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POTENTIAL ROLE OF SYLVATIC AND DOMESTIC AFRICAN MOSQUITO SPECIES IN DENGUE EMERGENCE

MAWLOUTH DIALLOInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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AMADOU A. SALLInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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ABELARDO C. MONCAYOInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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YAMAR BAInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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ZORAIDA FERNANDEZInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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DIANA ORTIZInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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LARK L. COFFEYInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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CHRISTIAN MATHIOTInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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ROBERT B. TESHInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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SCOTT C. WEAVERInstitut Pasteur de Dakar, Dakar, Senegal; Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas

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Dengue virus 2 (DENV-2) strains that circulate in sylvatic habitats of Senegal and other parts of west Africa are believed to represent ancestral forms that evolved into endemic/epidemic strains that now circulate widely in urban areas of the tropics. Previous studies suggested that the evolution of the endemic/epidemic strains was mediated by adaptation to the peridomestic mosquito vectors Aedes aegypti and Ae. albopictus. We conducted experimental infections using sylvatic and peridomestic Senegalese mosquitoes, and both sylvatic and urban DENV-2 strains to determine if endemic DENV-2 adaptation was vector species specific, and to assess ancestral vector susceptibility. Aedes furcifer and Ae. luteocephalus, probable sylvatic vectors, were highly susceptible to both sylvatic and urban DENV-2 strains. In contrast, sylvatic Ae. vittatus and both sylvatic and peridomestic populations of Ae. aegypti were relative refractory to all DENV-2 strains tested. These results indicate that adaptation of DENV-2 to urban vectors did not result in a loss of infectivity for some African sylvatic vectors. Implications for dengue emergence in west Africa are discussed.

INTRODUCTION

Dengue is a viral disease caused by four viral serotypes (dengue virus 1–4 [DENV1–4]) belonging to the genus Flavivirus of the family Flaviviridae. These viruses are transmitted to humans by mosquitoes of the genus Aedes and infect approximately 100 million people annually.1,2 Until the mid 1920s, dengue was considered a mild disease without a serious public health impact. This situation changed dramatically with its increasing prevalence after the Second World War and the appearance of dengue hemorrhagic fever (DHF) and associated shock syndrome, which can be fatal.3 Factors leading to dengue emergence include ecologic and demographic disruptions, increases in human travel resulting in DENV introduction into previously disease-free regions, and an expansion of the range of the principal epidemic mosquito vector Aedes aegypti.

Two distinct transmission cycles have been described for DENV: Endemic and epidemic cycles that occur in urban/periurban environments and involve human reservoir and amplification hosts. The peridomestic mosquito Ae. aegypti is the principal DENV vector, with Ae. albopictus and other anthropophilic Aedes mosquitoes serving as secondary vectors. Ecologically distinct, sylvatic, enzootic cycles of DENV occur in west Africa and Malaysia, probably involving non-human primate reservoir hosts and sylvatic Aedes spp. mosquito vectors.4 The two kinds of DENV cycles are also evolutionarily distinct, and all four serotypes of endemic/epidemic DENV are believed to have evolved independently from sylvatic progenitors during the past few 1,000 years.5

The sylvatic DENV transmission cycles have received little study. In contrast to Asia, Oceania, the Caribbean, and Latin America, where human-to-human transmission by peridomestic vectors (Ae. aegypti and Ae. albopictus) is the predominant form of DENV circulation and human infection, sylvatic circulation seems to be predominant in Africa.6 Although several DEN-2 epidemics have been documented in Burkina Faso,6 Kenya,7 Somalia,8 Sudan,9 and Djibouti,10 no DHF has been described and many of the etiologic strains may have been introduced from other regions of the world.11,12 Many different factors could play a role in the apparent lack of severe DEN in Africa, including: 1) differences in the virulence of DENV strains endemic to Africa; 2) human host-related factors; people of African descent may be less predisposed to severe DEN fever and DHF than persons of Caucasian or Asian background13 and thus human infections in west Africa may be less frequently recognized; 3) cross-protection from heterologous flavivirus antibodies might be more prevalent or stronger than in other parts of the world with different flaviviruses; and 4) the vectorial competence of African mosquitoes may differ from populations in other regions of the tropics.

The origins of endemic/epidemic DENV strains from sylvatic ancestors also raise important questions regarding host range changes and possible adaptation associated with the establishment of urban transmission. To determine if endemic emergence involved adaptation to more efficient infection of urban vectors, both urban (derived) and sylvatic (ancestral) DENV-2 strains were used to infect Ae. aegypti and Ae. albopictus from several geographic populations. Endemic/epidemic strains consistently infected both species more efficiently than sylvatic strains from Africa and Malaysia, supporting the hypothesis that the emergence of urban dengue was mediated by viral adaptation to peridomestic vectors.14

In the present study, we tested this hypothesis that adaptation to urban vectors was species specific by comparing the susceptibility to sylvatic versus endemic/epidemic DENV-2 strains of several mosquito species implicated in sylvatic DENV-2 transmission in Senegal. The prediction of this hypothesis is that the ancestral, sylvatic DENV-2 vectors are more susceptible to the ancestral, sylvatic virus strains than to the endemic strains. We also used these studies to test the hypothesis that insusceptibility of Senegalese mosquitoes to endemic DENV-2 strains explains, at least in part, the paucity of DEN epidemics in west Africa.

MATERIAL AND METHODS

Mosquitoes.

Except for one peridomestic population of Ae. aegypti from Koung Koung, all mosquitoes were collected in the Kedougou forest gallery, located in southeastern Senegal, where sylvatic DENV-2 circulation has been described.15 A total of five populations belonging to four species were tested: Ae. furcifer and Ae. luteocephalus, believed to be the principal DEN-2 sylvatic vectors in Senegal;15 both sylvatic and domestic populations of Ae. aegypti, the principal endemic/epidemic vector around the world;4 and Ae. vittatus, which was found infected by DENV-2 virus during the last epizootic amplification in Kedougou15 (Table 1). Adult female Ae. furcifer and Ae. luteocephalus were captured by human landing collections using volunteers vaccinated against yellow fever and taking malaria prophylaxis. The study protocol included informed consent and was reviewed and approved by the Conseil de Perfectionnement of the Pasteur Institute and by the Senegalese Ministry of Health. Larvae and pupae of Ae. aegypti and Ae. vittatus were collected from the field. The F1 generation adults used for experiments were reared in the laboratory using standard methods16 at 27 ± 1°C and a relative humidity of 70–75%, with a 12-hour photoperiod. The parental females were triturated and inoculated into Aedes pseudoscutellan’s (AP61) cell cultures and assayed by immunofluorescense to confirm that they were not naturally infected with DENV-2 or other cytolytic arboviruses.

Virus strains.

Four DENV-2 strains were used for experimental infections (Table 2): two epidemic strains (New Guinea C and 1349) and two sylvatic strains (PM33974 and DakAr2022). These strains were selected based on their low passage histories and their use in previous studies of urban vector susceptibility.14 Their classification as sylvatic or epidemic strains was based on their phylogenetic placements reported previously.5 Virus stocks were prepared by inoculating Ae. albopictus (C6/36) cell cultures maintained in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, nonessential amino acids, and antibiotics. After infection, the cells were incubated 14 days (the medium was changed at day 7). Cell culture media were aliquoted, frozen at −80°C, and used directly as viral stocks to prepare artificial, viremic blood meals as described previously.14 Although fresh DENV stocks are more infectious for oral infection of mosquitoes,17 we prepared blood meals from frozen virus stocks so that comparable and reproducible titers could be used. Virus stocks were titrated using serial dilutions in 96-well plates of C6/36 cells and infection was detected by immunofluorescence using DENV-2 ascitic fluids as described previously.14

Experimental infections.

Three to five-day-old F1 female mosquitoes were placed into 0.45-liter cardboard cages and sucrose-starved for 24 hours before being offered an infectious blood meal. The infectious meal consisted of a 33% volume of sheep erythrocytes (Colorado Serum Company, Denver, CO), a 20% volume of fetal bovine serum, 1% (w/v) sucrose, and 5 mM ATP. The blood meals were administered in glass membrane feeders using mouse or chicken skins as membranes. Mosquitoes were allowed to feed for 30 minutes, and a sample of the blood meal was then stored at −80°C before titration. The mosquitoes were cold-anaesthetized, and engorged specimens were incubated at 28°C and a relative humidity of 70–80%, and provided 10% sucrose for 14 days. After incubation, mosquitoes were cold-anaesthetized and their legs and wings were removed. Detection of DENV in the mosquito body without infection of the legs, which contain hemolymph, indicated a non-disseminated infection limited to the midgut, whereas the presence of virus in both the mosquito body and legs indicated an infection disseminated into the hemocoel. The infection (number of infected mosquito bodies/number of mosquitoes tested) and dissemination (number of mosquitoes with infected legs/number of infected mosquitoes) rates were compared among cohorts ingesting DENV titers within a range of one log10 using Fisher’s exact test and Epi-Info software (Centers for Disease and Control and Prevention, Atlanta, GA); P < 0.05 was considered significant.

RESULTS

Testing of field-collected mosquitoes in AP61 cells yielded no evidence of natural infection by DENV or other cytolytic arboviruses. All artificial blood meal titers tested for infection of mosquitoes were ≥ 5.5 log10 50% tissue culture infectiousdoses (TCID50/mL). This titer was selected as a threshold based on preliminary data indicating high infection rates of Ae. luteocephalus using the PM33974 sylvatic DENV-2 strain. Titers less than 5 log10 TCID50/mL generally yielded low rates of infection. All mosquito species tested were susceptible to DENV-2 infection, with infection rates ranging from 3% to 97% (Table 3). Aedes furcifer and Ae. luteocephalus were the most susceptible species.

Aedes furcifer.

Aedes furcifer infection rates ranged from 26% to 97%; when results were compared from cohorts receiving similar oral doses (within one log10 titer), no consistent differences were observed between epidemic (NGC and 1349) and sylvatic (PM33974 and DakAr2022) strains in either infection or dissemination. However, unlike the two epidemic strains tested, the two sylvatic strains differed in their infection and dissemination rates in Ae. furcifer, with strain PM33974 exhibiting more efficient infection (P < 0.0001) and dissemination (P < 0.01) than strain DakAr2022, after blood meals of comparable titer.

Aedes luteocephalus.

Aedes luteocephalus was also highly susceptible to three of the four DENV-2 strains tested, with no consistent difference in infection rates between the sylvatic strains and endemic strain 1349 (Table 3). The absence of infection by endemic strain NGC might be explained by the relatively low blood meal titer; however, a high (67%) infection rate was obtained with sylvatic strain PM33974 tested with the same infectious dose. Disseminated infections were obtained for all virus strains except NGC, with variable rates that were comparable between the two sylvatic strains. However, dissemination rates were significantly higher with epidemic strain 1349, compared with those obtained with the sylvatic strain DakAr2022 (P < 0.05) following comparable oral doses.

Aedes aegypti and Ae. vittatus. In contrast to Ae. furcifer and Ae. luteocephalus, Ae. aegypti from Kedougou and Koung Koung and Ae. vittatus from Kedougou were relatively refractory to DENV-2 infection after ingestion of high DENV titers. Infection rates ranged from 13% to 15% in Ae aegypti from Kedougou, 3% to 10% in Ae. aegypti from Koung Koung, and 6% to 18% in Ae. vittatus; all three populations were significantly less susceptible than Ae. furcifer and Ae. luteocephalus after ingestion of comparable blood meal titers (P ≤ 0.01). As with the more susceptible mosquito species, no consistent differences in infection or dissemination efficiency were observed between the endemic and sylvatic DENV strains in Ae. aegypti or Ae. vitattus after similar oral doses (P ≥ 0.05). The only exception was that Ae. aegypti from Kedougou were significantly more susceptible to epidemic strain 1349 than to sylvatic strain PM33974 (P < 0.05). However, because infection was inefficient, the numbers of infected Ae. aegypti and Ae. vitattus tested for dissemination were very low. Likewise, no differences in infection or dissemination were observed between the arboreal (Kedougou) and peri-domestic (Koung Koung) populations of Ae. aegypti. Although it exhibited relative refractoriness to infection, infected Ae. vittatus exhibited high rates of dissemination into the hemocoel, even higher than the relatively susceptible Ae. furcifer (P < 0.01) and Ae. luteocephalus (P < 0.05) infected with comparable DENV-2 doses.

DISCUSSION

Although sylvatic DENV-2 transmission cycles in west Africa were described many years ago, no experimental studies have investigated the vectorial competence of mosquitoes found to be infected in enzootic regions. We demonstrated that several Senegalese mosquitoes are susceptible and capable of supporting disseminated DENV-2 infections. However, significant variation in susceptibility and dissemination was found, both with respect to the mosquito species and virus strains evaluated.

One important finding was the difference in susceptibility between Ae. aegypti and other sylvatic mosquitoes. Regardless of their sylvatic or peridomestic origin, Senegalese Ae. aegypti were less susceptible than Ae. furcifer and Ae. luteocephalus. Our findings indicate low susceptibility of Ae. aegypti from Senegal. In contrast, higher susceptibility has been reported for populations from Asia and South America infected using the same conditions and the same virus strains.18 Variation in susceptibility among geographic populations of epidemic DENV vectors (Ae. aegypti and Ae. albopictus) has been identified previously, along with variation in sylvatic DENV vectors from Asia infected with different DENV strains.1922

Genetic variation among Ae. aegypti populations from different geographic areas may explain the differences between our results and others reporting greater susceptibility. Aedes aegypti includes two forms or subspecies that are distinguished by morphologic, ecologic, and behavioral differences; Ae. aegypti aegypti, the peridomestic form that transmits DENV in urban areas of the tropics, is believed to have evolved from Ae. aegypti formosus, the ancestral African tree hole form. Previous studies showed that only Ae. aegypti aegypti exists in Asia and the New World, whereas in west Africa, including Senegal, only the sylvatic form Ae. aegypti formosus was identified.23,24 This subspecies is less susceptible to DENV infection than Ae. aegypti aegypti.20,25 However, the absence of Ae. aegypti aegypti in Senegal is questionable because of the lack of reliable methods to distinguish the subspecies. Available morphologic keys are not sufficiently accurate to distinguish Ae. aegypti aegypti from Ae. aegypti formosus or to identify possible intermediate forms.26 Aedes aegypti populations from Kedougou and Koung Koung used in this study exhibit distinct ecologic features; mosquitoes from Kedougou colonize tree holes and other sylvatic breeding sites and are zoophilic, whereas the Koung Koung mosquitoes are found within human habitations, use artificial water containers as breeding sites, and are highly anthropophilic like Ae. aegypti aegypti. Despite these apparent differences (peridomestic or sylvatic), our data indicate that the two Senegalese Ae. aegypti populations are not significantly different in their susceptibility to DENV-2. Testing of other west African Ae. aegypti populations and genetic investigations are needed to better characterize these populations and assess their potential as DENV vectors.

Surprisingly, our results indicated a lack of correlation between infection and dissemination rates in most mosquitoes infected with a given DENV-2 strain. Aedes furcifer and Ae. luteocephalus exhibited high infection rates but low dissemination rates, whereas Ae. aegypti and Ae. vittatus exhibited low infection but high dissemination rates. This suggests a tradeoff between infection and dissemination rates leading to transmission potential by all of these vectors. These findings contrast with those from peridomestic DENV-2 vectors, where infection and dissemination rates were high for both sylvatic and epidemic strains.14

Previously, we hypothesized that endemic/epidemic DENV strains evolved from sylvatic, ancestral forms that adapted to peridomestic vectors. This hypothesis predicted that endemic DENV-2 strains are more efficient at infecting the urban vectors Ae. aegypti and Ae. albopictus than the ancestral, sylvatic strains. This hypothesis was supported experimentally by the higher susceptibility of these urban species to endemic/epidemic DENV-2 strains.14 Here, we tested the related hypothesis that adaptation to peridomestic vectors was species specific, which predicts that the sylvatic DENV-2 strains from Africa, representing the ancestral form, are more efficient at infecting sylvatic mosquito vectors than the derived, epidemic DENV-2 strains. However, our data do not support this hypothesis; we found no consistent difference in infectivity between the endemic and sylvatic strains in any of the sylvatic mosquitoes. Because a sylvatic transmission cycle of DENV was also described in Malaysia,27,28 additional studies using sylvatic vectors from that region are needed to fully evaluate this hypothesis.

Our results suggest that the evolution of endemic/epidemic DENV-2 occurred without a loss of infectivity to the ancestral sylvatic vectors, and that the adaptation of endemic/epidemic DENV to urban vectors was not species specific. Moreover, the high susceptibility of the sylvatic vectors Ae. furcifer and Ae. luteocephalus to all DENV-2 strains tested supports the hypothesis that DENV-2 has a long history of contact with these mosquitoes in west Africa. The relative lack of susceptibility to DENV-2 infection exhibited by Senegalese populations of Ae. aegypti also occurred regardless of the mosquito population origin. This suggests that Ae. aegypti is a recent DENV vector and that west African populations were not involved in adaptation to this species that led to urban transmission.

In west Africa, many mosquitoes in the Aedes subgenera Stegomyia and Diceromyia, most notably Ae. furcifer, Ae. luteocephalus, and Ae. aegypti, are suspected of DENV-2 transmission. Although susceptibility and ability to transmit are not the only important factors determining vectorial capacity,29 our results improve understanding of the role of each species in DENV-2 transmission cycles and emergence potential. The vectorial role of Ae. furcifer and Ae. luteocephalus, hitherto suspected based on frequent DENV-2 isolations in nature,15,3033 is supported by their high susceptibility to infection. In the light of our data and their bionomics, Ae. furcifer and Ae. aegypti are good candidate vector species for domestic DENV-2 transmission. As indicated in previous studies, only Ae. furcifer is a strong candidate for virus exchange between the forest and human habitations. Of the susceptible forest mosquitoes, it is the most common in villages and the only one found infected in a domestic environment.15 In contrast, Ae. luteocephalus appears to be confined to the forest habitat.34

Although less susceptible to DENV-2 infection, a vector role of Ae. aegypti and Ae. vittatus cannot be ruled out because they exhibited an efficient dissemination rate that may enhance their vectorial competence. However, despite its capacity to disseminate the DENV-2 infections, Ae. vittatus probably has little or no role in domestic transmission. Although susceptible to dengue infection and found infected in nature,35 it is zoophilic and exhibits little peridomesticity. The lack of Ae. vittatus involvement in DENV-2 transmission is further supported by its lack of infection in epidemic areas, where it occurs along with Ae. aegypti and Ae. albopictus.36

Table 1

Senegalese Aedes mosquitoes used for experimental dengue virus 2 infection

Species Geographic origin Collection habitat
Ae. furcifer Kedougou Gallery forest
Ae. luteocephalus Kedougou Gallery forest
Ae. vittatus Kedougou Gallery forest
Ae. aegypti KDG Kedougou Gallery forest
Ae. aegypti KK Koung Koung Peridomestic
Table 2

Dengue-virus 2 strains used for experimental mosquito infections

Virus strain Host Geographic origin Year of isolation Phylogenetic classification Passage history*
* C6/36 = Ae. albopictus cell culture; SM = suckling mouse.
New Guinae C (NGC) Human New Guinea 1944 Epidemic Monkey 1, mosquito 4, C6/361
1349 Human Burkina Faso 1982 Epidemic Mosquito 2, C6/362
PM33974 Aedes africanus Guinea 1981 Sylvatic Toxorhynchites amboinensis 1, C6/362
DakAr2022 Aedes africanus Burkina Faso 1980 Sylvatic SM6, C6/362
Table 3

Infection and dissemination rates of Senegalese Aedes mosquitoes with dengue virus 2 strains*

Mosquito species† Virus strain Blood meal titer (TCID50/mL) Infection rate (%) Dissemination rate (%)‡
* TCID/501 = 50% tissue culture infectious dose.
† KDG = Kedougou collection site; KK = Koung Koung collection site.
‡ Number with infected legs/number infected.
Ae. furcifer NGC (epidemic) 8.0 16/17 (94) 12/16 (75)
NGC (epidemic) 9.5 18/31 (58) 4/18 (22)
1349 (epidemic) 6.5 15/18 (83) 11/15 (73)
1349 (epidemic) 9.2 23/30 (77) 7/23 (30)
PM33974 (sylvatic) 8.0 29/30 (97) 14/29 (48)
DakAr2022 (sylvatic) 7.0 6/23 (26) 1/6 (17)
DakAr2022 (sylvatic) 7.8 13/37 (35) 0/13 (0)
Ae. luteocephalus NGC (epidemic) 5.5 0/18 (0.0)
1349 (epidemic) 8.2 16/18 (89) 8/16 (50)
PM33974 (sylvatic) 5.5 14/21 (67) 2/14 (14)
PM33974 (sylvatic) 8.0 8/12 (67) 1/8 (13)
DakAr2022 (sylvatic) 9.2 11/19 (58) 3/11 (27)
DakAr2022 (sylvatic) 8.2 15/19 (79) 2/15 (13)
Ae. aegypti KDG NGC (epidemic) 6.2 0/14 (0)
1349 (epidemic) 8.0 0/20 (0)
1349 (epidemic) 8.8 6/24 (25) 4/6 (67)
PM33974 (sylvatic) 8.5 0/12 (0)
PM33974 (sylvatic) 8.8 0/17 (0)
PM33974 (sylvatic) 8.2 0/17 (0)
DakAr2022 (sylvatic) 8.2 4/27 (15) 1/4 (25)
Ae. vittatus NGC (epidemic) 5.8 0/41 (0)
PM33974 (sylvatic) 8.8 2/36 (6) 2/2 (100)
DakAr2022 (sylvatic) 6.5 10/54 (19) 6/10 (60)
Ae. aegypti KK 1349 (epidemic) 8.8 4/38 (10) 4/4 (100)
DakAr2002 (sylvatic) 7.2 1/32 (3) 1/1 (100)

*

Address correspondence to Dr. Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. E-mail: sweaver@utmb.edu.

Authors’ addresses: Mawlouth Diallo, Amadou A. Sall, and Yamar Ba, Institut Pasteur de Dakar, 36 Avenue Pasteur Dakar, Senegal, E-mails: diallo@pasteur.sn, asall@pasteur.sn, and ba@pasteur.sn. Abelardo C. Moncayo, Tennessee Department of Health, Communicable and Environmental Services, Cordell Hull Building, 425 Fifth Avenue North, Nashville, TN 37247-4911, E-mail: a-moncayo@onu.edu. Zoraida Fernandez, Instituto Venezolano de Investigaciones Cientificas, Carretera Panamericana Km 11, Altos de Pipe, Apartado Postal 1040-A, Caracas, Venezuela, Fax: 0212-5041489, E-mail: zofernam@hotmail.com. Diana Ortiz, Lark L. Coffey, Robert B. Tesh, and Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, Fax: 409-747-2415, E-mails: diortiz@hotmail.com, llcoffey@utmb.edu, rtesh@utmb.edu, and sweaver@utmb.edu. Christian Mathiot, World Health Organization, 58 Avenue Debourg, 69007 Lyon, France, Fax: 33-4-72-71-64-71, E-mail: mathiotc@lyon.who.int.

Acknowledgment: We thank Wenli Kang for excellent technical assistance.

Financial support: This work was supported by grants TW01162, AI10984, and AI39800 from the National Institutes of Health. Lark L. Coffey was supported by the James W. McLaughlin Fellowship fund.

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Author Notes

Reprint requests: Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609.
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