in india , influenza surveillance has been carried out in multisite regional centres located at different parts of india ( pune and nagpur in west , delhi and lucknow in north , kolkata and dibrugarh in east , chennai , vellore and kerala in south)20 . the national institute of virology ( niv ) pune , monitors genetic variations and drug susceptibility in circulating influenza viruses received from regional centres . influenza viruses , isolated from 2004 to 2011 were analyzed genetically for known resistance markers by m2 and na gene sequencing . viruses : virus isolation and antigenic characterization was carried out at respective regional laboratories by inoculating nasal / throat swabs into madin darby canine kidney ( mdck ) cells21 . a total of a / h1n1 ( n=206 ) , a / h3n2 ( n=371 ) viruses were studied for amantadine resistance and a / h1n1 ( n=206 ) , a / h3n2 ( n=272 ) , pandemic ( h1n1 ) ( n= 493 ) and type b ( n=326 ) were studied for oseltamivir resistance . fluorescent neuraminidase inhibition assay ( ni ) : the half maximal inhibitory concentration ( ic50 ) to oseltamivir carboxylate was determined by a fluorescence - based neuraminidase inhibition assay22 . eleven sensitive and 50 resistant a / h1n1 virus isolates of 20072009 seasons , and 40 a / h3n2 sensitive viruses of 2009 - 2010 were tested along with reference viruses . the reference viruses were provided by who collaborating centre for reference and research on influenza , melbourne , australia . molecular detection of m2 and na gene rna extraction and rt - pcr - viral rna was extracted from culture supernatant using the qiaamp viral rna mini kit ( qiagen , usa ) . one step pcr [ reverse transcription ( rt)-pcr ) ] ( invitrogen superscript iii platinum kit country ) was performed to amplify m2 and na gene for detection of established molecular markers using published and gene - specific primers23242526 and ha1 gene using cdc ( unpublished ) for phylogenetic analysis . sequencing - the amplicons for m2 ( 270 bp covering amino acid positions 26 to 31 ) , n1 ( 253 bp covering 274 amino acid positions ) , n2 ( 1103 bp covering all known mutation sites from 118 to 294 amino acid position ) and type b na ( 1146bp covering all known mutation sites from 119 to 371 amino acid position ) genes were purified using pcr purification kits ( qiagen ) . dna sequencing was carried out using big dye terminator v 3.1 cycle sequencing ready reaction kit ( abi , foster city , ca , usa ) and unincorporated labelled ddntps ( dideoxy nucleotides ) were removed using dye - x removal column purification kit ( qiagen ) . the sequencing was done on abi 3730 dna analyzer ( abi , foster city , ca , usa ) and pair wise sequence alignment and protein translation was performed with mega 4 program27 . drug susceptibility markers : m2 gene sequencing of a / h1n1 ( n=206 ) and a / h3n2 ( n=371 ) and na gene sequencing of a / h1n1 ( n=206 ) , a / h3n2 ( n=272 ) , and type b ( n=326 ) were performed to analyze the established molecular markers . amantadine susceptibility of a / h1n1 and a / h3n2 viruses was examined for mutations at positions 26 , 27 , 30 , 31 or 34 in m2 gene ; for n1 subtype oseltamivir susceptibility was examined for amino acid position 274 ( n2 numbering ) and for n2 subtype amino acid positions 118 , 119 , 151 , 152 , 222 , 224 , 227 , 274 , 276 , 292 and 294 and 244 - 247 were examined . type b influenza positions 119 , 152 , 198 , 222 , 274 , 371 ( n2 numbering ) were checked . allelic real - time rt - pcr assay for detection of h274y in pandemic h1n1 - susceptibility to oseltamivir for pandemic ( h1n1 ) viruses was evaluated by allelic discrimination real - time rt - pcr . the primers and probes used in this assay were designed by south - east asia regional influenza reference laboratory , thailand28 . phylogenetic analysis of ha1 gene : phylogenetic analysis of a / h1n1 and a / h3n2 viruses used in this study along with who reference strains was done . mega version 4 was used for constructing neiughbour - joining ( nj ) trees using the kimura 's two - parameter distance model , with 1000 bootstrap replicates27 . seasonal a ( h3n2 ) viruses - as summarized in table i , a / h3n2 viruses circulating in 2004 were sensitive to amantadine . in 2005 , resistant viruses ( 23% ) , were introduced and were co - circulating along with sensitive viruses in pune , delhi and kolkata . at the same time , the viruses from chennai remained sensitive to amantadine . in 2006 , proportion of resistant viruses increased to 48 per cent in pune , delhi , chennai , kolkata and dibrugarh . in 2007 , it reached to 90 per cent and 2008 onwards it became 100 per cent in india . the most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . amantadine susceptibility testing of influenza a virus isolates ( 2004 - 2011 ) seasonal a ( h1n1 ) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 ( table i ) . a single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . in 2008 , three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b , a / brisbane/59/2007 with signature amino acid changes d45n , k149r ( h3 numbering ) . on the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y ( table ii ) . up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . in 2009 , resistant viruses were detected from delhi ( 13/14 ) , kolkata ( 7/7 ) , chennai ( 5/5 ) , pune ( 10/12 ) and similar findings were reported from kolkata . neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k , e119q , d151e , r152k , i222v , r224k , e227d , h274y , e276d , r292k , n294s and a  244 - 247 , and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . ( accession nos : kf 314400-kf314591 ; kf 314609-kf314682 ; kf014004,6,8,10 ) ( table ii ) . the analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . this mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain ( 0.6 - 3.0 nm ) allowed to confirm 15/23 ( 65.22% ) and 35/38(92.10% ) a / h1n1 virus isolates from 2008 and 2009 , respectively resistant to oseltamivir . the na sequence of oseltamivir - resistant viruses had t81n , d354 g when compared with a / brisbane/59/2007 . majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain ( 48 to 180 nm ) . table iii presents year - wise number of isolates tested along with mean ic50 ( nm ) , standard deviation ( nm ) , minimum ic50 ( nm ) and maximum ic50 ( nm ) . four isolates showed ic50 values 24.52 , 55.24 , 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . though asn(n ) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir ; our study showed that the sensitive h3n2 viruses had either asp ( d),/asn ( n ) or gly ( g ) at residue 151 . so we could not correlate the presence of d151 in four minor outliers with their ic50 values . ic50 ( half maximal inhibitory concentration ) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a , r152k , d198n / e , i222 t , h274y and r371k indicated that all type b isolates were sensitive to oseltamivir ( accession nos : kf314197- kf314399 ; kf364360-kf364482 ) ( table ii ) . pandemic ( h1n1 ) viruses - pandemic ( h1n1 ) isolates ( n=493 ) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir seasonal a ( h3n2 ) viruses - as summarized in table i , a / h3n2 viruses circulating in 2004 were sensitive to amantadine . in 2005 , resistant viruses ( 23% ) , were introduced and were co - circulating along with sensitive viruses in pune , delhi and kolkata . at the same time , the viruses from chennai remained sensitive to amantadine . in 2006 , proportion of resistant viruses increased to 48 per cent in pune , delhi , chennai , kolkata and dibrugarh . in 2007 , it reached to 90 per cent and 2008 onwards it became 100 per cent in india . the most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . amantadine susceptibility testing of influenza a virus isolates ( 2004 - 2011 ) seasonal a ( h1n1 ) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 ( table i ) . a single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . in 2008 , three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b , a / brisbane/59/2007 with signature amino acid changes d45n , k149r ( h3 numbering ) . on the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y ( table ii ) . up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . in 2009 , resistant viruses were detected from delhi ( 13/14 ) , kolkata ( 7/7 ) , chennai ( 5/5 ) , pune ( 10/12 ) and similar findings were reported from kolkata . neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k , e119q , d151e , r152k , i222v , r224k , e227d , h274y , e276d , r292k , n294s and a  244 - 247 , and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . ( accession nos : kf 314400-kf314591 ; kf 314609-kf314682 ; kf014004,6,8,10 ) ( table ii ) . the analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . this mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain ( 0.6 - 3.0 nm ) allowed to confirm 15/23 ( 65.22% ) and 35/38(92.10% ) a / h1n1 virus isolates from 2008 and 2009 , respectively resistant to oseltamivir . the na sequence of oseltamivir - resistant viruses had t81n , d354 g when compared with a / brisbane/59/2007 . majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain ( 48 to 180 nm ) . table iii presents year - wise number of isolates tested along with mean ic50 ( nm ) , standard deviation ( nm ) , minimum ic50 ( nm ) and maximum ic50 ( nm ) . four isolates showed ic50 values 24.52 , 55.24 , 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . though asn(n ) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir ; our study showed that the sensitive h3n2 viruses had either asp ( d),/asn ( n ) or gly ( g ) at residue 151 . so we could not correlate the presence of d151 in four minor outliers with their ic50 values . ic50 ( half maximal inhibitory concentration ) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a , r152k , d198n / e , i222 t , h274y and r371k indicated that all type b isolates were sensitive to oseltamivir ( accession nos : kf314197- kf314399 ; kf364360-kf364482 ) ( table ii ) . pandemic ( h1n1 ) viruses - pandemic ( h1n1 ) isolates ( n=493 ) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir seasonal a ( h3n2 ) viruses - as summarized in table i , a / h3n2 viruses circulating in 2004 were sensitive to amantadine . in 2005 , resistant viruses ( 23% ) , were introduced and were co - circulating along with sensitive viruses in pune , delhi and kolkata . at the same time , the viruses from chennai remained sensitive to amantadine . in 2006 , proportion of resistant viruses increased to 48 per cent in pune , delhi , chennai , kolkata and dibrugarh . in 2007 , it reached to 90 per cent and 2008 onwards it became 100 per cent in india . the most common mutation s31n responsible for amantadine resistance was observed in all a / h3n2 isolates . amantadine susceptibility testing of influenza a virus isolates ( 2004 - 2011 ) seasonal a ( h1n1 ) viruses - in contrast to a / h3n2 viruses most of a / h1n1 viruses remained sensitive to amantadine till mid 2007 ( table i ) . a single resistant h1n1 virus was first detected from delhi in august 2007 followed by vellore in november . in 2008 , three isolates from kolkata were found to be resistant to amantadine and two of these had common mutation s31n in the m2 gene and single virus a / k/0951720/2009 had less common l26f mutation in m2 gene along with h274y mutation in na gene leading to dual - resistant . phylogenetic analysis of ha1 gene of resistant a / h1n1 and a / h3n2 - phylogenetic analysis of ha1 coding region of a / h1n1 viruses showed that the amantadine resistance viruses grouped with clade 2b , a / brisbane/59/2007 with signature amino acid changes d45n , k149r ( h3 numbering ) . on the contrary sensitive h3n2 viruses amantadine resistant a / h3n2 viruses with double mutations at position s193f and d225n were grouped in clade n. a / h1n1 viruses - na gene sequencing of 206 a / h1n1 isolates was done to assess oseltamivir resistance mutation h274y ( table ii ) . up to november 2008 all a / h1n1 isolates were sensitive to oseltamivir while in december 2008 resistant seasonal a / h1n1 viruses were observed from chennai . in 2009 , resistant viruses were detected from delhi ( 13/14 ) , kolkata ( 7/7 ) , chennai ( 5/5 ) , pune ( 10/12 ) and similar findings were reported from kolkata . neuraminidase drug susceptibility testing of influenza a and influenza b a / h3n2 viruses - a total of 272 a / h3n2 isolates were studied for the presence of substitutions at positions r118k , e119q , d151e , r152k , i222v , r224k , e227d , h274y , e276d , r292k , n294s and a  244 - 247 , and none of the isolates had any of the reported oseltamivir - resistance conferring mutations . ( accession nos : kf 314400-kf314591 ; kf 314609-kf314682 ; kf014004,6,8,10 ) ( table ii ) . the analysis of the a / h1n1 na sequences revealed the presence of h274y mutation in 15/23 of 2008 isolates and 35/38 of 2009 isolates . this mutation is known to be associated with an oseltamivir resistance in na1 subtype virus . its identification in the na sequence of a / h1n1 virus isolates which exhibited high fluorescence ic50 values between 299.8 to 2513 nm when compared with wild type reference strain ( 0.6 - 3.0 nm ) allowed to confirm 15/23 ( 65.22% ) and 35/38(92.10% ) a / h1n1 virus isolates from 2008 and 2009 , respectively resistant to oseltamivir . the na sequence of oseltamivir - resistant viruses had t81n , d354 g when compared with a / brisbane/59/2007 . majority of a / h3n2 subtype viruses had ic50 values between 0.215 - 9.67 nm and remained susceptible to oseltamivir when compaired with ic50 values of resistant reference strain ( 48 to 180 nm ) . table iii presents year - wise number of isolates tested along with mean ic50 ( nm ) , standard deviation ( nm ) , minimum ic50 ( nm ) and maximum ic50 ( nm ) . four isolates showed ic50 values 24.52 , 55.24 , 50.36 and 27.64 nm resulting as minor outliers in fluorescent assay . though asn(n ) at position 151 has been associated with a reduction in the susceptibility of influenza a and b viruses to oseltamivir and/or zanamavir ; our study showed that the sensitive h3n2 viruses had either asp ( d),/asn ( n ) or gly ( g ) at residue 151 . so we could not correlate the presence of d151 in four minor outliers with their ic50 values . ic50 ( half maximal inhibitory concentration ) values for influenza viruses from india type b viruses - a total of 326 type b isolates were sequenced for na gene and lack of changes at positions e119a , r152k , d198n / e , i222 t , h274y and r371k indicated that all type b isolates were sensitive to oseltamivir ( accession nos : kf314197- kf314399 ; kf364360-kf364482 ) ( table ii ) . pandemic ( h1n1 ) viruses - pandemic ( h1n1 ) isolates ( n=493 ) were screened for h274y mutation in na gene by allelic discrimination and all were sensitive to oseltamivir the acquisition of a mutation in influenza a viruses conferring resistance to an antiviral agent may occur as a result of drug selection , spontaneous mutation or through genetic reassortment with another drug resistant influenza a virus . high prevalence of amantadine resistance in influenza a viruses was observed in countries , irrespective of its use56 . globally , a marked increase in amantadine drug resistant virus isolates was reported from asian countries especially for a / h3n2 subtype4 . however , majority of a / h1n1 viruses remained sensitive till 2006 season and 2 per cent of a / h1n1 viruses worldwide showed resistance to amantadine29 . in the present study , resistance to amantadine was mainly observed for a / h3n2 subtype , with identification of common mutation s31n of m2 gene . in the present study , resistant a / h1n1 viruses detected in india were 3 per cent in 2007 , 13 per cent in 2008 with s31n substitution and agreeing with global data4 . in 2009 , a single isolate from kolkata had less common mutation l26f in m2 gene and h274y mutation in na gene which conferred dual resistance . indian isolates with s31n mutation indicated that strains already harbouring drug resistant mutation were most likely introduced in the community . since in india the use of amantadine for influenza treatment was extremely rare during the study period ; the possibility of emergence of amantadine resistance due to drug pressure is remote . before 2008 , oseltamivir - resistance for any influenza type / subtype was not observed in india , due to negligible use of oseltamivir till may 2009 . widespread use of oseltamivir for suspected h1n1 patients in india started after june 2009 when cases of pandemic a ( h1n1 ) 09 were detected in india . prior to this , use of antivirals for influenza was negligible indicating that there was no potential for drug resistance due to selective pressure . however , no evidence was found to suggest that increased access to oseltamivir had promoted resistance or lack of it . a similar study from japan , where oseltamivir is more widely used , reported no significant effect on the occurrence of resistance30 . in india , a probable importation or detection of global 2008 oseltamivir - resistant seasonal a / h1n1 virus was documented nine months after it was first reported in europe in january 2008910 . oseltamivir resistance was observed for seasonal a / h1n1 influenza viruses with an increased frequency of 65.227 per cent in 2008 to 100 per cent in 2010 . these resistant viruses exhibited extremely high ic50 values , between 299.8 to 2513 nm and carried h274y mutation . it was well studied that oseltamivir susceptibility to influenza viruses differed significantly depending on the na subtypes . this was expected to occur due to minor structural differences between the na active sites of each na subtype viruses which could result in different oseltamivir binding affinities22 . the reduction in susceptibility to oseltamivir observed worldwide from 2005 - 2006 to 2007 - 2008 in a / h1n1 subtype was most probably a result of na antigenic drift from a / caledonia/20/1999 to a / brisbane/59/2007 . we detected oseltamivir resistance in n1 subtype from 2008 and it could be a result of same antigenic drift . oseltamivir was important for controlling the transmission and dissemination of pandemic viruses before a vaccine became widely available . no vaccine was available in india until one year after the first case of influenza a(h1n1 ) pdm09 was detected . this study showed that all 493 influenza a(h1n1 ) pdm09 viruses from 2009 and 2011 were sensitive to oseltamivir . even though indian isolates had permissive secondary mutations r194 g , e214 g , r222q and v234 m in na gene from 2005 - 2007 ; which were thought to have occurred before the emergence and spread of h274y nai resistant viruses31 ; a / h1n1 isolates remained sensitive to oseltamivir till 2007 . the role of observed additional mutations t81n and d354 g when compared with a / brisbane/59/2007 is still unclear . our findings were in agreement with the widespread and sustained transmission of oseltamivir resistant a / h1n1 viruses observed worldwide since 2007/2008910 . like influenza a viruses , influenza b viruses also undergo continuous evolution with accumulation of point mutations and reassortment events between co - circulating lineages . it was observed worldwide that the community acquired isolates of influenza b virus remained sensitive to nai drugs . however , sporadic cases of reduced nai susceptibility in the na inhibition assays have been reported from drug treated patients13 . our results showed that the influenza b viruses were sensitive to oseltamivir with agreement to global data . continued surveillance for antiviral drug resistance in influenza viruses is required to ensure that stockpiled neuraminidase inhibitors are effective and clinicians can be kept informed of the efficacy of neuraminidase inhibitors when treating patients for influenza .
background & objectives : recent influenza antiviral resistance studies in south east asia , europe and the united states reveal adamantane and neuraminidase inhibitor ( nais ) resistance . 
 this study was undertaken to evaluate antiviral resistance in influenza viruses isolated from various parts of india , during 2004 to 2011.methods:influenza viruses were analyzed genetically for known resistance markers by m2 and na gene sequencing . 
 influenza a / h1n1 ( n=206 ) , a / h3n2 ( n=371 ) viruses for amantadine resistance and a / h1n1 ( n=206 ) , a / h3n2 ( n=272 ) and type b ( n=326 ) for oseltamivir resistance were sequenced . 
 pandemic ( h1n1 ) ( n= 493 ) isolates were tested for h274y mutation by real time reverse transcription ( rrt)-pcr . randomly selected resistant and sensitive influenza 
 a / h1n1 and a / h3n2 viruses were confirmed by phenotypic assay.results:serine to asparagine ( s3 in ) mutation was detected in six isolates of 2007 - 2008 . 
 one dual - resistant a / h1n1 was detected for the first time in india with leucine to phenylalanine ( l26f ) mutation in m2 gene and h274y mutation in na gene . 
 a / h3n2 viruses showed an increase in resistance to amantadine from 22.5 per cent in 2005 to 100 per cent in 2008 onwards with s3 in mutation . 
 fifty of the 61 ( 82% ) a / h1n1 viruses tested in 2008 - 2009 were oseltamivir resistant with h274y mutation , while all a / h3n2 , pandemic a / h1n1 and type b isolates remained sensitive . 
 genetic results were also confirmed by phenotypic analysis of randomly selected 50 resistant a / h1n1 and 40 sensitive a / h3n2 isolates.interpretation & conclusions : emergence of influenza viruses resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for continuous monitoring of antiviral resistance .