a 16-year - old previously healthy girl had a fever at the hong kong international airport after her arrival from san francisco , california , usa , on june 11 , 2009 .
physical examination showed a temperature of 38.3c , a blood pressure of 117/66 mm hg , a pulse rate of 94 beats / min , and an oxygen saturation of 99% at room air .
she had a leukocyte count of 4.69  10 cells / l , an absolute neutrophil count of 2.36  10 cells / l , and a lymphocyte count of 1.74  10 cells / l .
a nasopharyngeal aspirate ( npa ) was positive for influenza a virus ( h1n1 ) nucleoprotein by immunofluorescence .
npa specimens on days 1 and 5 were positive for influenza a virus ( h1n1 ) m gene and swine - specific specific h1 gene by reverse transcription 
serum and midstream urine specimens obtained on day 2 were negative for influenza a virus ( h1n1 ) m gene by rt - pcr .
the patient refused antiviral therapy with oseltamivir because of fear of its potential side effects .
subsequent drug susceptibility testing showed that this isolate was resistant to oseltamivir ( 50% inhibitory concentration 197.5 nm ) , but susceptible to zanamivir , as determined by enzymatic assay ( table ) . *
50% inhibitory concentrations ( ic50s ) for oseltamivir and zanamivir were determined by using na - star influenza neuraminidase ( na ) inhibitor resistance detection kits ( applied biosystems , foster city , ca , usa ) according to the manufacturer s instructions
. ratios of 274h and 274y were evaluated by cloning neuraminidase ( na ) gene pcr products from nasopharyngeal aspirate ( npa ) samples or mdck cell  cultured virus into the ta vector ( invitrogen , carlsbad , ca , usa ) and sequencing clones containing the na gene .
mutations associated with resistance to na inhibitors , na sequences from the day 1 npa specimen and an mdck cell isolate were examined .
viral rna was extracted from npa and mdck cell supernatants by using reported procedures ( 7 ) .
rt - pcr was performed by using primers spanning position 274 of the na gene ( forward : 5-acacaagagtctgaatgtgcatgt-3 ; reverse : 5-gtctccgaaaatcccactgcatat-3 ) .
direct sequencing of pcr products was performed by using a bigdye terminator v3.1 cycle sequencing reaction kit on an abi prism 3730 dna analyzer ( applied biosystems , foster city , ca , usa ) .
sequences indicated that the na genes in the npa and mdck cell virus isolates contained an hy mutation at the na 274 ( h3 numbering , 275 in h1 numbering ) residue ( genbank accession no .
no other na mutations known to be associated with oseltamivir resistance were observed . further examination of sequences showed mixed populations ( t / c ) in the na gene from the npa specimen ( figure , panel a ) .
neuraminidase ( na ) 274y ( h3 subtype numbering ) gene mutation in influenza a pandemic ( h1n1 ) 2009 virus a / hong kong/2369/2009 isolated from a patient who arrived in hong kong , china , from san francisco , california , usa , on june 11 , 2009 .
a ) na sequence of virus amplified by reverse transcription  pcr and sequenced directly from a day 1 specimen of a nasopharyngeal aspirate from the patient .
nucleotide sequence represents identification of nucleotides by the sequencing machine , and the sequence trace represents the signal ( peak ) of each nucleotide in the sequencing reaction .
nucleotide coordinates ( 812 and 827 ) refer to the na gene sequence of pandemic ( h1n1 ) 2009 virus .
residue 274y encoded by the 3-nucleotide codon is indicated in boxes and the nucleotide substitution ( ct for amino acid change hy ) is indicated by arrows colors of curves match those of specific nucleotides .
estimation of 274h and 274y populations in the npa specimen was performed by cloning and sequencing pcr products .
the npa specimen contained approximately equal proportions of 274y and 274h ( 52.63% and 47.37% , respectively ) .
examination of sequences from the mdck cell isolate showed predominantly the 274y type , although a minor 274h peak was also observed ( figure , panel b ) .
cloning and sequencing of pcr products from the mdck virus isolate showed that 97.92% of the na genes were 274y , which suggests that the 274y population overtook the 274h population during mdck cell culture .
resistance to na inhibitors among seasonal strains of human influenza viruses ( a / h1n1 , a / h3n2 , and b ) has been rare until recently .
development of resistance after oseltamivir treatment has occurred in 0.33%5.5% of treated patients ( 8) .
oseltamivir resistance associated with the na 274y genotype was also observed in human infections with avian influenza a virus ( h5n1 ) ( 9,10 ) .
low levels of 274y quasi - species in avian influenza a viruses ( h5n1 ) from avian hosts has been reported ( 11 ) .
oseltamivir - resistant human influenza a viruses ( h3n2 and h1n1 ) have been found to replicate less efficiently than oseltamivir - susceptible strains in cell culture and animal models ( 1214 ) .
however , the na 274y resistant mutant in highly pathogenic avian influenza a virus ( h5n1 ) retained the high pathogenicity of wild - type virus in mammalian species ( 15 ) . in 2007 ,
an na h274y oseltamivir - resistant variant of seasonal influenza a virus ( h1n1 ) was detected in norway ( 6 ) .
this virus has now become the dominant virus population globally , overtaking oseltamivir - susceptible influenza a virus ( h1n1 ) .
the molecular basis for the 274y variant in seasonal influenza a virus ( h1n1 ) virus and the mechanism by which this resistant variant became the dominant population remain unknown .
lack of general immunity to pandemic ( h1n1 ) 2009 virus in the human population , combined with the inherent adamantane resistance of the virus , indicates that na inhibitors constitute the primary treatment regimen for susceptible patient groups and those in whom severe diseases develop during the current pandemic . there is great concern that an oseltamivir - resistant variant of pandemic ( h1n1 ) 2009 virus may emerge and circulate in a manner similar to oseltamivir - resistant seasonal influenza a virus ( h1n1 ) . the patient in this study
detection of mixed populations of 274y and 274h in the npa specimen before antiviral treatment suggests that the mutation occurs naturally , either before or during infection .
although no experimental data exist that show the growth properties of this resistant variant , examination of the quasi - species population in the cell culture  propagated virus isolate showed that the 274y variant has become the dominant population .
this finding implies that the 274y mutation does not compromise replication of pandemic ( h1n1 ) 2009 virus in vitro .
quarantine procedures adopted by the hong kong special administrative region in china during the early containment phase might have limited transmission of this variant virus .
knowledge of this virus is still limited , and characterization of transmission properties of this resistant variant in in vitro and in vivo models is needed .
moreover , pandemic ( h1n1 ) 2009 virus should be closely monitored for emergence of resistant variants .
 resistance to oseltamivir was observed in influenza a pandemic ( h1n1 ) 2009 virus isolated from an untreated person in hong kong , china . 
 investigations showed a resistant virus with the neuraminidase ( na ) 274y genotype in quasi - species from a nasopharyngeal aspirate . 
 monitoring for the naturally occurring na 274y mutation in this virus is necessary . 
The Study
Conclusions