to understand early events leading to the replacement of giii viruses by gi viruses in jev - endemic areas , we conducted virologic surveillance using mosquitoes and pig serum specimens collected from pig farms in multiple counties in taiwan during the 2009 and 2010 transmission seasons .
eight pig farms were selected as sites for virologic surveillance ( figure ) : 3 in the central counties of taichung and changhua ; 4 in the southern counties of yulin , chiayi , and tainan ; and 1 in the eastern county of hualien .
mosquito pools and swine serum specimens were collected every other week during the jev transmission season from march 2009 through october 2010 .
the mosquito and swine specimens were subjected to viral rna detection by using a multiplex reverse transcription pcr ( rt - pcr ) . a qiaamp viral rna kit ( qiagen , hilden , germany ) was used to extract viral rna from pooled ground mosquitoes or from plasma specimens following the manufacturer s protocol .
three primer pairs were used in rt - pcr to differentiate gi and giii jev ( table ) . amplifying and
sequence primers were used as was done in our previous study ( 10 ) to obtain e gene region and the full genomic sequence .
the dna fragment of the correct size was excised , purified with a viogene gel extraction kit ( viogene , sunnyvale , ca , usa ) , and sequenced directly by using a prism automated dna sequencing kit ( applied biosystems , foster city , ca , usa ) .
* jev , japanese encephalitis virus ; gi , genotype i ; giii , genotype iii . 
a total of 62,266 mosquitoes were collected at multiple sites in taiwan from march 2009 through october 2010 .
tritaeniorhynchus ( n = 59,386 ) . of the 787 mosquito pools collected , 37 pools were jev - positive by multiplex rt - pcr , and all positive pools contained cx .
the jev - positive mosquitoes were collected from central ( taichung and changhua counties ) , southern ( yulin , chiayi , and tainan counties ) , and eastern ( hualin county ) taiwan ( figure ) .
unfortunately , jev was not detected from pig serum specimens , but seroconversion , defined by plaque - reduction neutralizing assay at the 50% reduction titer of > 10 , was evident among these samples after jev detection in mosquitoes ( data not shown ) .
full - length jev sequences , including tc20091 , tc200913 , and yl20094 ( genbank accession nos .
jf499788jf499790 ) , were obtained from 3 positive mosquito pools , and a neighbor - joining phylogenetic tree confirmed all 3 viruses belong to gi ( data not shown ) .
additionally , the e gene was sequenced from 37 jev - positive mosquito pools ( genbank accession nos .
the full - length e gene sequence of these isolates was more closely related to the gi k94p05 strain than to the giii nakayama strain ; sequence identities ranged from 97.6% to 98.6% , compared with 87.4% to 88.1% , respectively .
the e gene tree supports that all 37 isolates detected in this study belong to gi ( figure a1 ) .
however , of jev detected by cdc - taiwan in 2008 , only tpc0806c and yilan0806f were classified as gi ; all others belonged to giii ( 9 ) .
gi jev can be classified into various subclusters based on phylogeny ( 8) . in this study , 21 of the 37 viruses could be classified as subcluster i and the other 16 as subcluster ii .
the tpc0806c and yilan0806f strains , reported by cdc - taiwan , belong to subcluster ii ( figure a1 ) ( 9 ) .
this result indicates that the gi jev introduced into taiwan originated from multiple sources . to understand the origin of the gi jev introduced into taiwan , we selected the k91p55 strain as the root virus and constructed a minimum - spanning tree by using bionumerics software version 5.00 ( applied maths ; austin , tx , usa ) . among the subcluster
i jev isolated in taiwan , the most pronounced genetic linkages appeared between viruses isolated in taichung in 2009 and in japan in 2007 .
the taichung isolates ( tc2009 - 2 , -5 , -6 , -8 , -9 , and -11 ) were most closely linked to a group of viruses ( janar06 , 14 , 15 , and 17 ) isolated from mosquitoes collected in nagasaki , japan , in 2007 . among subcluster ii jev in taiwan ,
the tc2009 - 3 taichung isolate and the tpc0806c taipei city isolate were most closely related to janar32 - 04 , which was isolated from mosquitoes collected in nagasaki , japan , in 2004 . in summary , the jev gi isolates from taiwan , most closely related to gi viruses isolated from nagasaki , japan , were introduced at least twice into central and once into northern taiwan .
gi jev first appeared in taiwan in 2008 when giii viruses were still the dominant circulating genotype in the region .
we conclude , on the basis of molecular evidence , that the dominant jev genotype in taiwan has switched from iii to i. also , our study suggests that 1 ) the genotype replacement may have been accomplished within 1 year ; 2 ) the jev ecology remains unchanged , as evidenced by the involvement of cx .
tritaeniorhynchus mosquitoes and of swine in maintaining and circulating gi virus ; 3 ) the introduction of gi jev strains occurred multiple times , resulting in the detection of subcluster i and ii viruses ; and 4 ) the gi jevs isolated in taiwan were most closely related to gi viruses isolated from nagasaki , japan .
thus , the systemic evaluation of the cross - neutralizing activity of vaccinated human , as well as swine , serum specimens should be used to estimate the protective efficacy of giii - based vaccine .
 genotype i of japanese encephalitis virus first appeared in taiwan in 2008 . 
 phylogenetic analysis of 37 viruses from pig farms in 20092010 classified these viruses into 2 unique subclusters of genotype i viruses and suggested multiple introductions and swift replacement of genotype iii by genotype i virus in taiwan . 
The Study
Conclusions