mosquitoes were collected from june to september 2003 at the ejido francisco villa , municipality of pesqueria , state of nuevo leon ( 2547n , 10003w ) , with cdc - type light traps baited with dry ice and mechanical aspiration from resting sites on vegetation and in houses . the area is located 40 km northeast of monterrey and consists of mixed suburban housing and agriculture . average annual rainfall in the region is 550 mm ; the mean annual temperature is 28c . after collection , the mosquitoes were placed on dry ice for transport back to the medical entomology laboratory , faculty of biological sciences , autonomous university of nuevo leon , monterrey , where they were separated into pools of 10 insects each , based on species , date , and method of collection ( table 1 ) . the mosquitoes were stored in a mechanical freezer at 70c and later transported on dry ice to the university of texas medical branch ( utmb ) to be processed for virus isolation . a total of 2,297 mosquitoes , representing 4 genera and 11 species , were tested in 238 pools ( table 1 ) . individual mosquito pools were titrated manually in sterile , ten broeck tissue grinders containing 1.0 ml of phosphate - buffered saline , ph 7.4 , containing 30% fetal bovine serum and antimicrobial agents ( penicillin , streptomycin , and amphotericin ) . the resultant suspension was centrifuged at 12,000 rpm for 5 min ; then 200 l of the supernatant was injected into a flask culture of vero cells . after the solution was absorbed for 1 h at 37c , maintenance medium ( 9 ) was added ; cultures were maintained in an incubator at 37c and examined daily for evidence of viral cytopathic effect ( cpe ) for 14 days . a single pool of cx . quinquefasciatus yielded a virus isolate , designated nl-54 , which produced cpe on approximately day 7 . the isolate was identified as wnv by immunofluorescence , hemagglutination - inhibition ( hi ) test , complement - fixation test , vectest wnv / sle antigen assay ( medical analysis systems , camarillo , ca , usa ) , and reverse transcription  polymerase chain reaction ( rt - pcr ) ( 9,10 ) . the wnv human isolate was from a 62-year - old mexican woman living in the municipality of etchojoa ( near ciudad obregon ) in sonora state . she visited a local hospital in july 2004 with symptoms of fever , headache , vomiting , arthralgias , and myalgia . an acute - phase blood sample was obtained , and a presumptive diagnosis of dengue fever was made . the patient was sent home and subsequently completely recovered . when rt - pcr using dengue primers was negative on the acute - phase serum , a culture was performed . wnv was isolated from the sample at the state public health laboratory in sonora and at utmb , upon culture in vero cells . hi tests conducted on the acute - phase serum at utmb with west nile , st . louis encephalitis , yellow fever , dengue 1 , and dengue 2 viral antigens were negative , which indicated that the patient had no preexisting flavivirus antibodies . an immunoglobulin ( ig ) m enzyme - linked immunosorbent assay ( 11 ) , performed on the acute - phase specimen and a 30-day convalescent - phase serum specimen in sonora , demonstrated seroconversion and the presence of wnv - reactive igm antibodies in the convalescent - phase serum sample . viral rna was extracted from the 2 wnv strains after a single vero cell passage directly from 140 l of the infected cell culture supernatants , using the qiaamp viral rna extraction kit ( 12 ) . rt - pcr was performed by using 3 primer pairs to amplify the entire prm - e genes of each wnv isolate as previously described ( 12 ) . pcr products were gel purified with the qiaquick kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's protocol , and the resulting template was directly sequenced with the amplifying primers . analysis and assembly of sequencing data were performed with the vector nti suite software package ( informax , frederick , md , usa ) . nucleotide and deduced amino acid sequences of the 2004-nucleotide region representing the prm - e genes from each isolate were aligned with the alignx program in the vector nti suite and compared to sequences of selected north american wnv isolates for which the prm - e genes were available in genbank . phylogenetic trees were constructed by bayesian analysis with the program mrbayes , version 2.0 ( 13 ) , with the metropolis - coupled , markov chain , monte carlo algorithm run with 4 chains over 150,000 generations under a general time - reversible model with a burn - in time of 50,000 generations . the bayesian consensus tree was compared to trees generated by neighbor - joining , maximum parsimony , and maximum likelihood analyses using paup , version 4.0b10 ( 14 ) , and each method generated trees with the same overall topology . the consensus phylogram of the 40 wnv isolates generated by bayesian analysis ( 13 ) is shown in the figure , with confidence values at relevant nodes to demonstrate statistical support for each clade . phylogram of 2 west nile viruses ( wnv ) isolated from a mosquito pool and human serum in mexico ( shown in bold ) . the phylogenetic tree was generated by bayesian analysis of a 2004-nucleotide region of the prm and e genes of 40 wnv isolates rooted by the most closely related old world strain , israel 1998 . ay963774 ) , to the prototypical north american wnv isolate , wn - ny99 ( genbank accession no . ay371271 ) ( 2 ) , and an isolate collected in harris county , texas , in 2002 ( genbank accession no . ay185906 ) ( 15 ) indicated nucleotide and deduced amino acid differences and similarities among each of the isolates . both the mexican mosquito and human isolates reported herein shared a nucleotide mutation at position 660 ( c to u ) of the prm gene and 2 mutations at positions 1442 ( u to c ) and 2466 ( c to u ) of the e gene . each of these 3 mutations was shared with a 2003 horse strain from nuevo leon ( mexnl-03 ) ( 7 ) and a 2002 bird isolate from harris county , texas ( tx-1 ) ( 15 ) . the mutation at nucleotide 1442 also represented a deduced amino acid substitution in the envelope protein ( v159a ) . the mexican mosquito and human isolates reported herein shared a unique mutation at genomic position 1320 ( a to g ) in the e gene . the human isolate also had 3 additional mutations in the e gene at positions 1074 ( g to a ) , 1656 ( u to c ) , and 1974 ( c to u ) . the nucleotide mutations at nucleotide positions 660 , 1442 , and 2466 have also been described in most wnv isolates sequenced from texas , illinois , and colorado in 2002 ( 12 ) . this finding suggests that isolates obtained from northern states of mexico ( i.e. , nuevo leon and sonora ) were derived from wnv strains circulating in the western united states . only a single mutation at position nucleotide 2466 was shared by these 2 isolates and a 2003 bird isolate from tabasco state ( tm171 - 03 ) . this finding supports results from earlier studies that suggest separate introductions of wnv into mexico ( 2,7 ) . phylogenetic trees generated by a number of methods indicate that the recent mexican mosquito and human isolates belong to the clade comprised of wnv isolates collected outside the northeastern united states after 2001 , with the exception of isolates collected along the southeast coast of texas . ( those isolates constitute a separate , sister clade relative to all other north american wnv isolates sequenced to date [ figure ] . ) . because of a shared mutation between the recent mexican mosquito and human isolates , these 2 virus strains constitute a distinct subclade within the larger us 2002 clade that is supported by strong bayesian confidence values ( 94% ) . the accumulation of 3 additional nucleotide mutations in the 2004 mexican human isolate is illustrated by longer branch lengths in comparison to the 2003 mosquito pool isolate nl-54 , which suggests the continued microevolution of wnv in mexico from year to year . * nucleotide numbers correspond to wn - ny99 ; amino acid substitutions are in brackets .  genbank accession number . our patient represents the first reported autochthonous human case of confirmed wnv infection in mexico . the paucity of human cases reported to date from mexico is curious for several reasons : 1 ) a large number of cases are reported from the united states , 2 ) available evidence indicates that wnv is now widely distributed in mexico ( 17 ) , 3 ) most of the wnv virus strains circulating in the republic are genetically similar to those in the united states ( figure ) . one explanation for this difference could be the failure of local health personnel to recognize the various clinical forms of wnv infection . as illustrated by our patient , west nile fever can easily be mistaken for dengue fever . a second reason may be the difficulty of making a serologic diagnosis of wnv infection among persons living in geographic regions where several different flaviviruses circulate , and people have multiple flavivirus infections ( 11 ) . a third and related possibility is that wnv infection may be less severe in persons with preexisting heterologous flavivirus antibodies ( 11 ) .
west nile virus has been isolated for the first time in mexico , from a sick person and from mosquitoes ( culex quinquefasciatus ) . partial sequencing and analysis of the 2 isolates 
 indicate that they are genetically similar to other recent isolates from northern mexico and the western united states .