a total of 1,217 dead birds were shipped at 4c to the tropical medicine institute " pedro kouri " and identified by ornithology experts . brain , heart , and kidneys were removed and tested for wnv by using reverse transcription  polymerase chain reaction ( rt - pcr ) ( 12 ) . briefly , rna was extracted by using the qiamp viral rna kit ( qiagen , inc . , valencia , ca , usa ) . primers wn212 ( 5-ttgtgttggctctcttggcgttctt-3  ) and wn619c ( 5-cagccgacagcactggacattcata-3  ) were used to detect viral rna . a second rt - pcr with primers wn9483 ( 5-cacctacgccctaaacactttcacc-3  ) and wn9794 ( 5-ggaacctgctgccaatcataccatc-3  ) was performed on the same rna preparation . serum specimens from horses in havana and havana province were tested for antibodies to wnv by using a competitive enzyme - linked immunosorbent assay ( elisa ) with monoclonal antibodies 3.1112 g and 6b6c-1 as described by blitvich et al . we tested 210 serum specimens from horses collected as part of an infectious anemia study . the immunoglobulin m ( igm ) test was not performed because horses were never suspected of having wnv and did not have any history of suspected viral encephalitis or other illness or symptoms . an inhibition value > 30% was used as the diagnostic criterion to identify flavivirus antibody ( table 1 ) . * wnv , west nile virus ; elisa , enzyme - linked immunosorbent assay ; prnt , plaque reduction neutralization test ; slev , saint louis encephalitis virus . the cuban health ministry and medical services division conducted surveillance for encephalitis of unknown origin in patients > 30 years of age . serum and cerebrospinal fluid specimens were shipped at 4c to the tropical medicine institute " pedro kouri . " human sera were screened for wnv igm and igg by using commercial igm and igg elisa kits ( focus technologies , cypress , ca , usa ) according to manufacturer 's instructions . hemagglutination - inhibition ( hi ) tests were also undertaken with wnv and saint louis encephalitis virus ( slev ) antigen ( 14 ) . reactive serum samples were further tested by a plaque reduction neutralization test ( prnt ) with wnv ( ny99 , ontario , canada , 2001 isolate ) , slev ( parton strain , american type culture collection catalog no . vr-1265 ) , and dengue virus ( dengue 2 , ng - c strain ) . prnt was performed to confirm wnv - specific antibody and was carried out as described previously ( 15 ) by using a neutral red double - overlay procedure . horses or human patients were considered seropositive for a particular flavivirus if the 90% prnt titer for that virus was > 4-fold greater than the neutralization titers determined for other viruses used in the assay . endpoint titrations were defined as the highest dilution of serum that reduced plaque formation by > 90% . most ( 58% ) of the 1,217 birds tested were resident species of cuba , primarily chestnut manakins ( lonchura malaccas ) , blue jays ( cyanocitta cristata ) , herring gulls ( larus argentatus ) , yellow - faced grassquits / olive finches ( tiaris olivacea ) , and northern parulas ( parula americana ) . nineteen ( 9.0% ) of the 210 horses had serum specimens with antibodies to flaviviruses ( table 1 ) . four and 8 animals had wnv- and slev - specific antibodies , respectively , in the prnt . two horses seropositive for wnv came from havana city , and 2 others came from havana province . seven serum samples had antibodies to undetermined flaviviruses on the basis of results of neutralization assays , and further virus characterization was not performed . none of the horses , including those that were positive for flavivirus antibodies , showed any signs of illness at the time of serum collection . both acute - phase and convalescent - phase serum specimens from these patients were positive for flavivirus antibody by igm and igg elisas ( table 2 ) . convalescent - phase serum samples were tested for wnv - specific antibody by prnt , and the neutralization titers were 320 and 160 , respectively . neutralizing antibodies against slev or dengue virus these persons are the first to have confirmed cases of wnv - associated illness in cuba . both patients had histories of febrile illness , muscle weakness , and encephalitis , and both were hospitalized . these persons had jobs that required them to spend large amounts of time outdoors , and they lived in communities in santi spiritus and villa clara in central cuba . , table 2 ) , who also resided in santi spiritus , had a low wnv titer by hi assay but had neutralizing antibodies to wnv , which suggests a past wnv infection . this patient was identified during surveillance in 2004 but may have been exposed to wnv in 2003 . * wnv , west nile virus ; elisa , enzyme - linked immunosorbent assay ; prnt , plaque reduction neutralization test ; ig , immunoglobulin ; hi , hemagglutination inhibition ; slev , saint louis encephalitis virus ; denv , dengue virus ; nd , not done .  acute - phase / convalescent - phase serum samples . serum specimens from the 10 remaining patients were negative for wnv igm but were positive for flavivirus igg by elisa ; most of these were also positive by hi assay ( table 2 ) . one person appeared to have been exposed to dengue virus , and 4 had antibodies to unidentified flaviviruses . seroconversions were not demonstrated in any of these persons , so we can not say whether their illnesses were associated with slev or dengue virus infections . we report the first evidence of antibodies to wnv in horses and humans in cuba . the fact that human and horse infections have been detected strongly suggests that a local amplification cycle has been established in cuba . the mode of entry of the virus into cuba is unknown . in north america , avian death from wnv infection has been well documented ( 35 ) . however , none of the dead birds collected during this study showed evidence of viral infection . although 1,217 animals were tested , a more intensive dead bird surveillance program may be needed to identify animals that die from wnv infection . resident bird species in cuba may be less susceptible to wnv infection , and death rates among birds in the caribbean may be lower than those observed in canada and the united states . this study also provides evidence that suggests wnv and slev may co - circulate in cuba . further studies are required to confirm these observations and to characterize the transmission cycles involved . finally , expansion of existing mosquito control programs in cuba , which currently focus on aedes aegypti and dengue prevention , may be required to respond to this new public health threat .
a surveillance system to detect west nile virus ( wnv ) was established in cuba in 2002 . 
 wnv infection was confirmed by serologic assays in 4 asymptomatic horses and 3 humans with encephalitis in 2003 and 2004 . 
 these results are the first reported evidence of wnv activity in cuba .