cerebellar tissue was taken from a dead 12-year - old stallion from a privately owned ranch in the municipality of juarez in nuevo leon state , mexico , approximately 240 km south of the texas border .
the horse was first observed with neurologic symptoms on june 20 , 2003 , and it was euthanized 7 days later .
the horse had never been outside the state of nuevo leon and had not been vaccinated against wnv .
the tissue sample was immediately placed on dry ice and transported to the biosafety - level-3 facilities at colorado state university for processing . although we were unable to isolate virus from the sample by passing brain homogenate in vero cells , we successfully amplified viral rna .
total rna was extracted from approximately 100 g of cerebellar tissue with trizol reagent ( invitrogen , carlsbad , ca ) , according to the manufacturer 's instructions .
the prm - e genes were amplified as two fragments by reverse transcription - polymerase chain reaction ( rt - pcr ) by using primers designed from the nucleotide sequence of the prototype wn - ny99 strain ( genbank accession no .
pcr amplifications were performed by using ex taq dna polymerase ( takara biomedicals , shiga , japan ) , which has 3   5  exonuclease activity .
amplification products were separated by agarose gel electrophoresis , visualized with crystal violet , and extracted by using the rapid gel extraction system ( invitrogen , carlsbad , ca ) .
the resulting dna fragments were reamplified by pcr because of the low rna copy number in the original material and purified by using the qiaquick pcr purification kit ( qiagen , valencia , ca ) .
purified dnas were sequenced on both strands with an abi 377 dna sequencer ( davis sequencing , davis , ca ) and eight pairs of wnv - specific primers .
the nucleotide sequence of the prm - e genes of the wnv from nuevo leon state , mexico ( designated mexnl-03 ) was submitted to genbank ( genbank accession no .
this region comprises 2004 nucleotides and corresponds to nucleotides 466 to 2469 of the genomic rna of the wn - ny99 strain ( 8) .
alignment of the mexnl-03 sequence with other known sequences in the genbank database showed that it was most closely related to the homologous regions of three wnv isolates collected in harris county , texas , in june 2002 ( strains 119 , 123 , and v1151 ; genbank accession numbers ay185908 , ay185909 , ay185911 respectively ) .
the mexnl-03 sequence differed from the harris county isolates in three nucleotide positions ( 0.15% divergence ; table ) . in all cases ,
one change was in the prm gene at position 549 , and two changes were in the e gene at positions 1179 and 1356 .
all substitutions were in the third codon position , and none resulted in an amino acid change .
nucleotide numbers correspond to those of the prototype new york strain ( wn - ny99 ) . 
isolated from a chilean flamingo ( phoenicopterus chilensis ) ( collection date : 06/01/99 ) .
isolated from a bluejay ( cyanocitta cristata ) and passaged once in vero cells ( collection date : 06/14/02 ) .
ay185908 . the nucleotide sequence of mexnl-03 differed from that of the wn - ny99 strain ( genbank accession no .
two mutations were in the prm gene ( positions 549 and 660 ) , and four mutations were in the e gene ( positions 1179 , 1356 , 1442 , and 2466 ) .
the u to c substitution at 1442 resulted in an amino acid change ( val  ala ) ; all other substitutions were silent .
the u to c substitution at 549 and a to g substitution at 1179 have not been reported in any wnv isolates from the united states . however , an isolate collected in illinois in 2002 ( genbank accession no .
several more divergent strains of wnv , such as a kunjin virus isolated in australia in 1960 ( genbank accession no .
the prm - e genes of mexnl-03 differed from tm171 - 03 ( genbank accession no .
five mutations were in the prm gene , and eight mutations were in the e gene .
a phylogenetic tree was constructed by bayesian analysis using the complete prm - e gene sequences of 49 wnv strains , including this wnv from nuevo leon state , mexico ( figure 1 ) .
phylogenetic trees were also generated using neighbor - joining ( nj ) , maximum parsimony ( mp ) , and maximum likelihood ( ml ) analyses ( data not shown ) . in the bayesian tree , the wnv isolates from north america formed a monophyletic group consisting of two sister clades ( denoted as clade 1 and 2 ) .
mexnl-03 shared a close phylogenetic relationship with isolates from inland texas , consistent with our nucleotide sequence alignments .
these viruses , along with isolates from colorado , illinois , alabama , and tabasco state , belonged to a nested clade ( denoted as 1a ) within clade 1 .
the statistical support for clade 1a by parsimony and distance bootstrap analyses were 64% and 66% , respectively .
the other wnv isolates that clustered in clade 1 were from the eastern united states .
clade 2 contained wnv isolates from the southeast coastal area of texas and the northeastern united states .
the wnv isolates from coastal texas formed a nested clade ( denoted as 2a ) , confirming previous results ( 13,14 ) .
phylogenetic analysis of west nile virus ( wnv ) from nuevo leon state , mexico .
sampling of trees from the posterior probability distribution used the metropolis - coupled markov chain monte carlo algorithm to allow running of multiple markov chains . a run with four chains
was performed for 90,000 generations , under a general time - reversible model ( all six types of substitutions occur at different rates ) with parameter value estimation for base frequencies , substitution matrix values , and rate heterogeneity .
rate heterogeneity was estimated by using a  distribution for the variable sites and assuming a certain portion of sites to be invariable .
the phylogenetic analysis is based on the 2004-nt fragment encoding the complete prm - e genes of 49 wnvs .
the tree is rooted by using the prototype wnv strain from uganda in 1937 ( genbank accession no .
the parsimony - informative sites , corresponding to nucleotide positions 660 , 1442 , and 2466 , were important in defining the topologic features of the tree ( figure 2 ) .
all 21 wnv strains in clade 1a contained a unique c to u substitution at 2466 .
all , except for the five most basal isolates , contained a unique c to t substitution at 660 and all , except for tm171 - 03 , contained a unique t to c substitution at 1442 .
tm171 - 03 was basal to the other wnv strains in clade 1a , and this topologic arrangement was due to the single nucleotide difference at 1442 .
however , we consider it unlikely that tm171 - 03 was the predecessor of the other viruses in clade 1a .
in addition , the parsimony and distance bootstrap analyses did not significantly support this topologic arrangement ( bootstrap values = 50% and 51% , respectively ) .
moreover , wnv isolates from the northeastern united states occupied the basal position of clade 1 .
similarly , the basal position of clade 2 was occupied by wnv isolates from the northeastern united states .
thus , our findings suggest that the wnv isolates circulating in the united states and mexico diverged from a common ancestor from the northeastern united states .
parsimony - informative sites in the premembrane and envelope genes of selected west nile viruses used in the phylogenetic analyses .
the trees generated by nj , mp , and ml analyses showed the same overall topologic features to the bayesian tree , except that all of the coastal texas isolates were basal to the israeli isolate ( data not shown ) .
furthermore , the wnv isolates from maryland and new jersey , as well as most of the isolates from new york , occupy the basal positions of clade 1 of the nj / mp / ml trees . as a result
, we have not shown the bootstrap values for clades 1 and 2 of the bayesian tree because their composition does not match exactly to the corresponding clades of the nj / mp / ml trees .
however , the bayesian analysis provides a more robust and efficient phylogenetic tool compared to more conventional phylogenetic techniques ( 15 ) .
the data presented here indicate that wnv was introduced into nuevo leon state , mexico , from inland texas
. a likely mode of introduction was by infected birds traveling for short distances ( 16 ) .
earlier studies have provided serologic evidence of wnv infection in horses or birds in the nearby mexican states of coahuila , tamaulipas , and chihuahua ( 4,5,17 ) . taken together ,
our sequence data and the findings from the serosurveys indicate that wnv activity is now widespread in northern mexico , as well as in other regions in mexico ( 5,6,18 ) .
the geographic distribution of wnv in the americas will likely continue to expand ; thus , enhanced wnv surveillance in mexico is warranted .
 west nile virus rna was detected in brain tissue from a horse that died in june 2003 in nuevo leon state , mexico . 
 nucleotide sequencing and phylogenetic analysis of the premembrane and envelope genes showed that the virus was most closely related to west nile virus isolates collected in texas in 2002 . 
The Study
Conclusions