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in late february 2006 , two horses died after encephalitis developed at 2 stud farms near san antonio de areco , 110 km north of the city of buenos aires ( figure 1 ) .
clinical signs included circling and acute onset ataxia , hypersensitivity to noise , hyperexcitability , and recumbency . both mares ( eq001 and eq002 ) died within 48 to 72 hours of onset of signs .
locations of dead horses reported from february to march 2006 in central argentina . in march ,
another horse death was reported at the jockey club horse training center in san isidro , province of buenos aires , within 48 hours after its arrival from a polo horse farm near victoria ( entre rios province ) ( figure 1 ) .
clinical symptoms of the polo mare ( eq003 ) were weakness , paralysis , and recumbency .
diagnostic tests for rabies virus ( direccin de laboratorios y control tcnico , senasa ) and for equine herpesvirus 1 ( inta laboratory , castelar , argentina ) were performed , with negative results . at inevh ,
virus was isolated in african green monkey ( vero ) cells , as described ( 2 ) .
briefly , 20% homogenates of brain tissues were prepared and cultured in vero cells for 14 days .
infected cells that showed cpes were harvested and evaluated by immunofluorescent assay ( ifa ) ( 3 ) for flavivirus antigen , by using fluorescein isothiocyanate  labeled flavivirus polyclonal antisera ( centers for disease control and prevention , puerto rico ) , and for wnv antigen by using the wnv - specific monoclonal immunoglobulin m ( igm ) antibody h5 - 46 and igg neutralizing monoclonal antibody 7h2 .
cultures that did not show cpes on the original isolation were blind passaged 1 more time onto fresh vero monolayers .
two cultures ( areq001 and areq003 ) showed cpes on day 7 postinoculation of the original isolation ; in the third culture ( areq002 ) , cpes were observed only after the blind passage on day 7 after inoculation .
negative results were obtained for saint louis encephalitis virus by using the monoclonal antibody 6b5a-2 .
negative results were also obtained for the alphaviruses western equine encephalitis , eastern equine encephalitis , and venezuelan equine encephalitis by using mouse hyperimmune ascitic fluid .
for molecular identification of the virus isolates , viral rna was extracted from 140 l of infected vero cell culture supernatant by using qiaamp viral rna extraction kit ( qiagen , inc . ,
nested reverse transcription ( nrt) pcr assay was performed as described by shi et al .
a dna band of the correct size was visualized by gel electrophoreses of the rt - pcr product . using the nrt - pcr previously described by johnson et al .
( 5 ) , the inta laboratory also detected wnv rna directly from brain tissue from the 3 necropsy specimens . in this case ,
total rna was extracted from 50 to 100 mg of tissue by using trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) .
dna fragments from c / prem and ns5 genes amplified by rt - pcr assays employing primers wn212/wn619c ( 5-ttgtgttggctctcttggcgttctt-3/5-cagccgacagcactggacattcata-3  ) and wn9483/wn9794c ( 5-cacctacgccctaaacactttcacc-3/5-ggaacctgctgccaatcataccatc-3  ) were sequenced .
dna products were analyzed on a 2% agarose gel , and the bands of the correct predicted size were excised and purified with the gene clean kit ( bio 101 , carlsbad , ca , usa ) .
the nucleotide sequences were determined by automatic dideoxy cycle sequencing techniques ( applied biosystems , foster city , ca , usa ) .
alignments were performed with bioedit program by the clustal wallis method ( available from http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .
phylogenetic analysis of ns5 fragments placed the argentinean sequences in the north american cluster of lineage ia ( figure 2a ) .
sequences areq001 and areq002 showed a 100% nucleotide ( nt ) identity with hny1999 and differed by only 1 nt from sequence areq003 .
phylogenetic analysis of the c / prem fragments also placed the argentinean sequences in lineage ia ( figure 2b ) . once again , areq001 was identical to areq002 but showed 9 nt differences from areq003 .
areq001 and areq002 grouped together because of 3 point mutations at nt 172 , 208 , and 245 in our fragments ( corresponding to positions 390 , 426 , and 463 of wnv hny1999 strain . whereas the first 2 mutations are silent , at position 463 the substitution of g for a results in a change of valine to isoleucine .
areq001 , areq002 , and areq003 : dq537383 , dq537385 , and dq811782 ( fragments ns5 ) , dq537382 , dq537384 , and dq811783 ( fragments c / prm ) ; ug37 : m12294 ; vol99 : af317203 , ari04 : dq164201 ; geo02 : dq164196 ; har04 : dq164206 ; hny1999 : af202541 ; kun60 : d00246 ; rum96 : af260969 ; eg51:af260968 .
since wnv was first detected in new york in the summer of 1999 , it spread from the atlantic coast to the pacific coast in the united states and has affected canada , mexico , and some locations in the caribbean basin , central america , and northern colombia ( 7,8 )
wnv was isolated and detected by molecular techniques in argentina from 3 horses that had no record of traveling outside the country during the preceding 4 months and that had not received vaccines against wnv .
san antonio de areco may provide ideal conditions for detecting an enzootic cycle , given that > 9,000 horses are raised there .
horses are probably " dead - end " or incidental hosts in the wnv transmission cycle ; 10%20% of the infections result in clinical disease , and the mortality rate in equines varies from 28% to 45% ( 9 ) .
victoria , a potential site of infection for 1 of the studied equines , is 100 km southeast of paran city , where human flavivirus encephalitis cases have been diagnosed since february 2006 .
cross - neutralization studies with a panel of flaviviruses are being conducted to identify the specific etiologic agent .
phylogenetic analysis of the ns5 fragment place them in the north american cluster , although an additional resolution of this cluster was not achieved because of high conservation levels and shortage of parsimony informative sites . a clear resolution of lineage
ia clades was not obtained from the nucleotide sequences of the c / prem fragments .
the substitution of valine to isoleucine , detected in sequences from san antonio de areco , was not observed in any sequence available from genbank database .
additional investigation is necessary to establish possible implications for this change in terms of virulence and tropism of wnv .
wnv could be detected in southern south america because of the great economic value of the horses , which are subjected to continual veterinary evaluations .
the results reported here emphasize the need for an integrated surveillance system for wnv in argentina with greater diagnostic capacity and the need for development of control strategies .
west nile virus ( wnv ) was isolated from the brains of 3 horses that died from encephalitis in february 2006 . 
 the horses were from different farms in central argentina and had not traveled outside the country . 
 this is the first isolation of wnv in south america .