Infection, Genetics and Evolution 16 (2013) 419–425

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Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

Full genome analysis and characterization of influenza C virus identified inEastern India

Tapasi Roy Mukherjee, Anupam Mukherjee, Satarupa Mullick, Mamta Chawla-Sarkar ⇑National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India

a r t i c l e i n f o

Article history:Received 13 November 2012Received in revised form 18 February 2013Accepted 22 February 2013Available online 20 March 2013

Keywords:Influenza CWhole genomeIndiaPhylogenetic analysisAcute respiratory illness

1567-1348/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.meegid.2013.02.014

⇑ Corresponding author. Address: Division of VirCholera and Enteric Diseases, P-33, C.I.T. Road, Sche700010, West Bengal, India. Tel.: +91 33 2353 7470;

E-mail addresses: sarkarmc@icmr.org.in, chawlamSarkar).

a b s t r a c t

In tropical countries of Asia, like India, approximately 0.5 million children of <5 years of age die annuallydue to acute respiratory illness (ARI). Of common respiratory pathogens, influenza viruses (A & B) areassociated with annual worldwide epidemics; while the information on influenza C virus is inadequate.During January 2011 through December 2012, 2737 nasal and/or throat swabs were collected frompatients reporting at outpatient department of hospitals in eastern India with ARI. Nucleotide sequencingwas carried out using gene specific primers followed by pair-wise sequence alignments, multiple align-ments, construction of phylogenetic tree and analysis of deduced amino acid sequences. Study revealsthat, out of 2737 samples, 1616 (59.04%) were positive for one or more respiratory viruses; of which23.72% were positive for influenza A and B viruses. From influenza A & B negative samples, influenza Cvirus was screened and detected with a frequency of 0.18%. Phylogenetic analysis showed that the HE,matrix, NS, PB1 and PB2 gene of the studied strain (C/Eastern-India/1202/2011) possessed a close relat-edness to C/Yamagata/26/81 like strains. The P3 gene shows proximity with C/Mississipi/80 like strainswhereas NP gene revealed similarity with C/Miyagi/1/93 like strains. The outcome of the whole genomeanalysis of the strain C/Eastern-India/1202/2011 provided useful information regarding genetic diversityof influenza C strains in India.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Influenza C (Inf C) virus is widely distributed throughout theworld (Homma et al., 1982; Nishimura et al., 1987) as an agentof upper respiratory tract infections (URTI) in young children(Katagiri et al., 1983, 1987) as well as in adults (Homma et al.,1982; Katagiri et al., 1983, 1987; Matsuzaki et al., 1990). The riskof complications with lower-respiratory-tract illness (LRTI) suchas bronchitis and pneumonia (Moriuchi et al., 1991) is high inchildren younger than 2 years of age (Matsuzaki et al., 2006).Majority of humans acquire antibodies to the virus early in theirlife (Homma et al., 1982; Nishimura et al., 1987; Jennings, 1968;Manuguerra et al., 1992, 1994; Motta et al., 2000), which againsuggests that infection with this virus is common in childhood.Katagiri et al. further confirmed this fact through their studies ondetection of three outbreaks of type C influenza during a 3-yearstudy in a children’s home (Katagiri et al., 1983, 1987); theyreported that most of the children were <3 years old and someemployees of that home also showed fever and mild upper

ll rights reserved.

ology, National Institute ofme-XM, Beliaghata, Kolkata

fax: +91 33 2370 5066.70@gmail.com (M. Chawla-

respiratory tract (URT) symptoms associated with antibodyresponse. Furthermore most children infected with Inf C virushad fever (temperature of >38 �C), and were actually diagnosedas Inf A or Inf B infection (Matsuzaki et al., 2006); since it is difficultclinically to differentiate the three subtypes.

The genome of the Inf C virus consists of seven RNA segments,which encode three polymerase proteins (PB2, PB1, and P3),hemagglutinin-esterase glycoprotein (HE), nucleoprotein (NP),matrix (M1) protein, and two nonstructural proteins (NS1 andNS2) (Lamb and Krug, 2001). Comparison of the RNA genomes ofvarious strains by oligonucleotide mapping (Kawamura et al.,1986) and nucleotide sequencing (Buonagurio et al., 1985, 1986)indicated that ecology of Inf C virus is characterized by the pres-ence of many co-circulating strains at a time (Buonagurio et al.,1985, 1986), though it changes much more slowly than humanInf A viruses. Moreover, Matsuzaki et al. identified some importantepidemiological features of Inf C virus; such as antigenically andgenetically different strains co-circulate in a community (Matsuzakiet al., 1994), genetic reassorting occurs frequently among strains(Matsuzaki et al., 2003) and newly emergent reassortant virusesbecome predominant (Matsuzaki et al., 2003, 2004). Thus, reas-sortment characterized by exchange of genome segments betweentwo or more different Inf C virus strains occur not only in vitro(Nishimura et al., 1994) but also in nature (Alamgir et al., 2000;Matsuzaki et al., 2000; Peng et al., 1996; Tada et al., 1997).

420 T. Roy Mukherjee et al. / Infection, Genetics and Evolution 16 (2013) 419–425

However, the epidemiological significance of genetic reassortmentin Inf C viruses is unknown. Although, it has been nearly 65 yearssince the Inf C virus was first isolated in 1947 (Taylor, 1949); butstill there is scarcity of information on Inf C viruses in Indiansubcontinent.

In this study we are reporting the whole genome analysis of atype C influenza strain (C/Eastern-India/1202/2011) detectedduring July 2011 in eastern India.

2. Materials and methods

2.1. Sample collection and screening

Nasal and/or throat swabs were collected from outpatientdepartment of hospitals in Kolkata, West Bengal; with influenzalike illness (ILI) (sudden onset of fever, nasal discharge, sorethroat/cough, fatigue/body ache, difficulty in breathing). The typeC influenza virus characterize in this study was identified from asample of a 2.6 years old female patient who was suffering fromfever and cough.

2.2. RNA extraction and screening of clinical samples for influenzavirus

Viral RNA was extracted using commercially available QIAampViral RNA Mini Kit (Qiagen GmbH, Hilden, Germany) as per manu-facturer’s instructions. Initial detection of Inf A and Inf B viruseswas carried out as described previously by our group (Agrawalet al., 2009; Roy et al., 2011). Detection of Inf C viruses was carriedout with conventional RT-PCR with HE (Hemagglutinin-esterase)gene specific primers (Table 1).

2.3. Full length amplification and sequencing of viral genes

Nucleotide (nt) sequencing was carried out in an ABI Prism3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA,

Table 1Primer pairs used for amplification of genes of influenza C strains.

Gene Primer name Sequence (50-30)

HE C/HE-F (+)TAA AGA TAT GCC TTC AAA AC/HE-PR (�)TTT TTC TTT CAT GTC AAA GC/HE-PF (+)AAG ATT CCT TTT AAT GCC TC/HE-R (�)AAT ATA TCA TCC ATT GTT C

Matrix C/M-F (+)ATT TCA AAA CAA TGG CAC AC/M1-R (�)TTA TAA CAA CTA ACC ATT TC/M2-R (�)TAG CAA GGG GAT TTT TTC

NP C/NP-F (+)ATT TTC AAA AAT GTC TGA CC/NP-PR (�)AAA TTG GTA AAC TCA TCTC/NP-PF (+)TTA AAG CCT CAG ATA ACC AC/NP-R (�)TAG CAA TAC AAC AGT TGA

NS C/NS-F (+)AAG CAG GGG TAC TTT TTC AC/NS1-R (�)TAA GAT CTT CAG AAT TTT GC/NS2-R (�)AGC AAG GGG TTT TTT AAC

P3 C/P-F (+)ATG TCG AAA ACT TTT GCC GC/P-PR (�)TCT GTT GTT ATT ACA TCT TC/P-PF (+)TAA AAA GAT CCC AGA GATC/P-R (�)AAG GGG ATT TTT TCT TAT A

PB1 C/PB1-F (+)AAG CAG AGG ATT ATG GAAC/PB1-PR1 (�)TTT TGC TTT CTT CTC ATT AC/PB1-PF1 (+)TAT GTG AGA AAT TAA AAGC/PB1-PR2 (�)TAT TTG TCT TAA TAA TTG AC/PB1-PF2 (+)TTG TAT CCA ATC TTG CTA TC/PB1-R (�)TAA CAA AAA TAT GTG CAA

PB2 C/PB2-F (+)TCT TCT ATT GAC AAT AGC AC/PB2-PR1 (�)AAA ACA GGC AAG AAT CTAC/PB2-PF1 (+)AAA GTA GAT TCT TGC CTG TC/PB2-PR2 (�)AAA TGC TTC AAG ATT ATCC/PB2-PF2 (+)AAA AGA AAA AAT TCA CCTC/PB2-R (�)AGT AGC AAG AGG ATT TTT

USA) using gene specific primers (Table 1). Nucleotide and proteinsequence BLAST search was performed using the National Centrefor Biotechnology Information (NCBI, National Institutes of Health,Bethesda, MD) Basic Local Alignment Search Tool (BLAST) server onGenBank database release 143.0 (Schäffer et al., 2001).

2.4. Phylogenetic analysis

Pairwise sequence alignments were performed using LALIGNsoftware. Multiple alignments were done with Clustal W programwhich is available at DDBJ software. Phylogenetic tree wasconstructed by the neighbor-joining method (Saitou and Nei,1987) using the MEGA program, version 5.10 and to cross analysethe phylogenetic trees, they were constructed separately usingphylogeny.fr online software (Dereeper et al., 2008). Sequencealignment was performed by MUSCLE alignment (Edgar, 2004).

2.5. GenBank accession number

The nucleotide sequences were submitted to the GenBank un-der the accession numbers JX947861, JX947862, JX947863,JX947864, JX947865, JX947866, and JX947867 for HE, NP, P3,PB1, PB2, matrix and non-structural genes respectively.

3. Results

3.1. Identification of the strain

During 2010 through 2011, 2737 samples were screened.Among them 1616 (59.04%) were positive for one or more respira-tory viruses and of which 23.72% were positive for Inf A and Inf Bviruses. From Inf A & Inf B negative samples (n = 2139), fivesamples were positive for Inf C virus (0.18%). After screening byPCR, the MDCK cells were infected with all five influenza C strains.Only C/Eastern-India/1202/2011 which detected during July 2011

Position Product size (bp)

GC AAG TG 1–26 917CT CC 917–895GA AAG 866–889 1084

1951–1933TG AAA TAC TG 1–29 753CA TTG CC 753–728

AAG 1161–1141 1161AG ACG 1–24 909

TTC AGA G 909–885AC AAA ATG AC 821–849 937

TCA TAA AAT ATG 1757–1728AA AT 1–23 806GA TTC CC 806–781

TTT G 924–903 924AA ATA G 1–25 1088

GT GTT AGA G 1088–1061TTC TCT AAC AC 1041–1069 1113AT GAT CAA TC 2153–2125ATC AAC 1–24 861

CC ACC 861–838AAA GCG G 803–827 813CA TTG CAG C 1615–1588

GG AAT TG 1517–1542 808GTA GGA GG 2324–2298AA GGA ATA C 1–28 678CTT TCC 678–655TT TCG 657–680 908

AGA GAG C 1564–1540TGA GCT C 1519–1543 818AGT TAG 2336–2313

T. Roy Mukherjee et al. / Infection, Genetics and Evolution 16 (2013) 419–425 421

showed a high titer in HA test; while the rest four has shown neg-ligible titer. Therefore further analysis was performed with C/East-ern-India/1202/2011 only.

3.2. Phylogenetic analysis of individual RNA segments of the strain C/Eastern-India/1202/2011

As the detection rate of Inf C virus in eastern India is very low(0.18%), complete coding region sequences of all the 7 genes of asingle representative strain (C/Eastern-India/1202/2011) wereperformed in order to obtain knowledge about the origin and todetermine the genome composition of Inf C virus. The phylogenetictrees of individual genes were constructed using the referencesequences from the genbank database.

From the phylogenetic analysis of the HE gene (Fig. 1A); weobserved that, the reference strains form five different clades (cladeI-V). Clade-I possesses a combination of older and recent strains.The oldest strain of this clade was C/Yamagata/64 and the recentstrain was C/Catalonia/2588/2010. Clade-II was represented by C/Paris/1/67 and C/Ann Arbor/1/50. As shown in the phylogeneticanalysis C/Sao Paulo/378/82 was the oldest representative ofclade-III. C/Johannesburg/4/67, C/Kansas/1/79, C/Aichi/1/81, C/Miy-agi/7/91 formed clade-IV, whereas C/Greece/1/79 and C/Saitama/2/2000 belonged to clade-V. The strain C/Eastern-India/1202/2011was a member of clade-I and possesses a close relatedness to C/Yamagata/26/81 like strains. Similar results were observed whenanalysis was performed with the online software phylogeny.fr

Fig. 1. Phylogenetic comparison based on nucleotide sequences of HE (Fig. 1A), Matrix (Ffrom Kolkata, eastern India. The Kolkata strain was marked as ‘d’. The tree was created

(Supplementary Fig. 1A). Maximum homology for the nucleotidesequences of C/Eastern-India/1202/2011 showed 94.5% and 95.2%whereas the amino acid exhibits only 91.2% and 89.8% with the Jap-anese strain C/Sapporo/71 and C/Yamagata/64, respectively.

Fig. 1B shows the phylogenetic analysis of matrix protein gene.The phylogenetic tree was divided into two distinct clades (clade I& II). The eastern Indian strain belonged to clade-I and shows closeproximity with C/Yamagata/26/81 like strains. Clade-II was repre-sented by the older strains like C/Ann Arbor/1/50, C/Johannesburg/66 and C/Aomori/74 etc. Similar type of phylogenetic tree wasgenerated when data was uploaded in the online software (phylog-eny.fr) (Supplementary Fig. 1B).

The phylogenetic analysis of the nucleoprotein gene (NP)(Fig. 1C and Supplementary Fig. 1C) shows that there were sevendifferent clades. Clade-I consisted of C/Nara/82 like strains. C/Yamagata/81 like strains forms clade-II whereas C/Johannesburg/1/66 was the only representative of clade-III. Clade-IV consists ofC/Kanagawa/1/76 like strains. Clade-V has C/Aichi/1/81 like strains.Strain C/Eastern-India/1202/2011was one of a member of clade-VI,which consists of C/Miyagi/1/93 like strains.

Fig. 1D represents the phylogenetic analysis of non-structuralgenes with 27 reference strains along with Inf C strain used in thisstudy. The tree consists of two distinct clades (clade-I & II). Clade-Iconsists of five distinct subclades, whereas clade-II contains fourdiscrete subclades. Clade-II mainly contains the older strains, likeC/Ann Arbor/1/50, C/Nara/82, C/Aichi/1/81, C/Kanagawa/1/76etc.; on the contrary clade-I have a combination of both older as

ig. 1B), NP (Fig. 1C) and non structural genes (Fig. 1D) of C/Eastern-India/1202/2011by using neighbor-joining method with 1000 bootstrap replicates.

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well as the recent strains. C/Eastern-India/1202/2011 belonged toclade-I, with non-structural gene from a C/Yamagata/26/81 likestrains. Phylogenetic analysis performed with phylogeny.frsoftware also revealed that the strain C/Eastern-India/1202/2011possessed a non-structural gene from a C/Yamagata/26/81 likestrains (Supplementary Fig. 1D).

The phylogenetic tree of the P3 gene (Fig. 2A, SupplementaryFig. 2A) was divided into two different clades (clade-I & II). A com-bination of older and recent strains clustered in clade-II, howeverclade-I has only the older strains. The strain C/Eastern-India/1202/2011 was in clade-II and revealed close proximity with C/Miyagi/1/97 and C/Yamagata/2/99, which were again C/Mississi-pi/80 like strains (Matsuzaki et al., 2003).

The phylogenetic analysis of the PB-1 and PB2 gene (Fig. 2Band C, respectively) showed that the strains were clusteredmainly in two distinct clades (clade-I & II). For PB1 gene C/East-ern-India/1202/2011 clustered with C/Yamagata/2/99 of clade-IIwhereas for PB2 gene it clustered in clade-I. The strain C/East-ern-India/1202/2011 acquires both the polymerase genes froma C/Yamagata/26/81 like strain. Similar results were observedwith the alternative analysis in phylogeny.fr software (Supple-mentary Fig. 2B and C).

Fig. 2. Phylogenetic comparison based on nucleotide sequences of P3 (Fig. 2A), PB1 (Fig. 2The Kolkata strain was marked as ‘d’. The tree was created by using neighbor-joining m

3.3. Comparison of amino acid sequences of individual segments of thestrain C/Eastern-India/1202/2011

Comparison of the amino acid sequences of the HE gene of theKolkata strains with C/Ann Arbor/50 revealed multiple changes inamino acids (Table 2A). Amino acid changes were observed atpositions 88, 135, 139, 155, 166, 180, 182, 202, 204, 206, 207, 208,212, 217, 292, 331, 337, 340, 361, 401, 402, 477, 507, 585, 597 and629. Amino acid changes were observed at three different positionsin NP gene (Table 2B). Position 298 and 415 has threonine instead ofalanine of C/Ann Arbor/50. At position 399 alanine (C/Eastern-India/1202/2011) replaced glutamic acid (C/Ann Arbor/50).

Comparison of the amino acid sequences of C/Eastern-India/1202/2011 with C/Ann Arbor/50 revealed amino acid changes at six differ-ent positions (aa 47, 64, 65, 67, 68, 69) of the NS gene (Table 2C).Table 2D represents comparison of amino acid changes of the P3 geneof C/Eastern-India/1202/2011 with that of C/Ann Arbor/50. Aminoacid changes were observed at six different positions. In PB1 genethere was five positions where amino acid changes observed in re-spect to C/Ann Arbor/50 (Table 2E). In PB2 gene changes in amino acidwas observed at positions 3, 311, 407, 436, 657, 674, 726 and 737(Table 2F). No amino acid changes were observed in the matrix gene.

B) and PB2 (Fig. 2C) genes of C/Eastern-India/1202/2011 from Kolkata, eastern India.ethod with 1000 bootstrap replicates.

Table 2Comparison of amino acid (aa) sequences of the HE (A), NP (B), NS (C), P3 (D), PB1 (E) and PB2 (F) gene of C/Eastern-India/1202/2011 with the oldest strain C/Ann Arbor/1/50. Theaa position corresponds to strain C/Ann Arbor/1/50. The ‘�’ represents unavailability of respective sequences in the database.

Strain Amino acid positions

88 135 139 155 166 180 182 202 204 206 207 208 212 217 292 331 337 340 361

2A.C/Ann Arbor/1/50 A H N N M A A E N S V E K F L I E H YC/Yamagata/26/81 A H D S M V K E K L E Q E F L I K Y YC/Eastern-India/1202/2011 E Q D S L V K K K L E N E S I V Q Y D

Amino acid positions

401 402 477 507 585 597 629

C/Ann Arbor/1/50 T E V I V N SC/Yamagata/26/81 T E E I I N SC/Eastern-India/1202/2011 A K E T I D R

2B.Amino acid positions

298 399 415

C/Ann Arbor/1/50 A E AC/Eastern-India/1202/2011 T A T

2C.Amino acid position

47 64 65 67 68 69

C/Ann Arbor/1/50 K G E L F NC/Yamagata/26/81 R G E L F NC/Eastern-India/1202/2011 R E I R R S

2D.Amino acid position

55 58 193 353 380 708

C/Ann Arbor/1/50 T S K V K IC/Yamagata/2/99 A N – – – –C/Eastern-India/1202/2011 A N E I R T

2E.Amino acid position

76 88 181 691 751

C/Ann Arbor/1/50 D S V M IC/Yamagata/26/81 N G – – –C/Eastern-India/1202/2011 N G I I L

2F.Amino acid position

3 311 407 436 657 674 726 737

C/Ann Arbor/1/50 F L K S L F R KC/Yamagata/26/81 – – – – – – – –C/Eastern-India/1202/2011 L P Q T F L G N

T. Roy Mukherjee et al. / Infection, Genetics and Evolution 16 (2013) 419–425 423

4. Discussion

Influenza virus is the major pathogen, causing serious concernfor the respiratory tract infection. Although the sample size islow but still this present study could suggest that infection dueto Inf C virus mainly occurred in children belonging to the agegroup 0–5 years. This corroborates with the previous reportswhere Matsuzaki et al. in 2006 suggested that apart from the infec-tion due to Inf A and Inf B viruses; Inf C virus also plays a role incausing respiratory tract diseases, mainly in children under 6 yearsof age. Previous reports also suggested that, humans acquire anti-bodies against Inf C virus by the age of 7–10 years (Homma et al.,1982; Jennings, 1968); moreover, newborns have maternal anti-bodies to Inf C virus, which disappear by the age of 6 months(Homma et al., 1982). Since, the nucleotide changes in the HE genedo not accumulate with time (Buonagurio et al., 1985); antigenic-

aly the virus became much more stable than Inf A and Inf B viruses(Matsuzaki et al., 2003; Chakraverty, 1978) and the evolution ratebecame slow. Although there is antigenic variation among Inf Cviruses isolated in different time, antigenic drift characterized bythe emergence of successive antigenic variants does not occur(Sugawara et al., 1986), and thus the virus can survive for a longperiod of time (P9 years) without changing its antigenicity(Ohyama et al., 1992) and genome composition. In this study, thestrain C/Eastern-India/1202/2011 revealed several amino acidchanges in its HE gene with respect to C/Ann Arbor/50. Suchchanges may lead to alteration in the side chain polarity as wellas the charge of side chain at ph. 7.4. At the position 88, 207 and477, a non-polar, neutral amino acid of C/Ann Arbor/50 has beenreplaced with an acidic, polar amino acid bearing negative chargein its side chain whereas non-polar, neutral to basic, polar, positiveshift was observed at 182nd position. A polar, neutral amino acid at

424 T. Roy Mukherjee et al. / Infection, Genetics and Evolution 16 (2013) 419–425

139, 361, 401 and 597 has been replaced with an acidic, polar andnegatively charged amino acid. The amino acid position 202 and402, C/Ann Arbor/50 has an acidic, polar, negatively charged aminoacid; but C/ Eastern-India/1202/2011 have a basic, polar, positivelycharged amino acid at that particular position. At position 212 anopposite shift occurred, where a basic, polar, positively chargedamino acid has been replaced with acid, polar and negativelycharged amino acid. Muraki et al. also reported that many escapemutants selected with anti-HE monoclonal antibodies, having ami-no acid substitutions in the immunodominant region on HE, exhib-ited a change in receptor-binding activity (Muraki et al., 1996;Matsuzaki et al., 1992). The Kolkata strain also reveals amino acids(206 and 212) conserved for decrease in hemagglutinin activity.However after the occurrence of lineage separation in 1996, im-mune selection does not seem to have played a significant role inthe evolution of HE protein. It is possible that HE protein can nolonger evolve in response to antibody pressure because of a highdegree of functional constraint on the change of its immunodomi-nant region. Muraki et al. suggested that Inf C virus possess multi-ple genetic variants of different evolutionary lineages whichcoexist in nature (Muraki and Hongo, 2010). Though Inf C virus issometimes isolated from pigs (Guo et al., 1983), the viruses are alsomaintained within the human population (Kimura et al., 1997) andreassortment event may take place in patients infected with differ-ent strains of Inf C virus (Matsuzaki et al., 2004). The strain C/East-ern-India/1202/2011 has NP gene like in C/Miyagi/1/93 like strain,P3 gene from a C/Miyagi/1/97 like strain which is again a C/Mis-sissipi/80 like strain and all other genes (HE, Matrix, NS, PB1 andPB2) from a C/Yamagata/26/81 like strain. Occurrence of such mul-tiple reassortment events suggest that strain may have acquiredgene segments during evolution. However the timeline of reassort-ment cannot be determined in this study due to low frequency ofInf C strain in this region. Although Inf C virus usually causes a mildupper respiratory illness in the general population, regular virolog-ical surveillance and complete genomic characterization wouldprovide valuable information about the incidence and the role ofInf C virus in acute viral respiratory infections. Furthermore, genet-ic lineage identification would offer additional data for epidemio-logical purpose.

Acknowledgements

The study was supported by financial assistance from IndianCouncil of Medical Research (ICMR), New Delhi and Centers forDisease Control and Prevention, USA (Co-operative agreementU50CI 02440704).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2013.02.014.

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