruses had three additional NA mutations, V241I, N369K, and N386S, that were suggested to compensate for the destabilizing effect of the H275Y mutation and could, therefore, be permissive for fixing the H275Y mutation in the population. All of the 2011–2012 season H1N1p viruses 350
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B/Florida/4/2006 (2008-2009)
clade 2
0.01
B/Sichuan-Anyue/139/2011 B/Zahedan/7/2009 Feb S150I B/Turkey/93/2011 M249V N165Y B/Riyadh/03/2010 Oct S227D 94 B/Lebanon/L69/2009 Jan K88R D196N B/Bangladesh/3333/2007 B/Brisbane/3/2007 B/Myanmar/12M373/2012 89 T181A 99 B/Estonia/55763/11
clade 3
Fig. 5. Phylogenetic analysis of the hemagglutinin (HA1) genes of influenza B viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. The trees are rooted in the middle and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2008–2009 and 2010–2011 seasons are shown in red and green, respectively.
isolated in Lebanon, despite lacking the H275Y mutation, had these potentially permissive mutations, and 55% of the 2010–2011 isolates had 2 of these mutations (V241I and N369K). Collectively, these data suggest that H1N1p viruses have the capacity to accommodate the H275Y with potentially minimal burden on their fitness. Zaraket et al.
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100
B/Iraq/177/2009 May B/Qatar/2311002267/2011 Feb B/Myanmar/10M295/2010 88 B/Lebanon/10L-15/2011 Jan B/Lebanon/10L-16/2011 Jan B/Lebanon/L6/2008 Dec T232P, I264M, R276K B/Myanmar/11M416/2011 B/Hong Kong/3514/2012 B/Darwin/11/2011 B/Niigata/10F120/2011 Jan B/Odessa/3886/2010 B/Oman/3047/2010 Oct B/St. Petersburg/44/2012 B/Malaysia/2506/2004 90 B/Lebanon/L36/2009 Jan R69M B/Lebanon/L21/2009 Jan B/Mashad/6/2009 Feb A146T B/Colorado/04/2011
We also detected one H1N1p isolate with a 4-fold reduced oseltamivir IC50, due to an NA-S247N mutation, in a patient prior to prescription of oseltamivir. This mutation was reported in 10 and 30% of H1N1p viruses isolated in Australia and Singapore, respectively, during the same period [20]. The Lebanese S247N isolate was phylogenetically distinct from those isolated in Australia and Singapore. Of note, this mutation was also reported in H5N1 viruses isolated in Laos, reducing their susceptibility to oseltamivir by 24-fold [29]. These reports suggest that the S247N mutation could be naturally selected without antiviral drug pressure and is transmissible. While the IC50 values of S247N viruses are well below the biological drug concentrations of oseltamivir [20], the possibility that this mutation gets selected in viruses with the H275Y mutation is of concern. An isolate with dual NA-S247N and -H275Y mutations has been already reported and was shown to result in a 6,000-fold increase in oseltamivir IC50 [20], rendering treatment with oseltamivir ineffective. In conclusion, given the diversity of influenza viruses and the potential for rapid emergence of antiviral drugresistant strains, it is essential to closely monitor influenza viruses both by phenotypic and genotypic assays to
quickly identify such emerging mutations. Since the worldwide distribution of antiviral drug-resistant influenza strains varies, it is important to strengthen influenza surveillance at local and regional levels. Such information is critical for maintaining the effectiveness of antiviral drugs for both prophylaxis and treatment of influenza infections.
Acknowledgements We thank Dr. George Araj at the American University Medical Center (AUBMC) for his help with sample collection. The present study was funded by the US-Japan Acute Respiratory Infection Panel (The Ministry of Health, Labor, and Welfare, Japan), the US Department of State Biosecurity Engagement Program (Grant #BEP22033), the American Lebanese Syrian Associated Charities and JSPS Core-to-Core Program, B. Asia-Africa Science Platforms. Ethical approval was obtained from the American University of Beirut Institutional Review Board (PED.GD.02).
Disclosure Statement The authors have no conflicts of interest to disclose.
References
Post-Pandemic Influenza in Lebanon
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Received: April 3, 2014 Accepted after revision: July 5, 2014 Published online: October 3, 2014
Characterization of Human Influenza Viruses in Lebanon during 2010–2011 and 2011–2012 Post-Pandemic Seasons Hassan Zaraket a, c Clyde Dapat d Soha Ghanem b Zainab Ali b Mireille Lteif b Hiroki Kondo d Isolde C. Dapat d Kousuke Saito d Ghazi Kayali c Hiroshi Suzuki d, e Ghassan Dbaibo b Reiko Saito d a
Department of Experimental Pathology, Immunology and Microbiology, and b Division of Pediatric Infectious Diseases, Department of Pediatrics and Adolescent Medicine and the Center for Infectious Diseases Research, American University of Beirut, Beirut, Lebanon; c Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tenn., USA; d Division of International Health, Graduate School of Medical and Dental Sciences, Niigata University, and e School of Nursing, Niigata Seiryo University, Niigata, Japan
Abstract Objective: To genetically characterize human influenza viruses and their susceptibilities to antivirals during two post-pandemic seasons in Lebanon. Methods: Influenza virus was isolated from nasopharyngeal swabs that were obtained from patients with influenza-like illness during 2010–2012 and further analyzed both phenotypically and genotypically. Results: During the 2010–2011 season, both 2009 pandemic H1N1 (H1N1p) and B viruses co-circulated with equal prevalence, while the H3N2 virus predominated during the 2011–2012 season. All H3N2 and H1N1 viruses were resistant to amantadine. Importantly, all viruses of the influenza A and B types were susceptible to the neuraminidase (NA) inhibitors oseltamivir, zanamivir, peramivir, and laninamivir. Nonetheless, all 2011–2012 H1N1p isolates had three mutations (V241I, N369K, and N386S) in the NA gene that were suggested to be permissive of the H275Y mutation, which confers resistance to
© 2014 S. Karger AG, Basel 0300–5526/14/0576–0344$39.50/0 E-Mail [email protected] www.karger.com/int
oseltamivir. We also detected one H1N1p virus during the 2010–2011 season with a 4-fold decrease in susceptibility to oseltamivir due to an NA-S247N mutation. This isolate was phylogenetically distinct from other H1N1p viruses that were isolated in other regions. Conclusions: Influenza A viruses with reduced susceptibility to oseltamivir and mutations permissive for acquiring NA resistance-conferring mutation with minimal burden on their fitness were isolated in Lebanon. © 2014 S. Karger AG, Basel
Introduction
Annual influenza outbreaks affect on average 5–20% of the world’s population, resulting in a significant burden on human health [1]. These outbreaks are caused by seasonal influenza viruses that undergo continuous antigenic changes [2]. Occasionally, pandemics can occur due to antigenic shifts in the HA protein. Until the 2008– 2009 winter season, two influenza A subtypes, H3N2 and H1N1, and influenza B circulated in humans. However, in spring 2009, a novel influenza virus (pandemic H1N1 2009; H1N1p) emerged and quickly spread around the Hassan Zaraket Department of Experimental Pathology, Immunology and Microbiology American University of Beirut PO Box 113/6044/B21, Beirut (Lebanon) E-Mail hassan.zaraket @ aub.edu.lb
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Key Words Influenza virus · Pandemic H1N1 · Neuraminidase inhibitors · Antiviral drug susceptibility · Lebanon · Middle East
Flu B A/H1N1p A/H3N2 ILI
70 Number of cases
world [3]. In the following season, the seasonal H1N1 virus continued to circulate at a minimal level but eventually became extinct. However, a return and, in some cases, dominance of seasonal H3N2 and influenza B viruses was observed worldwide [4, 5]. In addition to their antigenic evolution, influenza viruses evolve under drug pressure or natural selection, and new virus populations that are resistant to treatment occasionally evolve and sometimes prevail if viral fitness is maintained [6, 7]. As a result, the use of antiviral drugs like amantadine, an M2-channel inhibitor, has been largely limited due to the surge of amantadine-resistant H3N2 influenza viruses [8]. Neuraminidase inhibitors (NAIs), including oseltamivir and zanamivir, became the drugs of choice for treatment of H3N2 influenza infections [9]. The utility of oseltamivir in treating influenza was jeopardized by the worldwide rapid emergence of oseltamivir-resistant seasonal H1N1 viruses during 2007 through 2009 [6, 7]. Nonetheless, this virus was soon replaced by the H1N1p virus, which is largely sensitive to oseltamivir but resistant to amantadine [10]. This has revived the utility of oseltamivir in treating influenza. Surveillance studies aimed at investigating the evolution and characteristics of influenza viruses worldwide are essential to early detection of new antigenically variant viruses as well as those with decreased antiviral drug susceptibility to allow informed recommendations for vaccination strategies and therapeutic interventions. Influenza surveillance efforts in the Middle East, including Lebanon, the site of this study, have been very limited. Characterization of influenza viruses in this region is critical to compliment the global effort to monitor and control influenza. The H1N1p influenza virus was first detected in Lebanon in May 2009. One study showed that a large outbreak caused by the pandemic virus occurred in October of the same year in Lebanon [11]. The prevalence of H1N1p viruses in the post-pandemic seasons and their susceptibilities to antiviral drugs is unknown in Lebanon. The goal of this study was to characterize influenza viruses that circulated in Lebanon during two post-pandemic influenza seasons.
60 50 40 30 20 10 0
A S ON D J F MAM J J A S ON D J F MA M J 2010 2011 2012
Fig. 1. Graphical representation of influenza cases in Lebanon during the 2010–2012 period. The dotted line represents all ILI cases that were identified during the study period. The letters on the xaxis represent months of the year.
Virus Isolation and Characterization 100 μl of the swab suspension was inoculated onto Madin-Darby canine kidney (MDCK) cells and incubated at 37° and 5% CO2. Influenza virus type and subtype were characterized using cyclingprobe real-time PCR [12–14]. RNA Extraction, PCR, and Sequencing Following RNA extraction and reverse transcription [15, 16], the HA (HA1 subunit) and neuraminidase (NA) genes were sequenced and aligned using BioEdit software. Phylogenetic trees were constructed using the Neighbor-Joining (MEGA program). Clusters were defined using WHO-designated reference strains [17]. Sequences generated in this study were deposited in the Japanese GenBank database (http://www.ddbj.nig.ac.jp/; accession No. KF000008-62). Antiviral Drug Susceptibility Testing Genotypic analysis of the single-nucleotide polymorphisms M2-S31N and NA-H275Y, which confer resistance to amantadine and oseltamivir, respectively, was performed using cycling probe method [12, 13]. In vitro antiviral drug susceptibilities of the isolated viruses were determined by measuring the 50% inhibitory concentrations (IC50) of oseltamivir (Sequoia Research, UK), zanamivir (Sequoia Research, UK), peramivir (Shionogi, Japan), and laninamivir (Daiichi Sankyo, Japan) using a fluorescencebased NA inhibition assay with methylumbelliferone N-acetylneuraminic acid (MUNANA) as the substrate at a final concentration of 0.025 mM [18].
Materials and Methods
Post-Pandemic Influenza in Lebanon
Results
A total of 202 samples were collected from patients with ILI during August 2010 through June 2012 (fig. 1). These included 73 samples from the 2010–2011 season and 129 samples from the 2011–2012 season. ILI activity Intervirology 2014;57:344–352 DOI: 10.1159/000365758
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Sample Collection Nasal swabs were collected from patients presenting with influenza-like illness (ILI) symptoms (i.e. fever of at least 37.5°, cough, rhinorrhea, myalgia, and headache) at a pediatric clinic in Beirut. One swab was immediately tested using a rapid influenza diagnostic test, Quick-Ex Flu kit (Denka Seiken, Japan), and a second swab was stored in virus transport media at –80° until further analysis.
2010 –2011 H1N1p
2011 –2012 H1N1p
1,000
1,000 n=3
n = 10 100
100
IC50 (nM)
S247N 1
1 1.23 ± 0.28
a
1.07 ± 0.13
0.63 ± 0.91
0.1
0.01
10
0.14 ± 0.02
0.10 ± 0.04 Peramivir
Oseltamivir
Zanamivir
Laninamivir
0.37 ± 0.13
0.1
0.31 ± 0.11
0.01
b
Peramivir
2011 –2012 H3N2
Oseltamivir
Zanamivir
1,000
100
100
10
10
1
c
n = 11
25.98 ± 3.94 29.90 ± 14.73
IC50 (nM)
IC50 (nM)
n = 25
0.01
Laninamivir
2010 –2011 influenza B
1,000
0.1
0.23 ± 0.01
1
0.24 ± 0.32
0.05 ± 0.10
0.39 ± 0.13
0.42 ± 0.12
Peramivir
Oseltamivir
Zanamivir
Laninamivir
11.51 ± 6.19 2.92 ± 0.90
0.1
d
0.01
Peramivir
Oseltamivir
Zanamivir
Laninamivir
Fig. 2. Box plots of IC50 values of NAIs against 2010–2011 H1N1p (a), 2011–2012 H1N1p (b), 2011–2012 H3N2 (c), and 2010–2011 influenza B (d) viruses collected in Lebanon. IC50 values of peramivir, oseltamivir, zanamivir, and laninamivir were determined using a fluorescence-based assay with MUNANA as a substrate.
The black bars indicate the 75th percentile + 3 × interquartile range (3 IQR). Values below the box plot represent mean ± SD. The mean value was calculated from viruses with IC50 values below the cutoff limit (75th percentile + 3 IQR). Samples with IC50 greater than the cutoff limit are considered outliers.
was highest during the months of January and February. In the 2010–2011 season, 12 (17%) samples tested positive for influenza A and 15 (21%) samples tested positive for influenza B using the rapid test kit. Among these, 10 H1N1p viruses and 11 influenza B viruses were isolated.
The influenza B cases were detected in the earlier part of the season, followed by H1N1p viruses. No H3N2 viruses were detected during the 2010–2011 season. In the 2011– 2012 season, 42 (33%) samples tested positive for influenza A by the rapid kit, and no influenza B viruses were
346
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IC50 (nM)
10
detected. Of these, 23 H3N2 and 3 H1N1p viruses were isolated. Cycling probe real-time PCR analysis revealed that all viruses from both subtypes had the M2-S31N mutation. None of the H1N1p viruses from either the 2010–2011 or 2011–2012 season had the NA-H275Y mutation. Using an in vitro fluorescence-based NA inhibition assay, all H1N1p viruses collected in this study were found susceptible to all the tested NAIs with IC50 values of 0.06–0.19 nM for peramivir, 0.6–5.11 nM for oseltamivir, 0.46–1.01 nM for zanamivir, and 0.19–0.57 nM for laninamivir (fig. 2a, b). One H1N1p isolate (A/Lebanon/10L-21/2011) had a 4-fold higher oseltamivir IC50 (IC50 = 5.11 nM) than the average IC50 (1.18 nM) for the rest of the H1N1p isolates from both seasons. No difference was observed in IC50values of the other three NAIs against the 10L-21 isolate. For the H3N2 viruses, IC50 values were 0.17–0.30 nM for peramivir, 0.94–1.2 nM for oseltamivir, 0.26–0.51 nM for zanamivir, and 0.23–0.73 nM for laninamivir (fig. 2c). Influenza B isolates had IC50 values that were higher than the influenza A viruses at 1.85–4.27 nM for peramivir, 19.58–32.05 nM for oseltamivir, 11.10–49.18 nM for zanamivir, and 3.41–21.82 nM for laninamivir (fig. 2d). In summary, the IC50 values suggest that all influenza A viruses isolated in the study are highly susceptible to NAIs. Influenza B isolates had up to 50-fold higher IC50 values than influenza A isolates. Phylogenetic analysis of the HA genes from H1N1p viruses revealed that the 2010–2011 season belonged to five distinct clusters: 1, 5, 6, 7 and 9 (fig. 3a). Cluster 1 included isolates from the 2009 pandemic outbreak with the exception of one isolate (A/Lebanon/10L-21/2011) from the 2010–2011 season, which had 2 additional mutations (S134T and R265K) in the HA gene. Two isolates (A/Lebanon/10L-34/2011 and A/Lebanon/10L-38/2011) from the 2010–2011 season formed a small cluster, designated cluster 9, characterized by 4 unique mutations (N44D, A267V, G270S, and I272L). None of these mutations belonged to any of the reported antigenic sites. In addition to the 2010–2011 season isolates, cluster 6 also accommodated the 2011–2012 isolates, which accumulated 3 additional HA mutations, including an H144R mutation in the Ca antigenic site and a V158I mutation adjacent to the Sa antigenic site [19]. Overall, the H1N1p viruses analyzed in this study had 6 amino acid mutations in the antigenic sites: 3 in Ca, 2 in Sb, and 1 in Sa, compared with A/California/07/2009. Three of these mutations were not detected in the earlier H1N1p viruses. To determine the genetic basis of the reduced susceptibility to oseltamivir of the 10L-21 (A/Lebanon/10L-21/
2011) isolate from the 2010–2011 season and its genetic relatedness to other isolates, we sequenced the NA genes of all H1N1p viruses isolated in this study. In general, the Lebanese H1N1p NA genes had a clustering pattern that corresponded with their counterpart HA clusters and had 5–7 mutations compared with the A/California/07/2009 vaccine strain (fig. 3). The 10L-21 isolate with reduced susceptibility to oseltamivir belonged to cluster 1 in the NA tree similar to its position in the HA tree. This isolate was characterized by 3 mutations: S247N, N385T, and N386K. The N385T and N386K mutations were located away from the enzyme-active site, suggesting no role in the reduced susceptibility to oseltamivir. The S247N mutation interacts with amino acid residues that in turn interact with oseltamivir and could interfere with stability of the inhibitor in the enzyme-active pocket [20]. The 10L-34 and 10L-38 strains formed a small isolated cluster in the NA tree just like in the HA tree and were characterized by 4 unique mutations: Q313E, T383R, I396T, and S442I. The HA genes of 8 Lebanese H3N2 influenza viruses collected during the 2011–2012 season were sequenced. All the sequenced viruses were A/Perth/16/2009-like and belonged to the Vic/208 lineage in the HA phylogeny (fig. 4). Two of the isolates belonged to clade 3A, and the rest belonged to clade 3B, which also contained strains from other Middle Eastern countries. These isolates accumulated 9–10 mutations, including 2 antigenic site A mutations (K144N/D and N145S). In case of influenza B, all of the 11 isolates collected during the 2010–2011 season were sequenced. All of these isolates were Victorialike and belonged to the Brisbane/60 clade (fig. 5). The Lebanese strains had 1–2 mutations compared with the B/Brisbane/60/2008 vaccine strain (2009–2012).
Post-Pandemic Influenza in Lebanon
Intervirology 2014;57:344–352 DOI: 10.1159/000365758
In this study, we found that in the first post-pandemic season (2010–2011), both influenza B and A/H1N1p cocirculated at a similar rate. In the 2011–2012 season, the A/H3N2 virus dominated and the H1N1p virus co-circulated with a lower prevalence. Similar circulation patterns have been reported elsewhere in the world [4, 21]. The extinction of seasonal H1N1 viruses has been suggested to be a consequence of the original antigenic sin, whereby infection with the H1N1p virus boosted the HA stalk antibodies against the seasonal H1N1 virus [22]. A similar phenomenon has been observed when H2N2 pandemic viruses, which emerged in 1957, replaced previously circulating H1N1 viruses. The H2N2 virus circulated for 11 347
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Discussion
A/Lebanon/11L043/2012 Jan A/Lebanon/11L008/2011 Dec A/Lebanon/11L045/2012 Jan A/Lebanon/10L-45/2011 Feb D103N A/St. Petersburg/27/2011 S191T S90I A/Lebanon/10L-40/2011 Jan (Sb) G161E (Sa) 98 A/Lebanon/10L-41/2011 Jan A/England/04860492/2010 Nov A/Singapore/GP4588/2010 Dec ¥ 98 A/Darwin/74/2011 Mar ¥ H144Q A/Perth/29/2011 Mar ¥ A/Astrakhan/1/2011 R211K (Ca) , A/Archangelsk/CRIE-SPA/2011 Jan I222V, V255L 95 A/Izmir/12/2011 Feb A/Lebanon/10L-13/2011 Jan N103D A/Ankara/03/2010 Dec A/Ghom/198/2010 Dec A/Lebanon/10L-30/2011 Jan A/Lebanon/10L-48/2011 Feb A192T A/Brazil/AVS11/2011 Jun (Sb) A/Ankara/02/2011 Jan A/Texas/JMS415/2010 Feb A/Lebanon/09L-04/2009 May N131D 4 A/Sydney/DD3-10/2010 Jul A/Christchurch/16/2010 S191T A/Matara/2132/2011 Feb (Sb) A/Brunei/1/2011 Jan ¥ A/Ghom/215/2010 Dec A/Karaj/5327/2010 Dec A/Tehran/5958/2010 Dec A/Athens/INS564/2011 Feb 7 S149G A/Karaj/5924/2010 Dec A203T 82 S209T A/Qatar/2311001414/2011 Jan 73 (Ca) A/Lebanon/10L-53/2011 Feb A/St. Petersburg/100/2011 A/Jeddah/3657/2010 Nov 89 A/Jeddah/3669/2010 Nov D228E (Ca) P303S A/Jeddah/3673/2010 Nov A/Lebanon/09L-30/2009 Nov 2 A/Bahrain/N11659/2009 Oct A140T A/Ancona/01/2010 Jan S189P A/New York/10/2009 A/Amman/WRAIR3448N/2010 Dec 3 A/Hong Kong/3934/2011 A/Ghana/FS-11-1803/2011 A/Boston/DOA08/2011 Jan 8 P89S A/Lebanon/09L-15/2009 Oct I327V A/Abu Dhabi/1134/2009 June A/Kuwait/4536/2009 Kuwait A/California/05/2009 A/Lebanon/09L-06/2009 Oct A/Dakar/WRAIR0019T/2010 Jan L38I A/Lebanon/09L-28/2009 Nov 93 A/Lebanon/10L-21/2011 Jan ¥ S134T, R265K N44D, A267V, A/Lebanon/10L-34/2011 Jan G270S, I272L 100 A/Lebanon/10L-38/2011 Jan
NA
H144R V158I, V255L 99
A/California/07/2009 (2009-2013)
6
A/Lebanon/11L045/2012 Jan N386S D451G 97 A/Lebanon/11L043/2012 Jan A/Lebanon/11L008/2011 Dec A/Lebanon/10L-40/2011 Jan V453M A/Lebanon/10L-45/2011 Feb A/England/04860492/2010 Nov N44S A/Lebanon/10L-53/2011 Feb A/St. Petersburg/100/2011 A/Athens/INS564/2011 Feb A/Ghom/215/2010 Dec A/Karaj/5327/2010 Dec 85 A/Tehran/5958/2010 Dec A/Karaj/5924/2010 Dec V81A A/Ghom/198/2010 Dec A/Archangelsk/CRIE-SPA/2011 Jan V241I 88 A/Lebanon/10L-13/2011 Jan N369K N141D A/Brazil/AVS11/2011 06/4 D416E A/Lebanon/10L-48/2011 Feb A/Lebanon/10L-30/2011 Jan L40I, I46T, S95I, S247N A/Perth/29/2011 Mar ¥ A/Brunei/1/2011 Jan ¥ S247N S299A 96 A/Singapore/GP4588/2010 Dec ¥ 85 A/Darwin/74/2011 Mar ¥ A/Sydney/DD3-10/2010 Jul 4 A/Amman/WRAIR3448N/2010 Dec 3 V106I Q313E, T383R A/Lebanon/10L-34/2011 Jan I396T, S442I 99 A/Lebanon/10L-38/2011 Jan 9 A/New York/10/2009 A/Jeddah/3673/2010 Nov A/Lebanon/09L-15/2009 Oct A/Jeddah/3669/2010 Nov N248D A/Lebanon/09L-30/2009 Nov 2 A/Lebanon/09L-06/2009 Oct 81 A/Ancona/01/2010 Jan A/Texas/JMS415/2010 Feb A/Jeddah/3657/2010 Nov Y381F A/Lebanon/09L-04/2009 May A/Boston/DOA08/2011 Jan V106I S247N A/Dakar/WRAIR0019T/2010 Jan 84 N385T, N386K A/Lebanon/10L-21/2011 Jan ¥ 1 A/Lebanon/09L-28/2009 Nov A/California/05/2009 N44S, V53I, V62I, N386S
5
1
6
Color version available online
HA
7
5
A/California/07/2009 (2009-2013) 0.001
9
Fig. 3. Phylogenetic analysis of the hemagglutinin (HA1) and NA genes of influenza A/H1N1 pandemic 2009 viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. A/California/07/09 was used as the root for the tree and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated on the branches. The HA protein antigenic site
to which a mutation belongs is in parentheses. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2009 (previously reported by Zaraket et al. [11]), 2010–2011, and 2011–2012 seasons are shown in red, green, and blue, respectively. ¥ Viruses with the S247N mutation. In the NA tree, only reference samples for which the NA gene sequence was available in the GenBank database are included.
years until it was replaced by H3N2 virus in 1968 due to antibodies specific to the retained N2 subtype [23]. The H1N1p viruses continued to evolve, accumulating mutations in antigenic sites, but remained antigenically similar to the A/California/07/2009 vaccine strain.
In the post-pandemic 2010–2011 season, H1N1p viruses collected in Lebanon were diverse, belonging to multiple clusters, suggesting multiple introductions of this virus into Lebanon. This was in contrast to mostly homologous H1N1p viruses that we detected in previous
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0.001
A198S N145S (A)
V223I
S45N T48I 92 A/Victoria/361/2011
(2012-2013)
A/Myanmar/M019/2011
S312Q A/Hong Kong/3969/2011 N144D (A) A/Stockholm/18/2011 N145S (A) 99 A/Lebanon/11L007/2011 Dec
3A
A/Virginia/03/2010 A/Kuwait/4554/2009 Dec A/Hong Kong/1736/2010 A/Serbia/71/2011 A/Hiroshima-C/27/2010 A/Myanmar/M049/2009 Q312 A/Iraq/7-FSS/2011 Jan A/Alabama/04/2011 A/Victoria/208/2009 A/Vietnam/BN420/2010 T212A D53N (C) A/Tehran/181/2012 Jan 92 Y94H I230N 93 A/Tehran/8331/2011 Nov A/Esfahan/8343/2011 Nov E280A A/Iowa/19/2010 A/Myanmar/M417/2010 A/Afghanistan/2950/2009 Oct A/Thailand/CU-B1697/2009 A/Norway/1330/2010 T212A A/Tianjin-Nankai/1687/2010 K260M 77 A/Taiwan/42/2010 I261Q 97 A/Iran/755/2009 June
Vic/208
A/Lebanon/11L018/2012 Jan A/Lebanon/11L033/2012 Feb K173 85 A/Lebanon/11L040/2012 Feb A/Bahrain/40/2011 Dec A/Lebanon/11L046/2012 Feb 3B A/Lebanon/11L013/2011 Dec A/Lebanon/11L023/2012 Jan A/England/259/2011 A/Hormozgan/7981/2011 Nov A/Jordan/20110030780/2011 Dec A/Lebanon/11L002/2011 Nov A/Oman/916/2011 July A/Alborz/96/2012 Jan A/Khorasan8032//2011 Nov A/Niigata/11F526/2012 Mar A/Ghom/9677/2011 Dec N278K A/Hong Kong/5047/2011 3C A/Gunma/11G001/2011 Dec
Color version available online
G5E, E62I A198P, N145S (A)
Q312S
K158N N189K E62K N144K (A)
82
A/Lebanon/L34/2009 Jan A/Lebanon/L47/2009 Jan A/Lebanon/L82/2009 Feb A/Tehran/93/2008 Nov A/Kuwait/WRAIR1561P/2009 Jan A/Wisconsin/67/2005 I179V
A/Brisbane/10/2007 (2008-2010)
Perth/16
A/Victoria/210/2009 (2011-2012) A/Perth/16/2009 (2010-2011)
G50E K173Q
Brisbane/10
0.002
work during the pandemic [11]. In the case of H3N2, all viruses from the 2011–2012 season sequenced in this study belonged to the A/Victoria/208/2011-like lineage, and most of them fell into clade 3B. The Lebanese isolates closely resembled WHO-designated reference strains A/Stockholm/18/2011 and A/England/259/2011 which exhibited 64- and 32-fold reduction, respectively, in their HA inhibition (HAI) titers of sera raised against the A/Victoria/210/2009 vaccine strain [17]. These viruses accumulated several mutations, including 3–4 in the antigenic sites compared with the vaccine strain
which explains the antigenic drift marked reduction in their HAI titers. The prevalence of oseltamivir-resistant H1N1p viruses remained low worldwide in the post-pandemic period, albeit at a slight increase from their level during the pandemic period [24–26]. Nonetheless, the rate of oseltamivir resistance is much higher among high-risk groups such as immunocompromised patients, and clusters of high community transmission rates have been reported [25, 27]. Hurt et al. [28] reported a significant increase in the frequency H275Y oseltamivir-resistant H1N1p virus-
Post-Pandemic Influenza in Lebanon
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Fig. 4. Phylogenetic analysis of the hemagglutinin (HA1) genes of influenza A/H3N2 viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. A/Brisbane/10/2007 was used as the root for the tree and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated on the branches. The HA antigenic site to which a mutation belongs is in parentheses. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2008–2009 and 2011–2012 seasons are shown in red and blue, respectively.
Color version available online
B/Niigata/11F374/2012 B/Ireland/12M17522/2012 B/Myanmar/12M444/2012 B/Latvia/3-39620p/2011 B/Tehran/5277/2010 Nov 75 B/Shanghai-Baoshan/193/2011 B/Qatar/2310012781/2010 Dec B/Lebanon/10L-39/2011 Jan T197L B/Lebanon/10L-20/2011 Jan 87 B/Lebanon/10L-25/2011 Jan A154T 98 B/Lebanon/10L-28/2011 Jan B/Lebanon/10L-32/2011 Jan R118K B/Kyoto/10K470/2011 Feb B/Lebanon/10L-11/2011 Jan B/Lebanon/10L-37/2011 Jan 89 B/Lebanon/10L-47/2011 Feb B/Lebanon/10L-43/2011 Jan B/BRUNEI/1/2010 Jan 98 B/Nagasaki/10N089/2011 Feb 89 B/Texas/08/2011 77
Brisbane/60
V146I N75K K109N N165K S172P
B/Brisbane/60/2008 (2009-2012)
L58P
B/Wisconsin/01/2010 (2012-2013)
R48K P108A S227G
es in a community in Australia. These resistant viruses had three additional NA mutations, V241I, N369K, and N386S, that were suggested to compensate for the destabilizing effect of the H275Y mutation and could, therefore, be permissive for fixing the H275Y mutation in the population. All of the 2011–2012 season H1N1p viruses 350
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B/Florida/4/2006 (2008-2009)
clade 2
0.01
B/Sichuan-Anyue/139/2011 B/Zahedan/7/2009 Feb S150I B/Turkey/93/2011 M249V N165Y B/Riyadh/03/2010 Oct S227D 94 B/Lebanon/L69/2009 Jan K88R D196N B/Bangladesh/3333/2007 B/Brisbane/3/2007 B/Myanmar/12M373/2012 89 T181A 99 B/Estonia/55763/11
clade 3
Fig. 5. Phylogenetic analysis of the hemagglutinin (HA1) genes of influenza B viruses isolated in Lebanon. The trees were constructed based on the nucleotide sequences using the Neighbor-Joining method with bootstrap analysis of 1,000 replicates. The trees are rooted in the middle and bootstrap values greater than 70% are shown. Amino acid mutations characteristic of the main clusters or the Lebanese isolates are indicated. Vaccine strains are in bold italics and reference strains are in bold. Lebanese isolates from 2008–2009 and 2010–2011 seasons are shown in red and green, respectively.
isolated in Lebanon, despite lacking the H275Y mutation, had these potentially permissive mutations, and 55% of the 2010–2011 isolates had 2 of these mutations (V241I and N369K). Collectively, these data suggest that H1N1p viruses have the capacity to accommodate the H275Y with potentially minimal burden on their fitness. Zaraket et al.
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100
B/Iraq/177/2009 May B/Qatar/2311002267/2011 Feb B/Myanmar/10M295/2010 88 B/Lebanon/10L-15/2011 Jan B/Lebanon/10L-16/2011 Jan B/Lebanon/L6/2008 Dec T232P, I264M, R276K B/Myanmar/11M416/2011 B/Hong Kong/3514/2012 B/Darwin/11/2011 B/Niigata/10F120/2011 Jan B/Odessa/3886/2010 B/Oman/3047/2010 Oct B/St. Petersburg/44/2012 B/Malaysia/2506/2004 90 B/Lebanon/L36/2009 Jan R69M B/Lebanon/L21/2009 Jan B/Mashad/6/2009 Feb A146T B/Colorado/04/2011
We also detected one H1N1p isolate with a 4-fold reduced oseltamivir IC50, due to an NA-S247N mutation, in a patient prior to prescription of oseltamivir. This mutation was reported in 10 and 30% of H1N1p viruses isolated in Australia and Singapore, respectively, during the same period [20]. The Lebanese S247N isolate was phylogenetically distinct from those isolated in Australia and Singapore. Of note, this mutation was also reported in H5N1 viruses isolated in Laos, reducing their susceptibility to oseltamivir by 24-fold [29]. These reports suggest that the S247N mutation could be naturally selected without antiviral drug pressure and is transmissible. While the IC50 values of S247N viruses are well below the biological drug concentrations of oseltamivir [20], the possibility that this mutation gets selected in viruses with the H275Y mutation is of concern. An isolate with dual NA-S247N and -H275Y mutations has been already reported and was shown to result in a 6,000-fold increase in oseltamivir IC50 [20], rendering treatment with oseltamivir ineffective. In conclusion, given the diversity of influenza viruses and the potential for rapid emergence of antiviral drugresistant strains, it is essential to closely monitor influenza viruses both by phenotypic and genotypic assays to
quickly identify such emerging mutations. Since the worldwide distribution of antiviral drug-resistant influenza strains varies, it is important to strengthen influenza surveillance at local and regional levels. Such information is critical for maintaining the effectiveness of antiviral drugs for both prophylaxis and treatment of influenza infections.
Acknowledgements We thank Dr. George Araj at the American University Medical Center (AUBMC) for his help with sample collection. The present study was funded by the US-Japan Acute Respiratory Infection Panel (The Ministry of Health, Labor, and Welfare, Japan), the US Department of State Biosecurity Engagement Program (Grant #BEP22033), the American Lebanese Syrian Associated Charities and JSPS Core-to-Core Program, B. Asia-Africa Science Platforms. Ethical approval was obtained from the American University of Beirut Institutional Review Board (PED.GD.02).
Disclosure Statement The authors have no conflicts of interest to disclose.
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