mosquito samples were collected in vietnam in 2002 . each species was separated and pooled , and each mosquito pool was then homogenized and centrifuged .
the supernatant was filtrated through a 0.22-m filter and then inoculated onto a monolayer c6/36 mosquito cell culture and subsequently incubated at 28c for 7 days .
virus amplification in c6/36 cells was repeated twice , and the culture fluids were stored at 80c for further analysis .
rna was extracted from the second cell culture fluid by trizol ls reagent ( invitrogen , carlsbad , ca , usa ) .
reverse transcription was performed by using superscript iii reverse transcriptase ( invitrogen ) and random hexamers , after rna denaturation at a temperature of 95c for 5 minutes in the presence of 15% dimethyl sulfoxide .
pcr was conducted by using takara la taq dna polymerase ( takara bio , inc . ,
otsu , japan ) with bav targeting primers ( for segment 5 , 5-cagctgcagtggttattgga-3 and 5accgtgcatcttaacccttg-3 ; for segment 8 , 5-ttgcagtcgctgagctttta-3 and 5-cgcatttgatcgtatgcttg-3 ) .
these targeting primers were designed from sequences of bav strains from china and java ( genbank accession nos .
af134519af134527 , ay549307ay549309 , af052024af052035 , ay568287ay568290 , nc_004211 , nc_004217-nc_004221 , nc_004200-nc_004204 , nc_004198 , and af052008af052013 ) . the amplified cdnas were sequenced in the 3100-avant genetic analyzer ( applied biosystems , foster city , ca , usa ) .
the genomic sequences of all 12 genome segments for the 5 strains were determined ( genbank accession nos .
the sequences were translated to protein sequences by using the transeq program of the emboss package ( 8) , version 5.00 .
the protein sequences were aligned with the t - coffee program version 5.05 ( 9 ) with chinese and javanese bavs .
nucleotide sequences were then aligned , on the basis of protein alignment , with the tranalign program of the emboss package .
maximum likelihood phylogenetic trees were constructed from the protein - guided alignments of nucleotide sequences with dnaml software of the phylip package ( 10 ) , version 3.67 .
five bav strains were isolated in ha tay and quang bing provinces ( figure 1 ) in vietnam from culex annulus and cx .
phylogenetic analysis showed diversity within the phylogenetic clustering of each segment ( table 2 ) .
segments 7 and 9 formed 5 independent clusters , segment 12 formed 3 clusters , and the remaining segments formed 4 clusters .
phylogenetic trees for some representative segments ( segments 2 , 6 , 9 , and 12 ) are shown in figure 2 .
javanese strains formed a single cluster for segments 1 , 2 , 6 , 8 , 10 , and 12 . for segments 7 , 9 , and 11 , however , they diverged into 2 clusters , i and ii ( figure 2 , panel c ) .
segments 11 and 12 of the 2 vietnamese strains 02vn178b and 02vn018b were included in cluster iii with chinese bavs .
segments 6 and 9 of isolate 02vn178b belonged to cluster v , although segments 1 , 2 , 3 , 4 , and 8 belonged to cluster iv .
moreover , segments 5 and 7 of isolate 02vn178b and segments 8 and 11 of isolate 02vn009b showed a mixed electrogram pattern of clusters v and iv or iii .
mosquito collection sites in vietnam . * length , length of each segment in the reference sequence of banna virus ( bav ) from refseq ( nc_004198 , nc_004200nc_004204 , nc_004211 , nc_004217nc_004221 ) ; aligned area , the fragment used to make the alignment and phylogenetic tree .
the numbers are set following the sequences of strain jht-6423 , which is the reference strain in national center for biotechnology information reference sequences .
i , the cluster including the javanese jkt-6423 strain ; ii , the cluster specific to the jkt-6969 and jkt-7043 strains ; iii , the cluster including chinese bavs ; iv , the cluster including the vietnamese 02vn018b strain ; v , the cluster including the vietnamese 02vn180b strain .
we report isolation of bav in vietnam . the nucleotide sequences of the vietnamese isolates genomic rna segments diverged into 2 phylogenetically distant clusters .
our data indicate that 2 bav populations exist in the country and that both evolved independently .
two strains were clearly different from chinese and javanese bavs ( 02vn078b and 02vn180b ) .
two other strains ( 02vn018b and 02vn178b ) were phylogenetically close to chinese bavs when 2 shorter segments , segments 11 and 12 , were analyzed but not when the remaining 10 , longer segments were considered .
this finding implies that a recent reassortment event has occurred and that segments of chinese or vietnamese strains were replaced .
in addition , although some vietnamese strains were phylogenetically distant , they could produce a viable progeny by reassortment of segments 5 , 6 , 7 , 8 , 9 , and 11 .
this finding is clearly illustrated by the diversity in the phylogenetic clustering pattern and the mixed electrogram pattern of these segments ( table 2 ) .
analysis of segment 12 showed that cluster iii ( table 2 ) contained 2 vietnamese strains ( 02vn178b and 02vn018b ) and the chinese strains af052030 and yn-6 ( isolated in yunnan province ) and bj95 - 75 ( isolated in beijing ) .
this finding suggests the possibility of wide circulation of bavs containing segment 12 type iii not only in southeast asia but also in east asia . a regionwide genotype shift of japanese encephalitis virus ( jev ) , which is also carried by culex spp .
mosquitoes , from genotype iii to genotype i , was witnessed in east asia during the 1990s ( 11,12 ) .
these results and phylogenetic analysis of old jev strains in japan and southeast asia ( 11 ) strongly suggested that jev could be transferred frequently from southeast asia to east asia .
similarly , bavs with segment 12 belonging to cluster type iii were also distributed in both southeast and east asia .
therefore , we can speculate that bavs are actively circulating within the asian continent . to achieve a better understanding of the distribution dynamics of bavs in asia , more isolates are needed . in vietnam ,
a seasonal increase of viral encephalitis and meningitis cases is reported during the rainy season every year . among these cases ,
50%70% are diagnosed as japanese encephalitis , while the rest of them remain idiopathic ( p.t .
nga et al . , unpub . data ) . a clear association between bav and human diseases , as well as the prevalence of bav infection in humans , has not yet been established in vietnam .
we also believe that bav is a good candidate for the differential diagnosis of viral encephalitis and meningitis cases of unknown origin in tropical and subtropical asia , where culex mosquitoes are abundant .
 we isolated and characterized a banna virus from mosquitoes in vietnam ; 5 strains were isolated from field - caught mosquitoes at various locations ; banna virus was previously isolated from encephalitis patients in yunnan , china , in 1987 . 
 together , these findings suggest widespread distribution of this virus throughout southeast asia . 
The Study
Conclusions