as part of ongoing surveillance , respiratory samples are routinely collected from patients seeking treatment with respiratory illness in clinics and hospitals of hong kong .
virus isolation is performed according to a standard protocol ( 3 ) . from january 2006 through june 2008 , a total of 827 influenza viruses ( h1n1 ) were tested for oseltamivir resistance by an enzyme inhibition assay ( na - star ; applied biosystems , foster city , ca , usa ) or by nucleotide sequencing of the na gene to detect the h274y mutation .
the isolates were also tested for resistance to amantadine by an in - house  designed pcr ( protocol available on request ) and nucleotide sequencing of the matrix ( m2 ) gene segment .
a 575-nt fragment of the na gene and a 199-nt fragment of the m2 gene were amplified by the designed primers n11h and n12h , and ma 692 and ma 891 ( available on request ) , respectively , and sequenced by using a genetic sequencer 3100 or 3130xl ( applied biosystems ) .
sequences obtained were aligned by using simmonics ( 4 ) and analyzed by mega version 2.1 ( 5 ) .
increasing amantadine resistance , from 107 ( 48.0% ) of 223 isolates in 2006 to 12 ( 85.7% ) of 14 in 2007 , was associated with clade 2c a / hong kong/2652/2006-like viruses ( 6 ) ; none were resistant to oseltamivir .
a subtype h1n1 virus resistant to oseltamivir , but sensitive to amantadine , was first detected in hong kong in january 2008 , which coincided with the emergence of clade 2b a / brisbane/59/2007-like viruses . from january through june 2008 , 87 ( 12.5% ) of 697 isolates tested were resistant to oseltamivir .
they all carried the h274y mutation in the na gene and showed an 1,000-fold reduction in susceptibility to oseltamivir ( 50% inhibitory concentration values increased from 0.5 nmol / l to 500
analysis of the m2 genes showed that 3 ( 3.4% ) of 87 oseltamivir - resistant isolates also carried the s31n mutation associated with amantadine resistance .
these 3 viruses were isolated from 2 infants and an elderly woman in different hospitals during 2008 ( march , april , and june , respectively ) . to eliminate the possibility of mixed strains
, we designed a real - time pcr using single nucleotide polymorphisms to detect antimicrobial drug  susceptible strains ( with s31 and h274 ) and drug resistant strains ( with s31n and h274y ) in the samples ( method available on request ) .
results showed that the 3 isolates did not possess s31 and h274 . to further study the genetic mechanism of emergence of antiviral drug resistance among subtype h1n1 viruses , we performed nucleotide sequencing on a partial segment of the hemagglutinin ( ha ) gene on 84 ( 97% ) of 87 oseltamivir - resistant and 37 ( 5% ) of 697 oseltamivir - susceptible viruses isolated in our laboratory in 2008 .
pcr and dna sequencing of the ha gene were carried out by using the primers h11 and h12 , which flank a fragment of 612 nt of the ha segment ( 7 ) .
sequences were obtained from 121 virus isolates ( table ) . * ha , hemagglutinin ; na , neuraminidase ; m2 , matrix . 
clade 2b or clade 2c indicates the gene specified has a nucleotide homology > 99% to that of a / brisbane/59/2007 or a / hong kong/2652/2006 virus , respectively .
of 37 oseltamivir - susceptible viruses , analysis of the ha , na , and m2 genes showed that 20 were susceptible to amantadine and similar to the clade 2b a / brisbane/59/2007 virus ( genbank accession no .
fourteen were similar to clade 2c a / hong kong/2652/2006 virus ( genbank accession no .
cy031342 ) , an amantadine - resistant virus isolated in our laboratory in 2006 which remained prevalent in hong kong in 2007 and 2008 .
three isolates had ha and na genes similar to those of clade 2b but carried an amantadine - resistant m2 gene similar to that of the a / hong kong/2652/2006 virus . among 84 oseltamivir - resistant strains , 81 remained susceptible to amantadine and had ha and m2 genes similar to those of clade 2b a / brisbane/59/2007 virus but carried a single point mutation h274y in the na gene .
the other 3 isolates were resistant to oseltamivir and amantadine ; they had similar ha and m2 genes to those of clade 2c a / hong kong/2652/2006 virus but had the h274y mutation in their na gene .
one of the viruses had an na gene similar to that of the oseltamivir - resistant clade 2b a / brisbane/59/2007 virus , whereas the other 2 viruses had na genes similar to those of amantadine - resistant clade 2c a / hong kong/2652/2006 virus ( figure ) .
these results suggest that the 3 isolates acquired amantadine and oseltamivir resistance by different mechanisms .
a clade 2c a / hong kong /942/2008 virus acquired an na gene carrying the h274y mutation by reassortment with an oseltamivir - resistant clade 2b virus ; the other 2 clade 2c viruses , a / hong kong /1052/2008 and a / hong kong /1313/2008 , acquired oseltamivir resistance through a spontaneous h274y mutation in the na gene .
phylogenetic analysis of the hemagglutinin gene ( a ) , neuraminidase gene ( b ) , and matrix gene ( c ) of influenza virus ( h1n1 ) .
the 3 clade 2c viruses with double resistance are indicated by circles , the 3 clade 2b viruses with amantadine resistance are indicated by triangles , and a / brisbane/59/2007 and a / hong kong/2652/2006 are indicated by squares .
to study the effect of the resistant gene on the replication efficiency of the viruses , we conducted experiments using amantadine- and oseltamivir - resistant viruses in mdck cells .
briefly , we placed in quadruplicate on mdck cells one 50% tissue culture infectious dose per 0.1 ml of subtype h1n1 viruses with different antiviral drug  resistance patterns ( amantadine - resistant only , oseltamivir - resistant only , amantadine- and oseltamivir - resistant , and amantadine- and oseltamivir - susceptible ) , incubated the cultures at 33c , and examined the cytopathic effect ( cpe ) daily for 7 days . by observing the timing of cpe appearance and relative progressive rate of cpe
, we found no substantial difference in growth efficiency and similar replication patterns for the 4 different types of subtype h1n1 strains .
reassortment of gene segments likely occurred among clade 2b and clade 2c subtype h1n1 viruses while they were cocirculating in hong kong .
clade 2b a / brisbane/59/2007-like viruses also were cocirculating with clade 2c a / hong kong /2652/2006-like viruses in about the same proportion during the 2008 influenza season .
we identified 3 clade 2b a / brisbane/59/2007-like viruses ( isolated from 1 outbreak ) that were oseltamivir - sensitive in their ha and na genes but carried an amantadine - resistant m2 gene that was similar to clade 2c a / hong kong/2652/2006 virus .
the ha , na , and m2 gene sequences of the 3 oseltamivir- and amantadine - resistant subtype h1n1 isolates were deposited in genbank ( accession nos .
all 3 isolates were clade 2c a / hong kong/2652/2006-like viruses , but they acquired oseltamivir resistance through spontaneous mutation or reassortment with a clade 2b a / brisbane/59/2007-like virus .
gene sequencing results confirmed that no epidemiologic link existed among the 3 patients infected with these isolates .
monitoring of antiviral resistance among influenza isolates showed that resistance pattern changed among subtype h1n1 viruses when different lineages were introduced .
while oseltamivir resistance appears to be largely confined to clade 2b viruses and amantadine resistance to clade 2c viruses , oseltamivir- and amantadine - resistant viruses due to spontaneous mutation or reassortment were detectable when clade 2b and 2c viruses were cocirculating .
if antiviral resistance markers are combined with sequence data of ha , na , and m genes , systematic monitoring would make it possible to track the spread of influenza viruses globally and to clarify the underlying mechanism for the spread of such resistance .
 surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in hong kong in 2006 . in 2008 , while both a / brisbane/59/2007-like and a / hong kong/2652/2006-like viruses ( h1n1 ) were cocirculating , we detected amantadine and oseltamivir resistance among a / hong kong/2652/2006-like viruses ( h1n1 ) , caused by genetic reassortment or spontaneous mutation . 
The Study
Conclusions