serum samples were collected in 9 of 17 provinces in laos from healthy ducks and chickens in live - bird markets , village backyard flocks , and layer duck farms .
cloacal , tracheal , and environmental ( fecal and water ) swab specimens were also collected and placed immediately in transport medium ( figure ) .
swab specimens were screened in pools of 4 by using a real - time reverse transcription pcr for the matrix ( m ) gene segment ( 6 ) .
positive pools were reextracted individually , retested , tested for hemagglutinin 5 ( h5 ) by real - time reverse transcription pcr ( 7 ) , and injected into 1011-day - old embryonated chicken eggs .
areas sampled and location of subtyped avian influenza viruses ( h5n1 ) , laos , 20092010 .
provinces that had previous outbreaks of highly pathogenic avian influenza and were part of the survey are indicated in gray , the province that had a previous outbreak but was not part of the survey is indicated in blue , and the province that had not had an outbreak but was part of the survey is indicated in green .
colored dots indicate presence of viruses : light blue , anti - h5 ( clade 2.3.4 ) ; gray , anti - h9 lineage g1 ; red , clade 2.3.4.1 ; orange , clade 2.3.4.2 ; green , anti - h5 ( clade 2.3.2 ) ; black , anti - h9 lineage y280 ; white , anti - h4 ; yellow , anti - h6 ; purple , clade 2.3.2.1 or virus - specific antibodies .
sequencing was conducted by using an illumina ( san diego , ca , usa ) platform ( swabs and isolates ) ( 8,9 ) and conventional sanger sequencing ( isolates ) .
illumina reads were first mapped to a database of publicly available human , avian , and swine influenza virus reference sequences from the western hemisphere americas and eurasia , and then remapped against references with the highest number of reads and best average coverage .
final average coverage varied between samples and segments with ranges of 12324,163 ( 9 samples ) , 2118,671 ( 12 samples ) and 9436 for sample a / duck / lao/670/10 .
mixed infections could not be excluded for direct sequencing in the absence of an isolate .
phylogenetic analysis ( clustalw [ http://www.clustal.org/ ] , neighbor - joining analysis , 1,000 bootstrap tests , maximum composite likelihood , pairwise deletions ) was conducted by using mega version 5.02 ( 10 ) .
sequences are available in genbank ( cy098294cy098334 , cy098336cy098340 , cy098342cy098368 , and cy098370cy098464 ) .
serum samples were screened by using an elisa ( flockchek multis screen ; idexx laboratories , westbrook , me , usa ) , and antibody - positive serum samples were tested by using a hemagglutinin inhibition assay for subtypes h3 , h4 , h5 ( clades 2.3.2 and 2.3.4 ) , h6 , and h9 ( lineages g1 and y280 ) as described ( 11 ) . during march 2010 , a total of 3,695 swab specimens were collected ( 1,928 duck and 279 chicken cloacal samples , 446 duck tracheal samples , 675 fecal samples , and 367 water samples ) .
m gene prevalence was 4.0% ( ducks ) , 1.8% ( chickens ) , and 0.3% ( environment samples ) .
positive swab specimens were collected in 13 locations ( 8 backyards , 2 markets , and 3 farms ) ( table 1 ; figure ) sample protection was suboptimal , and only 21 samples could be subtyped by real - time rt - pcr and sequencing ( table 1 ) . *
i d , identification ; pb , polymerase basic ; pa , polymerase acidic ; h , hemagglutinin ; np , nucleoprotein ; n , neuraminidase ; m , matrix ; ns , nonstructural ; na , not available ; env , environmental . 
pink shading , a / guizhou/1/2009-like clade 2.3.4.1 ; blue shading , a / whooper swan / mongolia/6/2009-like clade 2.3.2.1 ; purple shading , a / tree_sparrow / jiangsu/1/08-like clade 2.3.4 ; gray shading , a / environment / guizhou/4/2009-like clade 2.3.4.2 . 
phylogenetic analysis identified 3 groups of viruses : clades 2.3.2.1 , 2.3.4.1 , and 2.3.4.2 ( figure a1 ) .
two samples were closely related to a / chicken / lao / lh1/2010like virus ( outbreak in vientiane in april  may 2010 ) and to a / chicken / laos / c100209194-ptk/2009like virus ( outbreak in phongsaly in february 2009 ) ( clade 2.3.4.1 ) ( figure a1 ; table 1 ) .
these viruses were closely related to a / guizhou/1/2009 and a / chicken / vietnam / ncvd-404/2010 ( figure a1 ) . sixteen surveillance viruses of clade 2.3.4.2 were highly homogeneous ( figure a1 ; table 1 ) and closely related to a / environment / guizhou/4/2009 and a / chicken / vietnam / ncvd-394/2010like viruses , although they contained the polymerase basic 2 ( pb2 ) gene of a clade 2.3.2.1 donor virus and were therefore interclade reassortants ( figure a1 ; table 1 ) . the genotype of the 4 isolates was verified by using sanger sequencing .
these reassortants were detected in 3 locations in northern ( backyard ) , central ( backyard ) , and southern ( farm ) laos , suggesting multiple introductions of reassortants into laos ( figure a1 ; table 1 ) .
three clade 2.3.2.1 viruses were detected in 2 ducks and 1 environmental sample ( same trader ) .
two ( 1 isolate and 1 direct sequence ) contained 6 or 7 segments that were a / whooper swan / mongolia/6/2009 like ( clade 2.3.2.1 ) , and the nucleoprotein gene was a / tree_sparrow / jiangsu/1/08 like ( clade 2.3.4 ) . the environmental sample ( direct sequence ) contained a / whooper swan / mongolia/6/2009like hemagglutinin and m genes and a / guizhou/1/2009like pb2 , nucleoprotein , and neuraminidase genes ( clade 2.3.4.1 ) ( table 1 ) .
this identity and the source of the 3 samples ( 1 trader ) suggest that reassortment occurred recently , likely in laos .
antibody titers to h5 and h9 and a subtype h3n8 virus isolate have been reported in laos ( 4,5 ) .
subtypes h4 and h6 also circulate in this region ( 1,2,12 ) . for this study , 2,148 serum samples ( 1,899 from ducks , 200 from chickens , and 49 from unspecified species )
were collected and 267 antibody - positive ( seroprevalence 14% ) duck and 15 ( 7.5% ) chicken serum samples were detected .
hemagglutination inhibition testing for specific antibodies against h3 , h4 , h5 , h6 , and h9 detected all antibodies but to h3 ( table 2 ) .
antibodies against h9 were detected at highest titers and most frequently ( 1.1% of ducks for each h9 lineage ) , although some cross - reactivity between g1 and y280 likely occurred ( table 2 ) .
these ducks were most widely distributed ( 18 [ 19% ] of 97 locations in all 9 provinces ) .
antibodies against h5 clade 2.3.4 were found at the detection limit in few serum samples ( 0.4% of ducks in 3 northern and 2 southern provinces ) .
antibodies against h5 clade 2.3.2 were detected in 0.4% of ducks in 4 northern provinces and the capital of vientiane . *
all titers for h3 ( a / duck / laos - xaythany / a0573/2007 ) were < 10 .
h4 , a / duck / czech/1956 ; h5 clade 2.3.2 , a / common magpie / hong kong/5052/2007 ; h5 clade 2.3.4 , a / duck / laos/3295/2006 ; h6 , a / turkey / massachusetts/1965 ; h9 lineage g1 , a / hong kong/33982/2009 ; h9 lineage y280 , a / duck / hong kong / y280/1997 .   values represent hi titers reciprocal to the highest dilution of serum that inhibited haemagglutination of 4 hemagglutination units of antigen by using 0.5% chicken erythrocytes . among ducks , 34 ( 1.7% )
were either shedding or had antibodies against avian influenza virus ( h5n1 ) , and there was > 1 virus - positive or antibody - positive duck in each of the 9 provinces sampled .
one village had 36% of sampled ducks exposed to this virus ; 18% shed 2.3.4 virus and 18% had antibody to 2.3.2 virus ( table 2 ) .
exposure to 2.3.2 and 2.3.4 viruses was evident in ducks from locations in 3 other provinces ( 1 district each in champasak and xiengkhoung , and several districts in luang prabang ) .
this study showed that 3 groups of avian influenza viruses ( h5n1 ) were likely introduced into laos in 20092010 , one of which resulted in 2 outbreaks ( 2009 , 2010 ) . in all 9 provinces where surveillance was conducted ,
several interclade reassortants were identified , demonstrating the high genetic mobility of these viruses in the region . since 2004 , laos has had repeated outbreaks of highly pathogenic avian influenza viruses , which have also been detected in china and vietnam .
the frequency of introduction , diversity , and extent of these viruses in laos suggests considerable movement of viruses into the country from surrounding territories ( china and vietnam , but not cambodia ) and within the country .
 avian influenza viruses ( h5n1 ) of clades 2.3.4.1 , 2.3.4.2 , and 2.3.2.1 were introduced into laos in 20092010 . to investigate these viruses , we conducted active surveillance of poultry during march 2010 . 
 we detected viruses throughout laos , including several interclade reassortants and 2 subgroups of clade 2.3.4 , one of which caused an outbreak in may 2010 . 
The Study
Conclusions