Journal of Medical Virology 87:1268–1275 (2015)
Genetic Changes in Influenza A(H3N2) Viruses Circulating During 2011 to 2013 in Northern India (Lucknow) Amita Jain,1* Tanushree Dangi,1 Bhawana Jain,1 Ajay Kumar Singh,1 J. V. Singh,2 and Rashmi Kumar3 1
Department of Microbiology, King George’s Medical University, Lucknow, India Department of Community, Medicine King George’s Medical University, Lucknow, India 3 Department of Paediatrics, King George’s Medical University, Lucknow, India 2
Genetic variability in the hemagglutinin (HA1) and the neuraminidase (NA) genes of influenza viruses results in the emergence of new strains which differ in pathogenicity and severity. The present study was undertaken for genotypic characterization of the HA1 and NA genes of the influenza A(H3N2) strains, detected during the 2011–2013. A total of fifty five influenza A(H3N2) positive samples [2011 (n ¼ 20), 2012 (n ¼ 4) and 2013 (n ¼ 31)] were studied. The 824 bp segment of HA1 gene and 931 bp segment of NA gene were amplified and sequenced by Big-Dye terminator kit on ABI3130, Genetic analyzer. Molecular and phylogenetic analysis was done by MEGA 5.05 software and PhyML program (v3.0). Mutations were determined by comparing the deduced amino acid sequences of study strains with that of 2009–2013 vaccine strains. The studied influenza A(H3N2) strains showed 98.1–99.6% similarity in HA1 and NA amino acid sequences with the influenza A/Victoria/ 361/2011 vaccine strain. Four mutations in the HA1 amino acid sequences (T128A, R142G, L157S and N278K) and three unique mutations in the NA amino acid sequences [D251V, S315G and V313A] were found. These mutations were observed only in strains from the year 2013 (cluster II). None of the strains showed the presence of mutations, N294S and R292K, markers of oseltamivir resistance. In conclusion, Lucknow strains have accumulated the significant number of mutations in the antigenic sites of the HA and the NA coding sequences and continue to be evolving from the 2013 vaccine strain [A/Victoria/361/ 2011], however, mutations specific for oseltamivir resistance were not detected. J. Med. Virol. 87:1268–1275, 2015. # 2015 Wiley Periodicals, Inc. C 2015 WILEY PERIODICALS, INC.
KEY WORDS:
influenza A(H3N2) virus; epidemic; HA1 gene; NA gene; phylogenetic; mutations
INTRODUCTION Influenza virus is the major leading cause of respiratory tract infections in humans. It has caused severe pandemics and epidemics during the last century. Few subtypes of influenza A virus were responsible for severe infections in the human population, i.e., H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, H5N1 in 1997, PdmH1N1 in 2009 and H7N9 in 2013 [Falchi et al., 2011; Taubenberger and Morens, 2013]. Though, the mortality associated with these pandemics remain quite high, seasonal epidemics also have a major role in significant morbidity and mortality, affecting approximately 5–15% population globally each year [Stohr, 2002; WHO, 2003]. Genetic and antigenic variations in the hemagglutinin (HA) and the neuraminidase (NA) surface glycoproteins of the influenza A virus mainly attribute to seasonal epidemics. Till date, 16 variants of the HA gene and 9 variants of the NA gene are known to exist.
Conflict of Interest: None Grant sponsor: Influenza surveillance network; Grant sponsor: Indian Council of Medical Research; Grant number: 5/8/7/14/ 2009-ECD-1(Vol.II). Correspondence to: Prof. Amita Jain, MD, Department of Microbiology, King George Medical University, Lucknow-226003, India. E-mail: [email protected] Accepted 15 October 2014 DOI 10.1002/jmv.24096 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Genetic Changes in Influenza A(H3N2) Viruses Circulating
Different combinations of the HA and the NA genes determine the influenza A virus subtypes. At present, three subtypes of influenza A virus (H1N1, H3N2 and pandemic H1N1) are circulating, which, though well adapted in the human populations, can cause local outbreaks of varying severity [Lin et al., 2004; Falchi et al., 2010]. The variability among subtypes of the influenza A virus is so high that the HA and NA genes from two different subgroups have only 40% and 62–68% amino acid similarities, respectively [Latorre-Margalef et al., 2013]. The HA protein is a major target of host neutralizing antibodies. The HA protein helps the influenza virus to penetrate into the host cells by binding with the sialic acid receptors on host’s respiratory epithelial cells. The HA1 domain of the HA protein mutates more frequently than the HA2 domain [Plotkin and Dushoff, 2003]. Various studies have also reported the importance of the HA1 gene in prediction of genetic drift [Lee and Chen, 2004; Smith et al., 2004]. Genetic variations in the antigenic sites of the HA gene coded proteins may change the behaviour of influenza virus strains by allowing the virus to escape host’s immunity. Enzymatic activity of the NA gene encoded proteins play important role in dissemination of the newly formed virus particles. Neutralizing antibodies are generated against the NA protein during infections. Frequent genetic changes in the NA protein facilitates the influenza virus to escape host immune system which may enhance the virus titre and infectivity [Kilbourne et al., 1968]. High genetic variability in the HA and NA genes leads to the emergence of new antigenic variants (called antigenic drift). Therefore, frequent updating of the composition of seasonal vaccine by the World Health Organization (WHO), on the basis of the HA and NA genes is required [Willy et al., 1981]. The present study analyzed the nucleotide and deduced amino acid sequences of the HA1 and the NA genes of influenza A(H3N2) viruses circulating in Lucknow, Uttar Pradesh (India) during 2011–2013 to determine the genetic variations occurring in circulating strains, to understand their evolution at molecular level. MATERIALS AND METHODS Nasal and Throat swabs were collected from the patients presenting as influenza-like illness (ILI) during January 2011–December 2013, at Viral Diagnostic Laboratory, King George’s Medical University, Lucknow (India). The samples were tested for the presence of influenza A(H3N2) virus RNA by realtime reverse-transcription polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR. Clinical specimens having high viral load [Ct < 25 in real-time PCR] and showing single sharp band in PCR amplification of the HA and NA genes, were selected for sequencing experiments.
1269
Sample Processing and RNA Extraction Nasal and throat swabs were collected in 3.5 ml viral transport media (VTM) and centrifuged at 1000 rpm for 5 min. The supernatant was transferred to nuclease free collection tubes. Viral RNA was extracted from the 200 ml aliquots of the supernatant 1 by using the Invitrogen PureLink Viral RNA/DNA Extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Detection by Real Time RT-PCR Real-time RT-PCR was performed from the freshly extracted RNA for confirmation of Influenza A, and subtype H3N2 according to the Centers for Disease Control and Prevention (CDC) protocol [Mishra et al., 2009; World Health Organization, 2009]. Briefly, a 25 ml PCR reaction mixture was prepared in eppendorf containing 12.5 ml of 2xABI RT-PCR buffer with 1 ml of 25 RT-enzyme (AgPath-IDTM One Step RTPCR Kit, Applied Biosystem, Foster city, California, USA), 0.5 ml forward primer (40mM), 0.5 ml reverse primer (40mM), 0.2 ml probe (10mM) and 5 ml of nuclease free water. The PCR was carried out on ABI 7500 thermal cycler (Applied Biosystems, USA) with following reaction conditions: reverse-transcription step at 50 ˚C for 30 min and 95 ˚C for 2 min, followed by 45 cycles of PCR amplification at 95 ˚C for 15 sec and 55 ˚C for 30 sec. Positive control, negative control and extraction control were run simultaneously with each RT-PCR run for validity of the experiment. RNaseP gene segments were amplified as internal control for human nucleic acid gene to test the RNA extraction procedure. Amplification of the HA and NA Genes by Conventional PCR One strand complementary DNA (c-DNA) was synthesized in two separate eppendorfs using the HA and the NA gene-specific primers by the method as described previously [Dangi et al., 2014]. Two step conventional reverse transcription PCR (RT-PCR) was done to amplify viral c-DNA specific for 824 bp fragment (nucleotide position 360–1184) in the HA1 domain and 931 bp fragments (nucleotide position 367– 1298) in the NA gene [Hoffmann et al., 2001; Chutinimitkul et al., 2008]. Briefly, 2 ml of c-DNA was added to the reaction mixture containing 2.5 ml Taq Buffer (10X), 1 ml Taq enzyme (2U/ml; DyNAzymeTM, Fisher Scientific, Bishop Meadow Road, Loughborough, United Kingdom), 0.5 ml dNTP (100mM; KomaBiotech, 19F, IS BIZ Tower, Seoul, Korea), 0.5 ml forward and reverse primers (10mM) and nuclease-free water to the final volume of 25 ml. Amplification was performed in an Applied Biosystems Veriti 96-well thermo cycler 7500 under the following conditions: Initial denaturation at 94 ˚C for 10 min, followed by 35 amplification cycles, comprising of denaturation for 30 sec, primer annealing at 55 ˚C [HA1 gene product]/52 ˚C [N2 gene J. Med. Virol. DOI 10.1002/jmv
1270
Jain et al.
product] for 45 sec followed by extension at 72 ˚C for 1 min, to be ended by a final elongation step of 10 min at 72 ˚C. The amplified products were run on 1.2% Agarose gel and visualized on a UV trans-illuminator after staining with ethidium bromide solution. Sequencing The PCR products seen as a single sharp band on conventional PCR were used directly for further purification process by Exo-Sap treatment to remove excess primers and un-incorporated dNTPs (Exonuclease I,1U/ml and Shrimp Alkaline Phosphatase, 20U/ml) (FastApTM, Fermentas, Fisher Scientific, Pittsburgh, PA) [Potdar et al., 2010]. Purified products were sequenced in both the directions (30 and 50 prime) by using forward and reverse primers in two separate wells for both the HA1 and NA gene segments. Sequencing was carried out using Big-Dye Terminator 3.1 Cycle-Sequencing Kit (Applied Biosystems, Foster City, USA) on ABI Prism 3,130 genetic analyzer (Applied Biosystems, Foster City, USA). Nucleotide sequences obtained in this study were submitted in GenBank database. Data Analysis The generated nucleotide sequences were edited manually and aligned by ClustalW version 1.83 [Thompson et al., 1994]. To determine the genetic variability in the circulating strains, their nucleotide sequences were compared to the vaccine strains of the years 2009 to 2013 (Influenza A/Victoria/361/2011, A/ Perth/16/2009 and A/Brisbane/19/2007 strains) recommended by World Health Organization (WHO). The vaccine strain A/Victoria/361/2011 was used during 2012–2013 in the Northern Hemisphere and during 2013 in the Southern Hemisphere. Influenza A/Perth/ 16/2009 strain was used as vaccine strain during 2010, 2011, and 2012 in both Northern and Southern Hemisphere, and A/Brisbane/19/2007 vaccine strain was used during 2009 in both the Northern and Southern Hemisphere. The HA1 and the NA gene sequences of the vaccine strains were retrieved from the Influenza Research Database (www.fludb.org) available on National Center for Biotechnology Information (www. ncbi.nlm.nih.gov). Phylogenetic tree was constructed based on the HA1 and the NA amino acid sequences using the maximum likelihood (ML) approach in the PhyML program (version 3.0) [Guindon and Gascuel, 2003] at 500 bootstrap replicates. This analysis utilized the HKY85 þ I þ G4 nucleotide substitution model to determine the evolutionary divergence analysis between the HA1 and the NA gene sequences. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Amino acid substitutions within the HA1 and partial NA amino acid sequences were determined by doing multiple sequence alignment of the fifty five strains with respect to the influenza A(H3N2) J. Med. Virol. DOI 10.1002/jmv
vaccine strains by ClustalW in Molecular Evolutionary Genetics Analysis version 5.05 (MEGA 5.05) software [Tamura et al., 2011]. RESULTS Of 4,227 clinical samples collected from ILI patients, a total of 140 samples tested positive for influenza A(H3N2) virus by real-time RT-PCR. Only 55/140 clinical samples were sequenced for the HA and the NA genes. These samples were from year 2011(20/59), 2012 (4/7), and 2013 (31/74). Molecular Analysis of HA1 Gene Nucleotide sequence analysis (HA1 gene) of the studied strains showed that all the strains were more closely related to the 2013 vaccine strain (A/Victoria/ 361/2011) than the vaccine strains of the previous years. Strains isolated during the years 2011 and 2012 showed 98.1%, 99%, and 99.6% similarity with vaccine strains of years, 2008–2009 (A/Brisbane/10/ 2007), 2010–2012 (A/Perth/16/2009), and 2013 (A/ Victoria/361/2011), respectively. Strains circulating during the year 2013 showed 96.6%, 97.0% and 98.5% similarity with A/Brisbane/10/2007, A/Perth/ 16/2009 and A/Victoria/361/2011 strains, respectively. Phylogenetic analysis of the Lucknow strains, based on the HA gene revealed that these strains drifted from the vaccine strains recommended for the years 2009–2012. The Lucknow strains matched more closely with the influenza A/Victoria/361/2011 the 2013 vaccine strain (Fig. 1). The presence of differences in gene sequences divided Lucknow strains into two major clusters, cluster I and cluster II. Influenza A(H3N2) strains circulating during 2011–2012 grouped into the cluster I, while all 2013 strains fell into the cluster II. Cluster II further divided into two subgroups; IIA and IIB. Amino Acid Variations in HA1 Domain Comparison of deduced amino acid sequences of the HA1 gene of Lucknow strains with respective sequences of vaccine strains showed twelve mutations at different positions (Table I). N145S mutation located in the A epitope of globular HA1 domain was found in all the studied strains. Other common mutations present in all Lucknow strains as well as in 2013 vaccine strain were A198S, T212A, V223I, and N312S. Five mutations found in isolates from year 2013 were T128A, R142G, L157S, S146G, and N278K. Less frequent mutations are detailed in Table I. Molecular Analysis of NA Gene Phylogenetic tree of the NA gene sequences showed a similar evolutionary pattern of the influenza A(H3N2) strains as observed in the phylogeny of the HA1 gene. All studied strains grouped into two distinct clusters [cluster I and cluster II] due to accumulation of the some unique substitutions in the
Genetic Changes in Influenza A(H3N2) Viruses Circulating
1271
Fig. 1. Phylogenetic tree of Influenza A(H3N2) strains and representative vaccine strains based on HA1 gene using Maximum Likelihood Method. Amino acid substitutions in HA1 domain that characterized a particular branch are indicated at branch node. Numbers at the nodes indicate confidence levels of bootstrap analysis with 500 replicates.
NA gene sequences and cluster II further separated into two subgroups [IIA and IIB] as shown in Figure 2. Strains from years 2011–2012 showed 98.5% similarity with vaccine strains used during that duration while strains from year 2013 showed only 96.2% similarity to the vaccine strain used during that year.
mivir [N294S and R292K] was not observed in the studied strains. N402D mutation was observed in all the strains from the year 2013 and some of the strains from the years 2012 (n ¼ 10) and 2011 (n ¼ 1). Other less frequent substitutions are shown in Table I. Accession Numbers
Amino Acid Variation in NA Domain Partial amino acid sequences of the NA gene was analyzed in context to the amino acid sequences of representative vaccine strains (Table I). Twelve amino acid substitutions were identified in the coding sequences of the NA gene of studied strains. All strains from the years 2011–2013 showed two major substitutions S367N and K369T corresponding to the antigenic site. Two substitutions [D251V (n ¼ 31) and S315G (n ¼ 21) were seen only in the strains from the year 2013. Some of the Lucknow strains from the years 2011(n ¼ 5) and 2012 (n ¼ 1) showed I397M mutation which was not detected in strains from the year 2013. Drug resistance marker specific to oselta-
The nucleotide sequences derived in the study were deposited in the GenBank database under accession numbers KF142486, KF142487, KF142488; F668185– KF668207; KJ667978–KJ668007 for H3 gene and KF668208–KF668232; KJ668008–KJ668037 for N2 gene (Table I). DISCUSSION Worldwide, Influenza A virus causes seasonal epidemics. Annual influenza immunization is recommended to prevent influenza virus infection due to rapid mutation rate in influenza viral strains. Therefore, every year vaccine is updated according to the circulating strains for the best fit. In this study, J. Med. Virol. DOI 10.1002/jmv
A/Victoria/361/2011 A/Perth/16/2009 A/Brisbane/10/2007 LKO799 LKO805 LKO810 LKO819 LKO827 LKO845 LKO851 LKO856 LKO864 LKO871 LKO875 LKO883 LKO892 LKO896 LKO911 LKO934 LKO940 LKO968 LKO974 LKO983 LKO1442 LKO1501 LKO3108 LKO3154 LKO3475 LKO3491 LKO3714 LKO3738 LKO3812 LKO3815 LKO3818 LKO3821 LKO3824 LKO3825 LKO3828 LKO3839 LKO3863 LKO3865 LKO3876 LKO3887 LKO3889
Strains
2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
KC306165 CY081428 CY116577 KF668185 KJ667978 KJ667979 KJ667980 KF668186 KF668187 KF668188 KF668189 KF142486 KF66890 KJ667981 KJ667982 KJ667983 KF66891 KJ667984 KJ667985 KF142487 KJ667986 KJ667987 KF66892 KF66893 KF66894 KF66895 KJ667988 KJ667989 KJ667990 KJ667991 KJ667992 KJ667993 KJ667994 KF66896 KJ667995 KF66897 KF66898 KJ667996 KF66899 KF66900 KJ667997 KF668201 KF668202 KF668203
T . . . . . . . . . . . . . . . . . . . . . . . . . A A A A A A A A A A A A A A
Accession Year of No of HA1 isolation gene segment 128 R . . . . . . . . . . . . . . . . . . . . . . . . . . G G G G G G G G G G G G G G G G
142 N . . S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S
145 S . . . . . . . . . . . . . . . . . . . . . . . . . . G G . . . . . . . . . . . . G . .
146 L . . . . . . . . . . . . . . . . . . . . . . . . . . S S S S S . . S S S S . . S . S
157 S A A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
198 A T T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212 I V V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
223 R . . . Q . . . . . . . . . . Q Q . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
Amino acid variations in the HA1 amino acid sequences
N . . . . . . . . . . . . . . . . . . . . . . . . . . K K K K K K K K K K K K K K K K K
J. Med. Virol. DOI 10.1002/jmv . . . . . . . . . .
I . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .
S N N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CY121079 CY081429 EU199420 KF668208 KJ668008 KJ668009 KJ668010 KF668209 KF668210 KF668211 KF668212 KF668213 KF668214 KJ668011 KJ668012 KJ668013 KF668215 KJ668014 KJ668015 KF668216 KJ668016 KJ668017 KF668217 KF668218 KF668219 KF668220 KJ668018 KJ668019 KJ668020 KJ668021 KJ668022 KJ668023 KJ668024 KF668221 KJ668025 KF668222 KF668223 KJ668026 KF668224 KF668225 KJ668027 KF668226 KF668227 KF668228
K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . .
D . . . . . . . . . . . . . . . . . . . . . . . . . . V V V V V V V V V V V V V V V V V
Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . .
V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A A . . . . . . A A . . .
S . . . . . . . . . . . . . . . . . . . . . . . . . . G G G G . . G G G G G G . . G G G
D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
M . . . . . . . . . . . . . . . . . . . . . . . . . . . . I I . . . . . . I . . . . . .
S . . N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
K . . T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T
P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S
I . . M . M M . . . . M . . . M M . . . . . . . . . . . . . . . . .
N . . D D D D D D D D D D D D D D D D D D D D D D D D D D D D
Accession No of NA 278 282 312 gene segment 199 251 284 313 315 339 362 367 369 386 397 402
Amino acid variations in the NA amino acid sequences
TABLE I. Changes in Amino Acid Sequences Encoded by HA1Gene and Partial NA Gene (Antigenic Regions) of Influenza A(H3N2) With Respect to Vaccine Strains
1272 Jain et al.
D D D D D D D D D D D D D D . . . . . . . . . . . . . . . . . . . . . . . . . . . . T T T T T T T T T T T T T T N N N N N N N N N N N N N N . . . . . . . . . I I . . . . N . N . . . . . . . . . . G G . G G G G . . G G . . . . . A . . . . A A . . A A A . . . . . . . . . . . . . H V V V V V V V V V V V V V V . . . . . . . . . . . . . . KJ668028 KF668229 KJ668029 KF668230 KF668231 KF668232 KJ668030 KJ668031 KJ668032 KJ668033 KJ668034 KJ668035 KJ668036 KJ668037 . . . . . . . . .
. . . . . . . . . . . . . . . . . . . K K K K K K K K K K K K K K
Amino acids variations at various positions similar to A/Victoria/361/2011 were denoted by dot (.).
261
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
223 212
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
198 157
S . . S . . . . S . . . . . G . . . . . . . G . . . . .
146 145
S S S S S S S S S S S S S S G G G G G G G G G G G G G G A A A A A A A A A A A A KJ667998 KF668204 KJ667999 KF668205 KF668206 KF668207 KJ668000 KJ668001 KJ668002 KJ668003 KJ668004 KJ668005 KJ668006 KJ668007 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
142 Strains
LKO3890 LKO3893 LKO3905 LKO3906 LKO3913 LKO3914 LKO3918 LKO3925 LKO3938 LKO3960 LKO3969 LKO4012 LKO4129 LKO4167
Accession No of NA 278 282 312 gene segment 199 251 284 313 315 339 362 367 369 386 397 402 Accession Year of No of HA1 isolation gene segment 128
Amino acid variations in the HA1 amino acid sequences
TABLE I. (Continued)
Amino acid variations in the NA amino acid sequences
Genetic Changes in Influenza A(H3N2) Viruses Circulating
1273
influenza A(H3N2) strains from Lucknow, Uttar Pradesh, India, were characterized on the basis of the HA and NA genes diversity. Molecular analysis of theses sequences suggest that all strains circulating in Lucknow were related more closely to the prototype strain, influenza A/Victoria/361/2011 than the earlier year’s vaccine strains; A/Brisbane/10/2007 and A/Perth/16/2009. This data is consistent with the WHO report on the influenza activity and characterization of the influenza A(H3N2) strains circulating during 2011–12 influenza seasons [WHO surveillance report, 2012]. In the HA1 domain, four substitutions; N198S, T212A, V223I, and N312S were identified that were found to be stable in all the studied strains. These mutations were absent in the vaccine strains A/ Brisbane/10/2007 and A/Perth/16/2009 but were present in A/Victoria/361/2011 strain. According to the European Centre for Disease Prevention and Control (ECDC) report 2014, influenza A(H3N2) virus is divided into seven genetic groups; 1–7 based on the certain mutations in the HA1 coding sequences [ECDC surveillance Report, April 2014]. The group 3 of influenza A(H3N2) virus is further subdivided in three subgroups; 3 A, 3 B and 3 C [ECDC Reports, December 2013; Pariani et al., 2013]. In present study, all studied strains contained four mutations; N145S; A198S; V223I, and N312S in the HA1 coding sequences which are characteristic of the group 3 of influenza A(H3N2) virus. Lucknow strains isolated during 2013 showed few additional mutations R142G, T128A along with N278K that fall predominantly within the genetic subgroup 3 C of the influenza A(H3N2) virus, mainly within the subgroup 3 C.3 [ECDC surveillance Report, April 2014]. Previous studies have documented the five antigenic sites [A–E] in the HA1 polypeptide chain that covers major portion of the receptor binding domain (RBD) in the HA1 protein. The HA1 protein is also responsible for drifting of seasonal influenza strains [Wiley et al., 1981 Wilson et al., 1981; Wilson and Cox, 1990]. The present study identified twelve amino acid mutations in the antigenic sites of the HA1 domain, of which five mutations (N145S, A198S, T212A and V223I and N312S) were found in all the studied strains. Similar mutations were also reported from Northern Italy and Canada [Pariani et al., 2013; Skowronski et al., 2013]. These mutations cover four of the five antigenic sites of the HA1 protein i.e., A, B, C, and D epitopes. One major change at 278 amino acid position [N–K] was found only in the year 2013 strains, which lie in the C antigenic site of the HA1 domain. This mutation may affect antibody affinity since it is located next to important antibody binding site [Wiley et al., 1981; Julie et al., 2012]. Strains of 2013 differ from those of years 2011 and 2012 by the presence of four important amino acid mutations [T128 A; R142G, L157S, and N278K], located either within or close to the A and/or C antigenic sites. This explains the J. Med. Virol. DOI 10.1002/jmv
1274
Jain et al.
Fig. 2. Phylogenetic tree of Influenza A(H3N2) strains and representative vaccine strains based on partial NA gene using Maximum Likelihood Method. Amino acid substitutions in NA domain that characterized a particular branch are indicated at branch node. Numbers at the nodes indicate confidence levels of bootstrap analysis with 500 replicates.
possible continuous drift in the circulating influenza A(H3N2) strains since 2011. Genetic analysis of the NA gene showed overall 12 amino acid mutations in the NA coding sequences of Lucknow strains. All strains were characterized by acquiring changes at 367 (S –N) and 369 (K–T) amino acid positions in the NA domain, which was also reported by WHO influenza centre, London [WHO London’s influenza report WHO, 2011]. Two mutations [D251V and S315G] were observed in majority of the 2013 strains, which make them diverge from 2011 influenza A(H3N2) strains. Majority of the Lucknow strains contained N402D mutation in the NA coding sequences which represents the loss of the N-linked glycosylation site in the NA domain [Sobolev et al., 2011]. Variation in the N-glycosylation sites at HA and NA domain may potentially alter the antigenic property of the glycoprotein and its function [Falchi et al., 2010]. Many studies reported the mutations associated with the NA protein (i.e. E119V, R292K, del244–247, N294S, Q136K, E119V þ J. Med. Virol. DOI 10.1002/jmv
I222V) of influenza A(H3N2) virus, typical of strains resistant to antiviral oseltamivir, the most commonly used inhibitor of influenza virus NA [Sobolev et al., 2011; Global Influenza Surveillance Network, WHO, 2011]. None of the studied strains contain mutations in the sequences responsible for oseltamivir drug resistance. Genetic diversity in the HA1 and NA genes emphasize the importance of continuous molecular surveillance for characterizing the emerging influenza variants. This study is limited to the sequencing of the partial HA and the NA genes from limited numbers of the samples. Mutations have a great impact on the antigenic structure but multiple lineages of influenza strains arise by genetic reassortment, which cannot be investigated here. Whole genome sequencing can give the better answer about the reassortmentrelated events and queries. In spite of these limitations, the present study contributes certain findings that facilitate the better understanding of the genetic evolution of the circulating strains in India and
Genetic Changes in Influenza A(H3N2) Viruses Circulating
confirmed the effectiveness of 2013 vaccine (recommended by WHO) for circulating strains in the population. The presence of mutations in the HA and the NA antigenic sites documented that in upcoming days, these strains may drift into the new antigenic variants of different pathogenicity. Therefore, careful influenza virus surveillance is required to determine the further genetic changes in the important antigenic sites of the circulating influenza strains for the update of annual vaccine. ACKNOWLEDGMENTS We are thankful to Dr. K.P. Singh, Dr. Anil Kumar Verma, Mr. Mukesh Dwivedi, Mr. Ramesh Chandra Upadhyaya, Ms. Preeti Sharma and staff of influenza project, and staff of Viral Diagnostic Laboratory (VDL) for laboratory support [5/8/7/14/2009-ECD-1(Vol.II)]. REFERENCES Ann J, Papenburg J, Bouhy X, Rhe´aume C, Hamelin ME, Boivin G. 2012. Molecular and antigenic evolution of human influenza A/ H3N2 viruses in Quebec, Canada, 2009–2011. J Clin Virol 53: 88–92. Chutinimitkul S, Chieochansin T, Payungporn S, Samransamruajkit R, Hiranras T, Theamboonlers A, Poovorawan Y. 2008. Molecular characterization and Phylogenetic analysis of H1N1 and H3N2 human influenza A viruses among infants and children in Thailand. Virus Res 132:122–131. Dangi T, Jain B, Singh AK, Singh JV, Kumar R, Dwivedi M, Verma AK, Chadha MS, Jain A. 2014. 2014. Molecular Characterization of Circulating Pandemic Strains of Influenza A Virus During 2012 to 2013 in Lucknow (India). J Med Virol 2014. DOI: 10.1002/jmv.23946 European Centre for Disease Prevention and Control (ECDC) Surveillance Report 2013, Stockholm. Influenza virus characterisation, summary Europe, December 2013. Available on: http:// www.ecdc.europa.eu/en/publications/Publications/influenza-viruscharacterisation-dec-2013.pdf. European Centre for Disease Prevention and Control (ECDC) Surveillance Report 2014, Stockholm. Influenza virus characterization, summary Europe, April 2014. Available on: http://www. ecdc.europa.eu/en/publications/Publications/ERLI-Net-report-April-2014.pdf. Falchi A, Amoros JP, Arena C, Arrighi J, Casabianca F, Andreoletti L, Turbelin C, Flahault Blanchon AT, Hanslik T, Varesi L. 2011. Genetic structure of human A/H1N1 and A/H3N2 influenza virus on Corsica Island: Phylogenetic analysis and vaccine strain match 2006–2010. PLoS ONE 6:e24471. DOI: 10.1371/journal. pone.0024471 Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289. Kilbourne E, Laver W, Schulman J, Webster R. 1968. Antiviral activity of antiserum specific for an influenza virus neuraminidase. J Virol 2:281–288. Latorre-Margalef N, Grosbois V, Wahlgren J, Munster VJ, Tolf C, Fouchier RAM, Osterhaus ADME, Olsen B, Waldenstro¨m J. 2013. Heterosubtypic immunity to influenza A virus infections in mallards may explain Existence of multiple virus subtypes. PLoS Pathog 9:e1003443. DOI: 10.1371/journal.ppat.1003443 Lee MS, Chen JS. 2004. Predicting antigenic variants of influenza A/H3N2 viruses. Emerg Infect Dis 10:1385–1390.
1275 Lin YP, Gregory V, Bennett M, Hay A. 2004. Recent changes among human influenza viruses. Virus Res 103:47–52. Mishra AC, Chadha MS, Choudhary ML, Potdar VA. 2010. Pandemic influenza (H1N1) 2009 is associated with severe disease in India. PLoS ONE 5:e10540. DOI: 10.1371/journal.pone.0010540 Pariani E, Amendola A, Ebranati E, Ranghiero A, Lai A, Anselmi G, Zehender G, Zanetti A. 2013. Genetic drift of influenza A(H3N2) virus hemagglutinin (HA) variants originated during the last pandemic turn out to be predominant in the 2011–2012 season in Northern Italy. Infect Genet Evol 13:252–260. Plotkin JB, Dushoff J. 2003. Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus. Proc Natl Acad Sci USA 100:7152–7157. Potdar VA, Chadha MS, Jadhav SM, Mullick J, Cherian SS, Mishra AC. 2010. Genetic characterization of the influenza A pandemic (H1N1) 2009 virus isolates from India. PLoS ONE 5:e9693.DOI: 10.1371/journal.pone.0009693 Skowronski DM, Janjua NZ, De Serres G, Dickinson JA, Winter A, Mahmud SM. 2013. Interim estimates of influenza vaccine effectiveness in 2012/13 from Canada’s sentinel surveillance network, January 2013. Euro Surveill 18:pii: 20394. Available online: http://www.eurosurveillance.org/ViewArticle. aspx?ArticleId ¼ 20394. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM. 2004. Mapping the antigenic and genetic evolution o f influenza virus. Science 305:371–376. Sobolev I, Kurskaya O, Susloparov I, Ilyicheva T, Shestopalov A. 2012. Molecular genetic analysis of influenza A/H3N2 virus strains isolated in Western Siberia in the 2010–2011 epidemic seasons. Infect Genet Evol 12:1694–1698. Stohr K. 2002. Influenza-WHO cares. Lancet Infect Dis 2:517. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. Taubenberger JK, Morens DM. 2013. Influenza viruses: Breaking all the rules. mBio 4:e00365–e00313. DOI: 10.1128/ mBio.00365–13 Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucl Acid Res 22:4673–4680. WHO. 2003. Fact sheet number 211. Influenza. Available on: http:// www.who.int/mediacentre/factsheets/fs211. World Health Organization (WHO). 2009. CDC protocol of real-time RTPCR for influenza A (H1N1). Available from: http://www.who. int/csr/resources/publications/swineflu/realtimeptpcr/en/index. htm. (accessed on April 30, 2009). WHO 2011. London Influenza Report 2011: Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Northern Hemisphere. 14th–17th February. Available on: http://www.who.cc.imr.mrc.ac.uk. WHO Global Influenza Surveillance Network. 2011. Manual for the laboratory diagnosis and virological surveillance of influenza. WHO surveillance Report: Summary of the influenza season in the WHO European region. Available on: http://www.who.int/ influenza/vaccines/virus/recommendations/2012_13_north/en/. World Health Organization (WHO). CDC protocol of real-time RTPCR for influenza A (H1N1). Available from:flu/realtimeptpcr/en/index html; http://www.who.int/csr/resources/publications/swine. (accessed on April 30, 2009). Wiley DC, Wilson IA, Skehel JJ. 1981. Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature 289:373–378. Wilson IA, Cox NJ. 1990. Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8:737–771. Wilson IA, Skehel JJ, Wiley DC. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolutions. Nature 289:366–373.
J. Med. Virol. DOI 10.1002/jmv
Genetic Changes in Influenza A(H3N2) Viruses Circulating During 2011 to 2013 in Northern India (Lucknow) Amita Jain,1* Tanushree Dangi,1 Bhawana Jain,1 Ajay Kumar Singh,1 J. V. Singh,2 and Rashmi Kumar3 1
Department of Microbiology, King George’s Medical University, Lucknow, India Department of Community, Medicine King George’s Medical University, Lucknow, India 3 Department of Paediatrics, King George’s Medical University, Lucknow, India 2
Genetic variability in the hemagglutinin (HA1) and the neuraminidase (NA) genes of influenza viruses results in the emergence of new strains which differ in pathogenicity and severity. The present study was undertaken for genotypic characterization of the HA1 and NA genes of the influenza A(H3N2) strains, detected during the 2011–2013. A total of fifty five influenza A(H3N2) positive samples [2011 (n ¼ 20), 2012 (n ¼ 4) and 2013 (n ¼ 31)] were studied. The 824 bp segment of HA1 gene and 931 bp segment of NA gene were amplified and sequenced by Big-Dye terminator kit on ABI3130, Genetic analyzer. Molecular and phylogenetic analysis was done by MEGA 5.05 software and PhyML program (v3.0). Mutations were determined by comparing the deduced amino acid sequences of study strains with that of 2009–2013 vaccine strains. The studied influenza A(H3N2) strains showed 98.1–99.6% similarity in HA1 and NA amino acid sequences with the influenza A/Victoria/ 361/2011 vaccine strain. Four mutations in the HA1 amino acid sequences (T128A, R142G, L157S and N278K) and three unique mutations in the NA amino acid sequences [D251V, S315G and V313A] were found. These mutations were observed only in strains from the year 2013 (cluster II). None of the strains showed the presence of mutations, N294S and R292K, markers of oseltamivir resistance. In conclusion, Lucknow strains have accumulated the significant number of mutations in the antigenic sites of the HA and the NA coding sequences and continue to be evolving from the 2013 vaccine strain [A/Victoria/361/ 2011], however, mutations specific for oseltamivir resistance were not detected. J. Med. Virol. 87:1268–1275, 2015. # 2015 Wiley Periodicals, Inc. C 2015 WILEY PERIODICALS, INC.
KEY WORDS:
influenza A(H3N2) virus; epidemic; HA1 gene; NA gene; phylogenetic; mutations
INTRODUCTION Influenza virus is the major leading cause of respiratory tract infections in humans. It has caused severe pandemics and epidemics during the last century. Few subtypes of influenza A virus were responsible for severe infections in the human population, i.e., H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, H5N1 in 1997, PdmH1N1 in 2009 and H7N9 in 2013 [Falchi et al., 2011; Taubenberger and Morens, 2013]. Though, the mortality associated with these pandemics remain quite high, seasonal epidemics also have a major role in significant morbidity and mortality, affecting approximately 5–15% population globally each year [Stohr, 2002; WHO, 2003]. Genetic and antigenic variations in the hemagglutinin (HA) and the neuraminidase (NA) surface glycoproteins of the influenza A virus mainly attribute to seasonal epidemics. Till date, 16 variants of the HA gene and 9 variants of the NA gene are known to exist.
Conflict of Interest: None Grant sponsor: Influenza surveillance network; Grant sponsor: Indian Council of Medical Research; Grant number: 5/8/7/14/ 2009-ECD-1(Vol.II). Correspondence to: Prof. Amita Jain, MD, Department of Microbiology, King George Medical University, Lucknow-226003, India. E-mail: [email protected] Accepted 15 October 2014 DOI 10.1002/jmv.24096 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Genetic Changes in Influenza A(H3N2) Viruses Circulating
Different combinations of the HA and the NA genes determine the influenza A virus subtypes. At present, three subtypes of influenza A virus (H1N1, H3N2 and pandemic H1N1) are circulating, which, though well adapted in the human populations, can cause local outbreaks of varying severity [Lin et al., 2004; Falchi et al., 2010]. The variability among subtypes of the influenza A virus is so high that the HA and NA genes from two different subgroups have only 40% and 62–68% amino acid similarities, respectively [Latorre-Margalef et al., 2013]. The HA protein is a major target of host neutralizing antibodies. The HA protein helps the influenza virus to penetrate into the host cells by binding with the sialic acid receptors on host’s respiratory epithelial cells. The HA1 domain of the HA protein mutates more frequently than the HA2 domain [Plotkin and Dushoff, 2003]. Various studies have also reported the importance of the HA1 gene in prediction of genetic drift [Lee and Chen, 2004; Smith et al., 2004]. Genetic variations in the antigenic sites of the HA gene coded proteins may change the behaviour of influenza virus strains by allowing the virus to escape host’s immunity. Enzymatic activity of the NA gene encoded proteins play important role in dissemination of the newly formed virus particles. Neutralizing antibodies are generated against the NA protein during infections. Frequent genetic changes in the NA protein facilitates the influenza virus to escape host immune system which may enhance the virus titre and infectivity [Kilbourne et al., 1968]. High genetic variability in the HA and NA genes leads to the emergence of new antigenic variants (called antigenic drift). Therefore, frequent updating of the composition of seasonal vaccine by the World Health Organization (WHO), on the basis of the HA and NA genes is required [Willy et al., 1981]. The present study analyzed the nucleotide and deduced amino acid sequences of the HA1 and the NA genes of influenza A(H3N2) viruses circulating in Lucknow, Uttar Pradesh (India) during 2011–2013 to determine the genetic variations occurring in circulating strains, to understand their evolution at molecular level. MATERIALS AND METHODS Nasal and Throat swabs were collected from the patients presenting as influenza-like illness (ILI) during January 2011–December 2013, at Viral Diagnostic Laboratory, King George’s Medical University, Lucknow (India). The samples were tested for the presence of influenza A(H3N2) virus RNA by realtime reverse-transcription polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR. Clinical specimens having high viral load [Ct < 25 in real-time PCR] and showing single sharp band in PCR amplification of the HA and NA genes, were selected for sequencing experiments.
1269
Sample Processing and RNA Extraction Nasal and throat swabs were collected in 3.5 ml viral transport media (VTM) and centrifuged at 1000 rpm for 5 min. The supernatant was transferred to nuclease free collection tubes. Viral RNA was extracted from the 200 ml aliquots of the supernatant 1 by using the Invitrogen PureLink Viral RNA/DNA Extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Detection by Real Time RT-PCR Real-time RT-PCR was performed from the freshly extracted RNA for confirmation of Influenza A, and subtype H3N2 according to the Centers for Disease Control and Prevention (CDC) protocol [Mishra et al., 2009; World Health Organization, 2009]. Briefly, a 25 ml PCR reaction mixture was prepared in eppendorf containing 12.5 ml of 2xABI RT-PCR buffer with 1 ml of 25 RT-enzyme (AgPath-IDTM One Step RTPCR Kit, Applied Biosystem, Foster city, California, USA), 0.5 ml forward primer (40mM), 0.5 ml reverse primer (40mM), 0.2 ml probe (10mM) and 5 ml of nuclease free water. The PCR was carried out on ABI 7500 thermal cycler (Applied Biosystems, USA) with following reaction conditions: reverse-transcription step at 50 ˚C for 30 min and 95 ˚C for 2 min, followed by 45 cycles of PCR amplification at 95 ˚C for 15 sec and 55 ˚C for 30 sec. Positive control, negative control and extraction control were run simultaneously with each RT-PCR run for validity of the experiment. RNaseP gene segments were amplified as internal control for human nucleic acid gene to test the RNA extraction procedure. Amplification of the HA and NA Genes by Conventional PCR One strand complementary DNA (c-DNA) was synthesized in two separate eppendorfs using the HA and the NA gene-specific primers by the method as described previously [Dangi et al., 2014]. Two step conventional reverse transcription PCR (RT-PCR) was done to amplify viral c-DNA specific for 824 bp fragment (nucleotide position 360–1184) in the HA1 domain and 931 bp fragments (nucleotide position 367– 1298) in the NA gene [Hoffmann et al., 2001; Chutinimitkul et al., 2008]. Briefly, 2 ml of c-DNA was added to the reaction mixture containing 2.5 ml Taq Buffer (10X), 1 ml Taq enzyme (2U/ml; DyNAzymeTM, Fisher Scientific, Bishop Meadow Road, Loughborough, United Kingdom), 0.5 ml dNTP (100mM; KomaBiotech, 19F, IS BIZ Tower, Seoul, Korea), 0.5 ml forward and reverse primers (10mM) and nuclease-free water to the final volume of 25 ml. Amplification was performed in an Applied Biosystems Veriti 96-well thermo cycler 7500 under the following conditions: Initial denaturation at 94 ˚C for 10 min, followed by 35 amplification cycles, comprising of denaturation for 30 sec, primer annealing at 55 ˚C [HA1 gene product]/52 ˚C [N2 gene J. Med. Virol. DOI 10.1002/jmv
1270
Jain et al.
product] for 45 sec followed by extension at 72 ˚C for 1 min, to be ended by a final elongation step of 10 min at 72 ˚C. The amplified products were run on 1.2% Agarose gel and visualized on a UV trans-illuminator after staining with ethidium bromide solution. Sequencing The PCR products seen as a single sharp band on conventional PCR were used directly for further purification process by Exo-Sap treatment to remove excess primers and un-incorporated dNTPs (Exonuclease I,1U/ml and Shrimp Alkaline Phosphatase, 20U/ml) (FastApTM, Fermentas, Fisher Scientific, Pittsburgh, PA) [Potdar et al., 2010]. Purified products were sequenced in both the directions (30 and 50 prime) by using forward and reverse primers in two separate wells for both the HA1 and NA gene segments. Sequencing was carried out using Big-Dye Terminator 3.1 Cycle-Sequencing Kit (Applied Biosystems, Foster City, USA) on ABI Prism 3,130 genetic analyzer (Applied Biosystems, Foster City, USA). Nucleotide sequences obtained in this study were submitted in GenBank database. Data Analysis The generated nucleotide sequences were edited manually and aligned by ClustalW version 1.83 [Thompson et al., 1994]. To determine the genetic variability in the circulating strains, their nucleotide sequences were compared to the vaccine strains of the years 2009 to 2013 (Influenza A/Victoria/361/2011, A/ Perth/16/2009 and A/Brisbane/19/2007 strains) recommended by World Health Organization (WHO). The vaccine strain A/Victoria/361/2011 was used during 2012–2013 in the Northern Hemisphere and during 2013 in the Southern Hemisphere. Influenza A/Perth/ 16/2009 strain was used as vaccine strain during 2010, 2011, and 2012 in both Northern and Southern Hemisphere, and A/Brisbane/19/2007 vaccine strain was used during 2009 in both the Northern and Southern Hemisphere. The HA1 and the NA gene sequences of the vaccine strains were retrieved from the Influenza Research Database (www.fludb.org) available on National Center for Biotechnology Information (www. ncbi.nlm.nih.gov). Phylogenetic tree was constructed based on the HA1 and the NA amino acid sequences using the maximum likelihood (ML) approach in the PhyML program (version 3.0) [Guindon and Gascuel, 2003] at 500 bootstrap replicates. This analysis utilized the HKY85 þ I þ G4 nucleotide substitution model to determine the evolutionary divergence analysis between the HA1 and the NA gene sequences. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Amino acid substitutions within the HA1 and partial NA amino acid sequences were determined by doing multiple sequence alignment of the fifty five strains with respect to the influenza A(H3N2) J. Med. Virol. DOI 10.1002/jmv
vaccine strains by ClustalW in Molecular Evolutionary Genetics Analysis version 5.05 (MEGA 5.05) software [Tamura et al., 2011]. RESULTS Of 4,227 clinical samples collected from ILI patients, a total of 140 samples tested positive for influenza A(H3N2) virus by real-time RT-PCR. Only 55/140 clinical samples were sequenced for the HA and the NA genes. These samples were from year 2011(20/59), 2012 (4/7), and 2013 (31/74). Molecular Analysis of HA1 Gene Nucleotide sequence analysis (HA1 gene) of the studied strains showed that all the strains were more closely related to the 2013 vaccine strain (A/Victoria/ 361/2011) than the vaccine strains of the previous years. Strains isolated during the years 2011 and 2012 showed 98.1%, 99%, and 99.6% similarity with vaccine strains of years, 2008–2009 (A/Brisbane/10/ 2007), 2010–2012 (A/Perth/16/2009), and 2013 (A/ Victoria/361/2011), respectively. Strains circulating during the year 2013 showed 96.6%, 97.0% and 98.5% similarity with A/Brisbane/10/2007, A/Perth/ 16/2009 and A/Victoria/361/2011 strains, respectively. Phylogenetic analysis of the Lucknow strains, based on the HA gene revealed that these strains drifted from the vaccine strains recommended for the years 2009–2012. The Lucknow strains matched more closely with the influenza A/Victoria/361/2011 the 2013 vaccine strain (Fig. 1). The presence of differences in gene sequences divided Lucknow strains into two major clusters, cluster I and cluster II. Influenza A(H3N2) strains circulating during 2011–2012 grouped into the cluster I, while all 2013 strains fell into the cluster II. Cluster II further divided into two subgroups; IIA and IIB. Amino Acid Variations in HA1 Domain Comparison of deduced amino acid sequences of the HA1 gene of Lucknow strains with respective sequences of vaccine strains showed twelve mutations at different positions (Table I). N145S mutation located in the A epitope of globular HA1 domain was found in all the studied strains. Other common mutations present in all Lucknow strains as well as in 2013 vaccine strain were A198S, T212A, V223I, and N312S. Five mutations found in isolates from year 2013 were T128A, R142G, L157S, S146G, and N278K. Less frequent mutations are detailed in Table I. Molecular Analysis of NA Gene Phylogenetic tree of the NA gene sequences showed a similar evolutionary pattern of the influenza A(H3N2) strains as observed in the phylogeny of the HA1 gene. All studied strains grouped into two distinct clusters [cluster I and cluster II] due to accumulation of the some unique substitutions in the
Genetic Changes in Influenza A(H3N2) Viruses Circulating
1271
Fig. 1. Phylogenetic tree of Influenza A(H3N2) strains and representative vaccine strains based on HA1 gene using Maximum Likelihood Method. Amino acid substitutions in HA1 domain that characterized a particular branch are indicated at branch node. Numbers at the nodes indicate confidence levels of bootstrap analysis with 500 replicates.
NA gene sequences and cluster II further separated into two subgroups [IIA and IIB] as shown in Figure 2. Strains from years 2011–2012 showed 98.5% similarity with vaccine strains used during that duration while strains from year 2013 showed only 96.2% similarity to the vaccine strain used during that year.
mivir [N294S and R292K] was not observed in the studied strains. N402D mutation was observed in all the strains from the year 2013 and some of the strains from the years 2012 (n ¼ 10) and 2011 (n ¼ 1). Other less frequent substitutions are shown in Table I. Accession Numbers
Amino Acid Variation in NA Domain Partial amino acid sequences of the NA gene was analyzed in context to the amino acid sequences of representative vaccine strains (Table I). Twelve amino acid substitutions were identified in the coding sequences of the NA gene of studied strains. All strains from the years 2011–2013 showed two major substitutions S367N and K369T corresponding to the antigenic site. Two substitutions [D251V (n ¼ 31) and S315G (n ¼ 21) were seen only in the strains from the year 2013. Some of the Lucknow strains from the years 2011(n ¼ 5) and 2012 (n ¼ 1) showed I397M mutation which was not detected in strains from the year 2013. Drug resistance marker specific to oselta-
The nucleotide sequences derived in the study were deposited in the GenBank database under accession numbers KF142486, KF142487, KF142488; F668185– KF668207; KJ667978–KJ668007 for H3 gene and KF668208–KF668232; KJ668008–KJ668037 for N2 gene (Table I). DISCUSSION Worldwide, Influenza A virus causes seasonal epidemics. Annual influenza immunization is recommended to prevent influenza virus infection due to rapid mutation rate in influenza viral strains. Therefore, every year vaccine is updated according to the circulating strains for the best fit. In this study, J. Med. Virol. DOI 10.1002/jmv
A/Victoria/361/2011 A/Perth/16/2009 A/Brisbane/10/2007 LKO799 LKO805 LKO810 LKO819 LKO827 LKO845 LKO851 LKO856 LKO864 LKO871 LKO875 LKO883 LKO892 LKO896 LKO911 LKO934 LKO940 LKO968 LKO974 LKO983 LKO1442 LKO1501 LKO3108 LKO3154 LKO3475 LKO3491 LKO3714 LKO3738 LKO3812 LKO3815 LKO3818 LKO3821 LKO3824 LKO3825 LKO3828 LKO3839 LKO3863 LKO3865 LKO3876 LKO3887 LKO3889
Strains
2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
KC306165 CY081428 CY116577 KF668185 KJ667978 KJ667979 KJ667980 KF668186 KF668187 KF668188 KF668189 KF142486 KF66890 KJ667981 KJ667982 KJ667983 KF66891 KJ667984 KJ667985 KF142487 KJ667986 KJ667987 KF66892 KF66893 KF66894 KF66895 KJ667988 KJ667989 KJ667990 KJ667991 KJ667992 KJ667993 KJ667994 KF66896 KJ667995 KF66897 KF66898 KJ667996 KF66899 KF66900 KJ667997 KF668201 KF668202 KF668203
T . . . . . . . . . . . . . . . . . . . . . . . . . A A A A A A A A A A A A A A
Accession Year of No of HA1 isolation gene segment 128 R . . . . . . . . . . . . . . . . . . . . . . . . . . G G G G G G G G G G G G G G G G
142 N . . S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S
145 S . . . . . . . . . . . . . . . . . . . . . . . . . . G G . . . . . . . . . . . . G . .
146 L . . . . . . . . . . . . . . . . . . . . . . . . . . S S S S S . . S S S S . . S . S
157 S A A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
198 A T T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212 I V V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
223 R . . . Q . . . . . . . . . . Q Q . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
Amino acid variations in the HA1 amino acid sequences
N . . . . . . . . . . . . . . . . . . . . . . . . . . K K K K K K K K K K K K K K K K K
J. Med. Virol. DOI 10.1002/jmv . . . . . . . . . .
I . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .
S N N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CY121079 CY081429 EU199420 KF668208 KJ668008 KJ668009 KJ668010 KF668209 KF668210 KF668211 KF668212 KF668213 KF668214 KJ668011 KJ668012 KJ668013 KF668215 KJ668014 KJ668015 KF668216 KJ668016 KJ668017 KF668217 KF668218 KF668219 KF668220 KJ668018 KJ668019 KJ668020 KJ668021 KJ668022 KJ668023 KJ668024 KF668221 KJ668025 KF668222 KF668223 KJ668026 KF668224 KF668225 KJ668027 KF668226 KF668227 KF668228
K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . .
D . . . . . . . . . . . . . . . . . . . . . . . . . . V V V V V V V V V V V V V V V V V
Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . .
V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A A . . . . . . A A . . .
S . . . . . . . . . . . . . . . . . . . . . . . . . . G G G G . . G G G G G G . . G G G
D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
M . . . . . . . . . . . . . . . . . . . . . . . . . . . . I I . . . . . . I . . . . . .
S . . N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
K . . T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T
P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S
I . . M . M M . . . . M . . . M M . . . . . . . . . . . . . . . . .
N . . D D D D D D D D D D D D D D D D D D D D D D D D D D D D
Accession No of NA 278 282 312 gene segment 199 251 284 313 315 339 362 367 369 386 397 402
Amino acid variations in the NA amino acid sequences
TABLE I. Changes in Amino Acid Sequences Encoded by HA1Gene and Partial NA Gene (Antigenic Regions) of Influenza A(H3N2) With Respect to Vaccine Strains
1272 Jain et al.
D D D D D D D D D D D D D D . . . . . . . . . . . . . . . . . . . . . . . . . . . . T T T T T T T T T T T T T T N N N N N N N N N N N N N N . . . . . . . . . I I . . . . N . N . . . . . . . . . . G G . G G G G . . G G . . . . . A . . . . A A . . A A A . . . . . . . . . . . . . H V V V V V V V V V V V V V V . . . . . . . . . . . . . . KJ668028 KF668229 KJ668029 KF668230 KF668231 KF668232 KJ668030 KJ668031 KJ668032 KJ668033 KJ668034 KJ668035 KJ668036 KJ668037 . . . . . . . . .
. . . . . . . . . . . . . . . . . . . K K K K K K K K K K K K K K
Amino acids variations at various positions similar to A/Victoria/361/2011 were denoted by dot (.).
261
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
223 212
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
198 157
S . . S . . . . S . . . . . G . . . . . . . G . . . . .
146 145
S S S S S S S S S S S S S S G G G G G G G G G G G G G G A A A A A A A A A A A A KJ667998 KF668204 KJ667999 KF668205 KF668206 KF668207 KJ668000 KJ668001 KJ668002 KJ668003 KJ668004 KJ668005 KJ668006 KJ668007 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
142 Strains
LKO3890 LKO3893 LKO3905 LKO3906 LKO3913 LKO3914 LKO3918 LKO3925 LKO3938 LKO3960 LKO3969 LKO4012 LKO4129 LKO4167
Accession No of NA 278 282 312 gene segment 199 251 284 313 315 339 362 367 369 386 397 402 Accession Year of No of HA1 isolation gene segment 128
Amino acid variations in the HA1 amino acid sequences
TABLE I. (Continued)
Amino acid variations in the NA amino acid sequences
Genetic Changes in Influenza A(H3N2) Viruses Circulating
1273
influenza A(H3N2) strains from Lucknow, Uttar Pradesh, India, were characterized on the basis of the HA and NA genes diversity. Molecular analysis of theses sequences suggest that all strains circulating in Lucknow were related more closely to the prototype strain, influenza A/Victoria/361/2011 than the earlier year’s vaccine strains; A/Brisbane/10/2007 and A/Perth/16/2009. This data is consistent with the WHO report on the influenza activity and characterization of the influenza A(H3N2) strains circulating during 2011–12 influenza seasons [WHO surveillance report, 2012]. In the HA1 domain, four substitutions; N198S, T212A, V223I, and N312S were identified that were found to be stable in all the studied strains. These mutations were absent in the vaccine strains A/ Brisbane/10/2007 and A/Perth/16/2009 but were present in A/Victoria/361/2011 strain. According to the European Centre for Disease Prevention and Control (ECDC) report 2014, influenza A(H3N2) virus is divided into seven genetic groups; 1–7 based on the certain mutations in the HA1 coding sequences [ECDC surveillance Report, April 2014]. The group 3 of influenza A(H3N2) virus is further subdivided in three subgroups; 3 A, 3 B and 3 C [ECDC Reports, December 2013; Pariani et al., 2013]. In present study, all studied strains contained four mutations; N145S; A198S; V223I, and N312S in the HA1 coding sequences which are characteristic of the group 3 of influenza A(H3N2) virus. Lucknow strains isolated during 2013 showed few additional mutations R142G, T128A along with N278K that fall predominantly within the genetic subgroup 3 C of the influenza A(H3N2) virus, mainly within the subgroup 3 C.3 [ECDC surveillance Report, April 2014]. Previous studies have documented the five antigenic sites [A–E] in the HA1 polypeptide chain that covers major portion of the receptor binding domain (RBD) in the HA1 protein. The HA1 protein is also responsible for drifting of seasonal influenza strains [Wiley et al., 1981 Wilson et al., 1981; Wilson and Cox, 1990]. The present study identified twelve amino acid mutations in the antigenic sites of the HA1 domain, of which five mutations (N145S, A198S, T212A and V223I and N312S) were found in all the studied strains. Similar mutations were also reported from Northern Italy and Canada [Pariani et al., 2013; Skowronski et al., 2013]. These mutations cover four of the five antigenic sites of the HA1 protein i.e., A, B, C, and D epitopes. One major change at 278 amino acid position [N–K] was found only in the year 2013 strains, which lie in the C antigenic site of the HA1 domain. This mutation may affect antibody affinity since it is located next to important antibody binding site [Wiley et al., 1981; Julie et al., 2012]. Strains of 2013 differ from those of years 2011 and 2012 by the presence of four important amino acid mutations [T128 A; R142G, L157S, and N278K], located either within or close to the A and/or C antigenic sites. This explains the J. Med. Virol. DOI 10.1002/jmv
1274
Jain et al.
Fig. 2. Phylogenetic tree of Influenza A(H3N2) strains and representative vaccine strains based on partial NA gene using Maximum Likelihood Method. Amino acid substitutions in NA domain that characterized a particular branch are indicated at branch node. Numbers at the nodes indicate confidence levels of bootstrap analysis with 500 replicates.
possible continuous drift in the circulating influenza A(H3N2) strains since 2011. Genetic analysis of the NA gene showed overall 12 amino acid mutations in the NA coding sequences of Lucknow strains. All strains were characterized by acquiring changes at 367 (S –N) and 369 (K–T) amino acid positions in the NA domain, which was also reported by WHO influenza centre, London [WHO London’s influenza report WHO, 2011]. Two mutations [D251V and S315G] were observed in majority of the 2013 strains, which make them diverge from 2011 influenza A(H3N2) strains. Majority of the Lucknow strains contained N402D mutation in the NA coding sequences which represents the loss of the N-linked glycosylation site in the NA domain [Sobolev et al., 2011]. Variation in the N-glycosylation sites at HA and NA domain may potentially alter the antigenic property of the glycoprotein and its function [Falchi et al., 2010]. Many studies reported the mutations associated with the NA protein (i.e. E119V, R292K, del244–247, N294S, Q136K, E119V þ J. Med. Virol. DOI 10.1002/jmv
I222V) of influenza A(H3N2) virus, typical of strains resistant to antiviral oseltamivir, the most commonly used inhibitor of influenza virus NA [Sobolev et al., 2011; Global Influenza Surveillance Network, WHO, 2011]. None of the studied strains contain mutations in the sequences responsible for oseltamivir drug resistance. Genetic diversity in the HA1 and NA genes emphasize the importance of continuous molecular surveillance for characterizing the emerging influenza variants. This study is limited to the sequencing of the partial HA and the NA genes from limited numbers of the samples. Mutations have a great impact on the antigenic structure but multiple lineages of influenza strains arise by genetic reassortment, which cannot be investigated here. Whole genome sequencing can give the better answer about the reassortmentrelated events and queries. In spite of these limitations, the present study contributes certain findings that facilitate the better understanding of the genetic evolution of the circulating strains in India and
Genetic Changes in Influenza A(H3N2) Viruses Circulating
confirmed the effectiveness of 2013 vaccine (recommended by WHO) for circulating strains in the population. The presence of mutations in the HA and the NA antigenic sites documented that in upcoming days, these strains may drift into the new antigenic variants of different pathogenicity. Therefore, careful influenza virus surveillance is required to determine the further genetic changes in the important antigenic sites of the circulating influenza strains for the update of annual vaccine. ACKNOWLEDGMENTS We are thankful to Dr. K.P. Singh, Dr. Anil Kumar Verma, Mr. Mukesh Dwivedi, Mr. Ramesh Chandra Upadhyaya, Ms. Preeti Sharma and staff of influenza project, and staff of Viral Diagnostic Laboratory (VDL) for laboratory support [5/8/7/14/2009-ECD-1(Vol.II)]. REFERENCES Ann J, Papenburg J, Bouhy X, Rhe´aume C, Hamelin ME, Boivin G. 2012. Molecular and antigenic evolution of human influenza A/ H3N2 viruses in Quebec, Canada, 2009–2011. J Clin Virol 53: 88–92. Chutinimitkul S, Chieochansin T, Payungporn S, Samransamruajkit R, Hiranras T, Theamboonlers A, Poovorawan Y. 2008. Molecular characterization and Phylogenetic analysis of H1N1 and H3N2 human influenza A viruses among infants and children in Thailand. Virus Res 132:122–131. Dangi T, Jain B, Singh AK, Singh JV, Kumar R, Dwivedi M, Verma AK, Chadha MS, Jain A. 2014. 2014. Molecular Characterization of Circulating Pandemic Strains of Influenza A Virus During 2012 to 2013 in Lucknow (India). J Med Virol 2014. DOI: 10.1002/jmv.23946 European Centre for Disease Prevention and Control (ECDC) Surveillance Report 2013, Stockholm. Influenza virus characterisation, summary Europe, December 2013. Available on: http:// www.ecdc.europa.eu/en/publications/Publications/influenza-viruscharacterisation-dec-2013.pdf. European Centre for Disease Prevention and Control (ECDC) Surveillance Report 2014, Stockholm. Influenza virus characterization, summary Europe, April 2014. Available on: http://www. ecdc.europa.eu/en/publications/Publications/ERLI-Net-report-April-2014.pdf. Falchi A, Amoros JP, Arena C, Arrighi J, Casabianca F, Andreoletti L, Turbelin C, Flahault Blanchon AT, Hanslik T, Varesi L. 2011. Genetic structure of human A/H1N1 and A/H3N2 influenza virus on Corsica Island: Phylogenetic analysis and vaccine strain match 2006–2010. PLoS ONE 6:e24471. DOI: 10.1371/journal. pone.0024471 Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289. Kilbourne E, Laver W, Schulman J, Webster R. 1968. Antiviral activity of antiserum specific for an influenza virus neuraminidase. J Virol 2:281–288. Latorre-Margalef N, Grosbois V, Wahlgren J, Munster VJ, Tolf C, Fouchier RAM, Osterhaus ADME, Olsen B, Waldenstro¨m J. 2013. Heterosubtypic immunity to influenza A virus infections in mallards may explain Existence of multiple virus subtypes. PLoS Pathog 9:e1003443. DOI: 10.1371/journal.ppat.1003443 Lee MS, Chen JS. 2004. Predicting antigenic variants of influenza A/H3N2 viruses. Emerg Infect Dis 10:1385–1390.
1275 Lin YP, Gregory V, Bennett M, Hay A. 2004. Recent changes among human influenza viruses. Virus Res 103:47–52. Mishra AC, Chadha MS, Choudhary ML, Potdar VA. 2010. Pandemic influenza (H1N1) 2009 is associated with severe disease in India. PLoS ONE 5:e10540. DOI: 10.1371/journal.pone.0010540 Pariani E, Amendola A, Ebranati E, Ranghiero A, Lai A, Anselmi G, Zehender G, Zanetti A. 2013. Genetic drift of influenza A(H3N2) virus hemagglutinin (HA) variants originated during the last pandemic turn out to be predominant in the 2011–2012 season in Northern Italy. Infect Genet Evol 13:252–260. Plotkin JB, Dushoff J. 2003. Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus. Proc Natl Acad Sci USA 100:7152–7157. Potdar VA, Chadha MS, Jadhav SM, Mullick J, Cherian SS, Mishra AC. 2010. Genetic characterization of the influenza A pandemic (H1N1) 2009 virus isolates from India. PLoS ONE 5:e9693.DOI: 10.1371/journal.pone.0009693 Skowronski DM, Janjua NZ, De Serres G, Dickinson JA, Winter A, Mahmud SM. 2013. Interim estimates of influenza vaccine effectiveness in 2012/13 from Canada’s sentinel surveillance network, January 2013. Euro Surveill 18:pii: 20394. Available online: http://www.eurosurveillance.org/ViewArticle. aspx?ArticleId ¼ 20394. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM. 2004. Mapping the antigenic and genetic evolution o f influenza virus. Science 305:371–376. Sobolev I, Kurskaya O, Susloparov I, Ilyicheva T, Shestopalov A. 2012. Molecular genetic analysis of influenza A/H3N2 virus strains isolated in Western Siberia in the 2010–2011 epidemic seasons. Infect Genet Evol 12:1694–1698. Stohr K. 2002. Influenza-WHO cares. Lancet Infect Dis 2:517. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. Taubenberger JK, Morens DM. 2013. Influenza viruses: Breaking all the rules. mBio 4:e00365–e00313. DOI: 10.1128/ mBio.00365–13 Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucl Acid Res 22:4673–4680. WHO. 2003. Fact sheet number 211. Influenza. Available on: http:// www.who.int/mediacentre/factsheets/fs211. World Health Organization (WHO). 2009. CDC protocol of real-time RTPCR for influenza A (H1N1). Available from: http://www.who. int/csr/resources/publications/swineflu/realtimeptpcr/en/index. htm. (accessed on April 30, 2009). WHO 2011. London Influenza Report 2011: Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Northern Hemisphere. 14th–17th February. Available on: http://www.who.cc.imr.mrc.ac.uk. WHO Global Influenza Surveillance Network. 2011. Manual for the laboratory diagnosis and virological surveillance of influenza. WHO surveillance Report: Summary of the influenza season in the WHO European region. Available on: http://www.who.int/ influenza/vaccines/virus/recommendations/2012_13_north/en/. World Health Organization (WHO). CDC protocol of real-time RTPCR for influenza A (H1N1). Available from:flu/realtimeptpcr/en/index html; http://www.who.int/csr/resources/publications/swine. (accessed on April 30, 2009). Wiley DC, Wilson IA, Skehel JJ. 1981. Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature 289:373–378. Wilson IA, Cox NJ. 1990. Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8:737–771. Wilson IA, Skehel JJ, Wiley DC. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolutions. Nature 289:366–373.
J. Med. Virol. DOI 10.1002/jmv
