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West Nile virus (WNV) is a flavivirus (Flaviviridae) distributed throughout Africa, the Middle East, Australasia, Europe, and North America, and is maintained in nature between birds and ornithophilic Culex species mosquitoes.1,2 Since the discovery of the virus in New York City in 1999, WNV has spread throughout the United States, Canada, and Mexico.3–5 Equines and numerous species of Neotropical resident birds also have been found with WNV neutralizing antibodies, indicating local transmission, in Central America, Jamaica, the Dominican Republic, and Guadeloupe.5–9
Birds have been implicated in WNV dispersal during migration in Europe, the Middle East, Asia, and Africa.10–13 Therefore, we hypothesized that WNV transmission might be initiated on the overwintering grounds of Nearctic-Neotropical migratory birds. In 2001, we established a network of sampling sites in the American tropics, specifically Jamaica, Mexico, Puerto Rico, and in 2004 Cuba to monitor for WNV incursion. In Jamaica, 2002, 18 blood samples from 12 year-round resident species were found with neutralizing antibodies to WNV.6 In 2003, 32 samples from resident birds collected in the Yucatan Peninsula of Mexico (n = 7) and Jamaica (n = 25) were positive for WNV neutralizing antibody (Dupuis AP II, unpublished data). Resident bird samples collected in Puerto Rico in 2002 and 2003, were negative for antibodies to WNV, although sampling was equally intense with respect to number of samples collected, geographical extent, and habitat diversity.
Two sites in the Caribbean, Cuba and Puerto Rico, where WNV had not been documented previously were monitored during the dry season in 2004. Fifteen 12-meter mist nets were erected daily for four weeks during the period January–March 2004 in both locations. More than 1,900 avian blood specimens were collected from resident (n = 1,071) and migratory (n = 868) birds. Migratory/resident status was assessed according to Raffaele and others.14 A taxonomically diverse range of birds was sampled. Fifty-six species representing nine orders were collected in Puerto Rico and Cuba. In Cuba, 734 (364 residents and 370 migrants) samples were collected at the United States Naval Base at Guantanamo Bay (19°54'N, 75°9'W). In Puerto Rico, 1,205 (710 residents and 495 migrants) samples were collected at Roosevelt Roads Naval Station (18°4'N, 65°7'W), Cabo Rojo National Wildlife Reserve (17°55'N, 71°38'W), and Vieques National Wildlife Reserve (18°13'N, 65°40'W). At the time of capture, all migratory birds were banded with an aluminum United States Fish and Wildlife Service band, and 3–5 breast feathers and one tail feather were removed for isotope analysis. Resident birds had the lowest portion of the right outer rectrix cut. Blood was collected into microcapillary tubes by lancing the brachial vein with a 27-gauge needle. All birds were aged and sexed if possible and then released. Samples were processed as described,6 and sent on dry ice to the Arbovirus Laboratories (Wadsworth Center, New York State Department of Health, Slingerlands, NY) for serologic analysis. All avian work was approved by animal use protocol 03-355.
Specimens were screened at 1:100 for antibodies against flaviviruses using an indirect enzyme-linked immunosorbent assay (ELISA).15 Samples with a positive:negative ratio > 2.0 were tested further by a plaque-reduction neutralization test (PRNT) against St. Louis encephalitis virus (SLEV) and WNV as described.16 The virus strains used for the PRNTs were Parton (SLEV) and 3100365 (WNV), an isolate from a pool of Culex sp. mosquitoes collected in Staten Island, New York. The indirect ELISA was used to screen the samples to take advantage of its ability to detect antibodies against a wide range of flaviviruses in a great variety of avian species. The PRNT was used as a confirmatory assay to differentiate among recognized flaviviruses.17,18 The antibody titer reported is the dilution of serum that inhibited 90% of the test virus inoculum (i.e., PRNT90). The ELISA results indicated 18 of 1,939 serum specimens tested from the two study sites contained IgG antibody against a flavivirus. Seven of the 18 reactive samples representing 11 avian species were collected in Cuba, and 11 were collected in Puerto Rico. The PRNTs on the Cuban bird samples indicated three WNV infections (two residents and one migrant) and four undetermined flavivirus infections (one resident and three migrants). Results on the sera collected in Puerto Rico indicated 10 WNV infections (one resident and nine migrants) and one SLEV infection in a resident bird (Table 1
). Overall WNV seroprevalence rates are 0.55% and 0.14%, among the resident bird populations in Cuba and Puerto Rico respectively. The rates reported are lower than those seen in Mexico and Jamaica, i.e., 1.85% and 4.13%, respectively (Dupuis AP II, unpublished data), indicating a lower level of virus activity in the sampled bird populations at Roosevelt Roads and Guantanamo Bay Naval Stations.
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Puerto Rican and Cuban resident birds that had evidence of neutralizing antibodies to WNV are conspecific to WNV antibody-positive birds found in other parts of the Caribbean. Bananaquits (Coereba flaveola) and red-legged thrushes (Turdus plumbeus) are abundant and ubiquitous throughout their distributions.14 The role of these species in WNV maintenance is unknown, but experimentally infected American robins (Turdus migratorius), a similar member of the thrush family (Turdidae), have been shown to be susceptible to WNV infection, produce high viral titers, and maintain the viremia for up to five days.20 Bananaquits belong to a single species family, Coerebidae, and have not been subjected to experimental inoculations. Green herons (Butorides virescens) and little blue herons (Egretta caerulea) have Caribbean populations that are permanent residents as well as populations that are transient. Being conservative, we considered these species migratory. Birds from similar species of the wading bird family (Ardeidae) have been found with antibodies to WNV during a study in India.21
The first evidence of SLEV in Puerto Rico was reported in 2003 at the Roosevelt Roads Naval Station study site.6 Here we report evidence of SLEV transmission on the southwestern coast of Puerto Rico (Cabo Rojo National Wildlife Reserve), suggesting multiple foci or a widespread distribution of the virus.
Migratory birds are considered to be prime candidates for dispersal of WNV across North America and the Caribbean. This work demonstrates 10 birds representing six species of Nearctic-Neotropical migrants were exposed to WNV, survived infection, and continued migratory behavior. However, these migrants may have been infected in the tropics, in which case there is a potential for annual reseeding of the virus to the United States mainland. Great numbers of warblers (Parulidae) overwinter on the islands of the Caribbean in coastal mangroves and mangrove/scrub forest ecotone. Birds of this family and other passerines as well as waders (herons), raptors (falcons, hawks), and shorebirds (gulls, sandpipers) should be considered as potential dispersal agents of WNV.
Results from this study emphasize the need for more thorough ecologic studies of WNV and other arboviruses in tropical ecosystems. To date, WNV has not been isolated from mosquitoes, equines, or other vertebrates in the Caribbean. There has been a conspicuous lack of WNV-associated morbidity and mortality in vertebrates in this region until the recently reported three human cases in Cuba (February 2005).22 At least three projects are critical to begin to address this difference. First, the strains of WNV circulating in the Caribbean need to be isolated and genotypic and phenotypic comparisons made with strains collected from the United States. Increased surveillance activities (i.e., testing of mosquitoes and dead birds, as well as conducting avian serosurveys) where seropositive birds were found, should produce a virus isolate provided transmission is still occurring. Second, the host competence of resident avifauna for WNV should be evaluated using both local and geographically distinct viral strains. And finally, the vectorial capacity of Caribbean mosquito populations should be assessed. These three endeavors will help clarify the apparent differences in ecology of WNV in the western hemisphere between temperate and tropical ecosystems.
Received February 4, 2005. Accepted for publication March 15, 2005.
Acknowledgments: We thank Brian Gibbons, Sara Campbell, Ryan Peters, Bill Deluca, Nora Diggs, Dr. Lynn Cassell, Joe Smith, Jim Goetz, Colin Studds, Crista Cramer, and Pierre Goulet for the collection of field samples; Joe Maffei and Alex Ciota for laboratory support; and Chris Eberly for logistical support in Cuba. We also thank the Department of Defense and the U.S. Fish and Wildlife Service for access and cooperation at the Naval Stations and wildlife refuges.
Financial support: This work was supported by grant agreement number U50/CCU320544-01 from the Centers for Disease Control and Prevention (CDC). Its contents are solely the responsibility of the authors and do not represent the official views of the CDC.
* Address correspondence to Mr. Alan P. Dupuis, II, Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159. E-mail: dupuis{at}wadsworth.org 
Authors’ addresses: Alan P. Dupuis, II, Matthew J. Jones, Karen L. Louie, and Laura D. Kramer, Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159, Telephone: 518-869-4531, Fax: 518-869-4530, E-mail: dupuis{at}wadsworth.org. Peter P. Marra and Robert Reitsma, Smithsonian Environmental Research Center, 647 Contees Wharf Road, Edgewater, MD 21037, Telephone: 443-482-2224, E-mail: marrap{at}si.edu.
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