during 20092010 , serum samples from patients with suspected dengue were received by the fdoh for dengue diagnostic testing ; the samples came from 16 of florida s 67counties . specific , real - time rt - pcr ( 8) and igm anti - denv elisa ( 9 ) . samples with highly positive rt - pcr results were spread onto cultured ae . albopictus c6/36 cells , and the presence of virus and genome were confirmed by immunofluorescence ( 10 ) and rt - pcr , respectively ( 11 ) . isolates were further propagated and viral rna was extracted from culture supernatants by using the universal biorobot system ( qiagen , valencia , ca , usa ) . the envelope glycoprotein ( e ) gene was amplified ( technical appendix ) , and the e gene open - reading frame ( 1,485 bp ) was sequenced . multiple sequence alignments were performed by using the muscle module available in mega 5 ( www.megasoftware.net ) . evolutionary history was inferred by using maximum likelihood and phylogenetic trees constructed by using neighbor - joining methods . evolutionary distances were computed , and several e gene sequences from genbank were included in the phylogenetic tree to support tree topology by genotype ( technical appendix ) . in 2009 , five denv-1positive cases were identified by rt - pcr in key west . subsequently , in 2010 , the fdoh tested 195 serum samples by real - time rt - pcr . fifty - six ( 29% ) samples were positive for denv rna : denv-1 ( 37 [ 66% ] samples ) , denv-2 ( 13 [ 23% ] samples ) , denv-3 ( 3 [ 5% ] samples ) , and denv-4 ( 3 [ 5% ] samples ) . monroe county submitted 73 serum samples , of which 31 ( 42% ) had results positive for recent dengue infection : denv-1 was detected in 22 by rt - pcr , and 9 had positive igm anti - denv elisa results . none of the denv-1 patients from monroe county had a history of recent travel to a dengue - endemic region before the onset of symptoms . fifteen other florida counties submitted serum samples : 13 counties submitted < 10 specimens , 1 submitted 2030 specimens , and 1 submitted > 60specimens . denv-1 was found in 15 serum samples from 6 of these counties ; however , all the patients had a history of recent international travel . we sequenced the e gene of 12 denv-1 strains isolated in florida during 20092010 to determine their genetic relatedness ; of the 12 strains , 8 were from key west and 1 each was from dade , broward , orange , and pinellas  counties . in addition , 23 denv-1 e sequences published in genbank , including the 2010 key west isolates obtained from a blood donor and a mosquito pool ( 6,7 ) , were used to construct a maximum - likelihood phylogenetic tree . this phylogenetic analysis showed that all the florida denv-1 isolates belong to the american  african genotype ( genotype v ) ( 12,13 ) together with other viruses isolated throughout the americas ( figure 1 ) . maximum - likelihood phylogenetic tree of dengue virus type 1 , including isolates from key west , florida , usa , and representative isolates from 5 genotypes with global geographic distribution . solid circles , 8 key west viruses ( monroe county ) isolated during 20092010 ; solid diamonds , isolates from other florida counties ( dade , pinellas , orange , and broward counties ) . each taxon represents a single virus isolate and is labeled with the geographic origin and collection year . thirty - six envelope glycoprotein gene sequences obtained from genbank were included to support tree topology and identify genotypes . all genotypes except the american  african genotype ( v ) have been collapsed . key west denv-1 viruses grouped among central american viruses , which configure a distinct lineage separate from the caribbean viruses . this divergence between the central american and caribbean lineages is well supported by high bootstrap values . moreover , the key west and monroe county viruses grouped together and indicated a distinct sublineage supported by a high bootstrap value ( 99% ) , separating them from viruses isolated in dade , orange , pinellas , and broward counties that were more closely related to other central american viruses ( figure 1 ) . one 2009 isolate ( iq425062 ) from a key west patient is related to this group , suggesting a separate introduction of denv-1 in key west . the sequence similarity between the 2009 and 2010 key west strains was < 0.9% ; however , the evolutionary distance and taxa positions between the 2009 and 2010 strains presented in the phylogenetic tree suggests that the 2010 strains diverged from the 2009 strains . the observed differences between e gene sequences for the key west strains ( 20092010 ) and the rest of the strains in this phylogeny were < 2.1% with the other florida strains , < 1.2% with central american strains , and < 4.8% with the rest of the american  african genotype . evolutionary distances and the topology of the central american lineage suggest this lineage is the genetic origin of the florida denv-1 strain . most viruses isolated in monroe county diverged from the central american lineage into a distinct sublineage  the key west denv-1 strain associated with the 20092010 outbreak . the high level of genetic similarity among the viruses isolated in monroe county , their close evolutionary distances , and the lack of recent international travel for the case - patients suggest endemic transmission and microevolution of this denv . conversely , the scattered and separate phylogenetic positioning of virus strains from patients with travel - associated cases from other florida counties indicates a different origin from the majority of key west isolates . although the 2009 broward isolate ( jq425067 ) is positioned near the central american lineage , the low bootstrap value ( 53% ) does not support lineage ancestry . the epidemiologic and phylogenetic evidence suggests that the 2010 cases appeared to be a continuation of the 2009 outbreak . unlike cases along the texas  mexico border ( 14 ) , in addition , denv-1 was detected in a blood donation from the monroe county in 2010 , further supporting local transmission of denv ( 7 ) . collectively , these findings indicate that endemic denv-1 was transmitted in key west over a period of > 2 years . table showing the dengue virus type 1 ( denv-1 ) envelope gene reverse transcription pcr and another showing denv-1 strains used in a study of the genetic relatedness of dengue viruses in key west , florida , usa , 20092010 .
sequencing of dengue virus type 1 ( denv-1 ) strains isolated in key west / monroe county , florida , indicate endemic transmission for > 2 years of a distinct and predominant sublineage of the american  african genotype . 
 denv-1 strains isolated elsewhere in florida grouped within a separate central american lineage . 
 findings indicate endemic transmission of denv into the continental united states .