Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 9, Number 7—July 2003

Serologic Evidence of West Nile Virus Transmission, Jamaica, West Indies

Article Metrics
citations of this article
EID Journal Metrics on Scopus
Alan P. Dupuis*Comments to Author , Peter P. Marra†, and Laura D. Kramer*
Author affiliations: *New York State Department of Health, Slingerlands, New York, USA; †Smithsonian Environmental Research Center, Edgewater, Maryland, USA

Cite This Article


In spring 2002, an intensive avian serosurvey was initiated in Jamaica, Puerto Rico, and Mexico. We collected >1,600 specimens from resident and nonresident neotropical migratory birds before their northerly migrations. Plaque reduction neutralization test results indicated specific neutralizing antibodies to West Nile virus in 11 resident species from Jamaica.

West Nile virus (WNV) is maintained in nature between birds and Culex species mosquitoes (1,2). Unlike other viruses maintained in bird and mosquito transmission cycles (for example, St. Louis encephalitis, western equine encephalomyelitis, and eastern equine encephalomyelitis), the WNV strain responsible for the current epizootic in the Western Hemisphere is associated with a high number of avian deaths (3). Although crows and other corvids appear to be especially susceptible to disease (4), WNV has been documented in >190 bird species, including neotropical migratory species and exotic zoo specimens (5).

Birds have been implicated in spreading WNV during migratory events in Europe, Asia, Africa, and the Middle East (69). Because of the apparent ease of infecting a multitude of avian hosts, WNV can potentially be introduced during annual migratory events in the Western Hemisphere. Predicting the incursion of WNV into the tropics is complicated by our incomplete knowledge regarding geographic connectivity in populations of migratory birds between winter and summer. We do not know where most species of migratory birds from North America spend their winter, where wintering birds spend their summer, or the routes they use while in transit. Evidence from mark-and-recapture efforts suggests that birds from the northeastern United States tend to winter in the southeastern United States and Greater Antilles (e.g., Puerto Rico, Jamaica) whereas birds from the western United States migrate to Mexico and Central America (1012). In response to the potential introduction of WNV in tropical America during the fall migrations, we established a network of monitoring sites on the overwintering grounds of neotropical migratory birds in Jamaica, Puerto Rico, and Mexico.

The Study

The primary goal of the monitoring system was to obtain a large number of blood specimens from birds belonging to as many species as possible across the Caribbean. At seven study sites in Jamaica, Puerto Rico, and the Yucatan Peninsula of Mexico, we erected 15 mist nets (12-m) daily for 3 to 4 weeks during January through March 2002. We collected 1,619 avian blood specimens, which represented 98 species, 25 families, and eight orders. In Jamaica, 542 samples were collected from Westmoreland, Manchester, and St. Catherine Parishes; 649 samples were collected at Roosevelt Roads Naval Station in Puerto Rico; and 430 samples were collected from the states of Yucatan and Campeche in Mexico (Table 1). At the time of capture, all migratory birds were banded with an aluminum U.S. Fish and Wildlife Service band and 3–5 breast feathers were removed for isotope analysis. Resident birds had outer rectrices cut. All birds were evaluated for age and gender if possible, bled with microcapillary tubes from the brachial vein, and released. Blood was added immediately to BA-1 medium, consisting of M199 medium with Hank’s salts, 1% bovine albumin, TRIS base (tris [hydroxymethyl] aminomethane), sodium bicarbonate, 20% fetal bovine serum (FBS), and antibiotics. The samples were placed in a cooler on ice packs until storage in a –20°C freezer. Samples were sent on dry ice or hand-carried on ice packs to the Arbovirus Laboratories, Wadsworth Center, New York State Department of Health for serologic analysis and virus isolation attempts.

Specimens were screened at 1:100 for antibodies against flaviviruses by using an indirect enzyme-linked immunosorbent assay (ELISA) (13). Samples with a P/N ratio >2.0 were tested further by a plaque reduction neutralization test (PRNT) for Ilhéus virus (ILHV), St. Louis encephalitis virus (SLEV), and WNV, as described (14). The particular virus strains used for the PRNTs were ILHV (original), SLEV 59268 Parton, and WNV (3100365), an isolate from a pool of Culex sp. mosquitoes collected in Staten Island, New York. The indirect ELISA was chosen to screen the samples in order to take advantage of its ability to detect antibodies against a wide range of flaviviruses. PRNT was used as a confirmatory assay to differentiate among recognized flaviviruses (15,16). Briefly, serial dilutions of test samples were mixed with an equal amount of virus suspension containing 200 PFU/1 mL and incubated at 37°C for 1 h. We then added 0.05 mL of each virus-diluted blood sample onto 1 well of a 12-well tissue culture plate containing confluent monolayers of African green monkey kidney cells (Vero). The plate was incubated for 1 h at 37°C, after which an agarose overlay was added and incubation was continued. When virus plaques became visible, we added a second overlay containing neutral red and counted plaques. The antibody titer reported is the dilution of serum that inhibited 90% of the test virus inoculum. For virus isolation attempts using confluent Vero cell monolayers, 0.1 mL of each serum specimen was added onto one well of a six-well tissue culture plate, incubated for 1 h, rinsed with phosphate-buffered saline, and then the cells were refed with minimum essential medium containing 2% FBS. Cells were monitored once a day for 5 days for cytopathic effect. Cell cultures showing any abnormal cell morphology were then blind passaged after 5 days.

ELISA results indicated 34 of 1,413 serum specimens tested from the three study sites contained immunoglobulin G antibody against a flavivirus. Of the 34 reactive samples representing 20 bird species, 27 were collected in Jamaica (26 residents, 1 migrant), 5 in Puerto Rico (3 residents, 2 migrants), and 2 in Mexico (1 resident, 1 migrant). PRNTs on the Jamaican bird samples indicated 18 WNV infections, 3 SLEV infections, 5 undetermined flavivirus infections (positive results for two or more viruses without a fourfold difference in antibody titer); one additional reactive serum sample was negative for the three viruses tested. Results on the serum samples collected in Puerto Rico indicated one WNV infection in a migratory bird and one SLEV infection in a resident bird; three additional reactive serum samples were negative for all three viruses tested. In Mexico, we found evidence of WNV infection in one migrant bird and SLEV infection in one resident bird (Table 2). Virus isolation attempts were negative for all 1,603 specimens tested (16 samples destroyed). Negative isolation results were not entirely unexpected, considering that birds are viremic for a short period of time (17) and maintaining a proper cold chain (i.e., temperatures) to preserve virus is difficult when working in the tropics.


We detected neutralizing antibodies to WNV in resident birds from two parishes in Jamaica. This detection marks the earliest evidence of WNV introduction into the neotropics; WNV antibodies have been demonstrated in birds and horses in Mexico (late 2002, spring 2003) (18,19) and detected in resident birds from the Dominican Republic (spring 2003) (20). This evidence of WNV in the neotropics may be an important development in the spread of the virus. The tropics provide all the necessary components (i.e., high temperatures, dense avian populations, and large numbers of Culex sp. mosquitoes) to maintain an enzootic focus. Furthermore, the climates of Mexico and the Caribbean are suitable for year-round transmission of the virus. No dead birds have been reported in Jamaica, but surveillance activity there is less intensive than in the United States; the study sites, being rural in nature, are not conducive to observing dead birds. Another contributing factor to the lack of reports of dead birds may be the rapid decomposition of dead birds as a result of the heat, humidity, and detritivore foragers, such as ants.

Arbovirus activity, particularly of flaviviruses, is well documented in the Caribbean and Mexico. Dengue and yellow fever viruses are recurring public health threats in these areas. SLEV is endemic to Mexico and has been isolated from mosquitoes and one Northern mockingbird (Mimus polyglottos) nestling in Jamaica (21). This disease is still active in the region, and its known range may have expanded into Puerto Rico, considering the one seropositive Caribbean elaenia (Elaenia martinica) sampled during this study, although the antibody may have resulted from infection with yet another flavivirus. Neutralizing antibodies to WNV in migratory birds collected in Mexico and Puerto Rico, coupled with the apparent absence of antibody to WNV in the resident bird population, indicate that infection likely occurred in an enzootic area of the United States, but this observation shows that individual birds from at least three species of neotropical migratory birds have survived WNV infection and may serve as hosts for spreading the virus. The results from this study suggest that WNV now appears to be established in Jamaica, on the basis of the neutralizing antibodies to WNV found in the resident bird population.

Mr. Dupuis is an assistant research scientist in the Arbovirus Laboratories, Wadsworth Center, New York State Department of Health. His research focuses on the role of birds in the maintenance of arbovirus transmission cycles and dispersal of the virus.



We thank Brian Gibbons, Karin Roux, Javier Salgado-Ortiz, Joe Smith, and Jenn Barg for collecting field samples and Robert Reitsma, Kim Kent, and Donna Young for tremendous logistical and laboratory support.

This work was supported by Grant Agreement Number U50/CCU320544-01 from the Centers for Disease Control and Prevention (CDC) and the National Science Foundation (NSF), Grant Number DEB 0089565. Its contents are solely the responsibility of the authors and do not represent the official views of CDC or NSF.



  1. Hayes  CG. West Nile fever. In: Monath TP, editor. The arboviruses: epidemiology and ecology. Vol. V. Boca Raton (FL): CRC Press, Inc.; 1989. p. 59–88.
  2. Kramer  LD, Bernard  KA. West Nile virus in the western hemisphere. Curr Opin Infect Dis. 2001;14:51925.PubMedGoogle Scholar
  3. Bernard  KA, Maffei  JG, Jones  SA, Kauffman  EB, Ebel  GD, Dupuis  AP II, West Nile virus infection in birds and mosquitoes, New York State, 2000. Emerg Infect Dis. 2001;7:67985. DOIPubMedGoogle Scholar
  4. Bernard  KA, Kramer  LD. West Nile virus activity in the United States, 2001. Viral Immunol. 2001;14:31938. DOIPubMedGoogle Scholar
  5. U.S. Department of the Interior, U.S. Geological Survey, National Wildlife Health Center. Species found positive for WNV in surveillance efforts. [Cited April 5, 2003.] Available from: URL:
  6. Malkinson  M, Banet  C, Weisman  Y, Pokamunski  S, King  R, Drouet  MT, Introduction of West Nile virus in the Middle East by migrating white storks. Emerg Infect Dis. 2002;8:3927. DOIPubMedGoogle Scholar
  7. Malkinson  M, Banet  C. The role of birds in the ecology of West Nile virus in Europe and Africa. Curr Top Microbiol Immunol. 2002;267:30922.PubMedGoogle Scholar
  8. Ernek  E, Kozuch  O, Nosek  J, Teplan  J, Folk  C. Arboviruses in birds captured in Slovakia. J Hyg Epidemiol Microbiol Immunol. 1977;21:3539.PubMedGoogle Scholar
  9. Rappole  JH, Derrickson  SR, Hubalek  Z. Migratory birds and spread of West Nile virus in the Western Hemisphere. Emerg Infect Dis. 2000;6:31928. DOIPubMedGoogle Scholar
  10. Johnson  LS, Wise  J. Wintering grounds of North American house wrens as revealed by band recoveries. J Field Ornithol. 2000;71:5015.
  11. Webster  MS, Marra  PP, Haig  SM, Bensch  S, Holmes  RT. Links between worlds: unraveling migratory connectivity. Trends Ecol Evol. 2002;17:7682. DOIGoogle Scholar
  12. Elphick  J. The atlas of bird migration. New York: Random House; 1995.
  13. Ebel  GD, Dupuis  AP, Nicholas  D, Young  D, Maffei  J, Kramer  LD. Detection by enzyme-linked immunosorbent assay of antibodies to West Nile virus in birds. Emerg Infect Dis. 2002;8:97982.PubMedGoogle Scholar
  14. Lindsey  HS, Calisher  CH, Matthews  JH. Serum dilution neutralization test for California group virus identification and serology. J Clin Microbiol. 1976;4:50310.PubMedGoogle Scholar
  15. DeMadrid  AT, Porterfield  JS. The flaviviruses (group B arboviruses): a cross-neutralization study. J Gen Virol. 1974;23:916. DOIPubMedGoogle Scholar
  16. Calisher  CH, Karabatsos  N, Dalrymple  JM, Shope  RE, Porterfield  JS, Westaway  EG, Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol. 1989;70:3743. DOIPubMedGoogle Scholar
  17. Komar  N, Langevin  S, Hinten  S, Nemeth  N, Edwards  E, Hettler  D, Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis. 2003;9:31122.PubMedGoogle Scholar
  18. West Nile virus, equine, 2002—Mexico (Tamaulipas). ProMed mail archive no. 20030325.0739. [Cited 25 March 2003]. Available from: URL
  19. West Nile virus, birds—Mexico (North). ProMed mail archive no.: 20030315.0640. [Cited 15 March 2003]. Available from: URL:
  20. West Nile virus, birds—Dominican Republic. ProMed mail archive no.: 20030315.0645. [Cited 15 March 2003]. Available from: URL:
  21. Belle  EA, King  SD, Griffiths  BB, Grant  LS. Epidemiological investigation for arboviruses in Jamaica, West Indies. Am J Trop Med Hyg. 1980;29:66775.PubMedGoogle Scholar




Cite This Article

DOI: 10.3201/eid0907.030249

Table of Contents – Volume 9, Number 7—July 2003

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Alan P. Dupuis II, Griffin Laboratory, 5668 State Farm Rd., Slingerlands, NY 12159, USA; fax: (518) 869-4530

Send To

10000 character(s) remaining.


Page created: December 22, 2010
Page updated: December 22, 2010
Page reviewed: December 22, 2010
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.