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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The DEN viruses are a group of four viruses in the genus Flavivirus designated DEN-1, DEN-2, DEN-3, and DEN-4.1 Although the viruses are closely related, they are antigenically distinct, and previous infection by one serotype does not confer protection against infection by a second serotype. Antibodies raised against the primary infection serotype are cross-reactive in the secondary infection, but not cross-protective.8,9 Thus, an individual may be infected with a second DEN virus serotype, and cross-reactive antibodies raised against the primary DEN virus infection may function to enhance the entry of the second DEN serotype virus into cells in a process called antibody-dependent enhancement,9,10 which is hypothesized to contribute to DHF/DSS.11 Therefore, in regions where all four DEN serotypes are co-circulating, a person may be exposed to all four serotypes in his or her lifetime. The cross-reactivity, antibody-dependent enhancement of infection, and pan-distribution of all four DEN virus serotypes are factors that have frustrated attempts to develop a DEN vaccine, which will have to be administered so that it simultaneously protects against all four DEN viruses.
Acambis, Inc. has developed recombinant vaccine viruses based on using the attenuated yellow fever (YF) vaccine virus 17D (YF-VAX) as a live vector.12 The genes encoding the premembrane (prM) and envelope (E) proteins of the YF-VAX virus have been replaced with those of heterologous flaviviruses, such as Japanese encephalitis (JE) virus;12–14 DEN-1, DEN-2, DEN-3, and DEN-4 viruses (Figure 1
);15,16 and West Nile (WN) virus.15–20
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A vaccine consisting of a tetravalent mix of ChimeriVaxTM-DEN-1, ChimeriVaxTM-DEN-2, ChimeriVaxTM-DEN-3, and ChimeriVaxTM-DEN-4 viruses, which will protect against infection by all four DEN virus serotypes, is currently in development (Acambis, Inc.). In nonhuman primates vaccinated with the ChimeriVaxTM-DEN 1, 2, 3, 4 tetravalent mixture, viremia is very low (peak titer of primary immunization 1.5 log10 plaque-forming units [PFU]/mL), yet good immune response is elicited against all four DEN virus serotypes.27 However, despite the low viremia, the potential for a competent vector mosquito to become infected with the chimeric virus through taking a blood meal on a viremic vaccinee needed to be evaluated.
In previous studies, mosquitoes were shown to be poorly infected orally with ChimeriVaxTM-JE, ChimeriVaxTM-DEN-2, and ChimeriVaxTM-WN viruses and growth was limited compared with the wt viruses in vector mosquito species when virus was introduced directly by inoculation.24–26 We report here growth kinetics of the ChimeriVaxTM-DEN 1, 2, 3, 4 tetravalent mixture of viruses in vitro in Ae. albopictus C6/36 cell culture and in vivo in Ae. aegypti mosquitoes, the primary vector of both YF and DEN viruses. Single infections of ChimeriVaxTM-DEN-1, ChimeriVaxTM-DEN-3, and ChimeriVaxTM-DEN-4 viruses were also carried out. The low infection and transmission potential of ChimeriVaxTM-DEN-2 virus by Ae. aegypti and Ae. albopictus mosquitoes was determined in earlier studies.25 Growth of the chimeric viruses in C6/36 cells and in mosquitoes either intrathoracically (IT) inoculated with virus or offered virus in an artificial infectious blood meal was compared with that of the corresponding wt DEN and YF-VAX viruses. As in the previous vector competence studies with ChimeriVaxTM viruses, the chimeric DEN viruses assessed in this report were found to be phenotypically similar to YF-VAX, which is attenuated in mosquitoes, and their ability to infect mosquitoes via oral infection was reduced compared with the parental DEN viruses.
A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method of virus detection and quantification (Taqman) was developed to separately assay growth of each of the ChimeriVaxTM-DEN serotypes in the tetravalent mix. The Taqman assay detected virus titer both in vitro in the C6/36 cell culture growth curves, and also in individual, triturated mosquitoes.
The stability of the chimeric virus nucleotide sequence after replication in the mosquito was also determined. The prM and E gene regions of chimeric virus isolated from IT inoculated mosquitoes were sequenced and compared with the corresponding gene region of the chimeric virus inoculum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Virus titer. Individual mosquitoes were triturated by pestle in 1.7-mL tubes (Kontes Glass Co., Vineland, NJ) in 1 mL of BA-1 diluent (1x M-199 medium with Hanks’ salts, 0.05 M Tris, pH 7.6, 1% bovine albumin, 0.35 g/L of sodium bicarbonate, 100 units/mL of penicillin, 100 µg/mL of streptomycin, 1 µL/mL of fungizone). Mosquito suspensions were clarified by centrifugation at 8,000 rpm for 5 minutes. Virus titer was determined by double-overlay plaque assay in Vero cells as previously described.28
Real-time reverse transcription-polymerase chain reaction (TaqMan).
Primers and probes were designed with the PrimerExpress software package (PE Applied Biosystems, Foster City, CA) (Table 1). The TaqMan probes were labeled at the 5' end with the FAM reporter dye and at the 3' end with dark quencher. Taqman primers and probes were designed either to detect a unique, serotype-specific sequence in the prM and E region of a DEN virus, or a region in the YF 17D backbone that was common to all chimeric DEN viruses and to YF-VAX (Figure 1
). Thus, each ChimeriVaxTM-DEN primer-probe set detected a single wt DEN virus and the corresponding chimeric DEN virus serotype. Heterologous chimeric DEN virus RNA was included as negative control in all single infection real-time RT-PCR assays. The YF 17D primers and probe detected all ChimeriVaxTM-DEN virus serotypes and YF-VAX virus. Wild-type DEN virus RNA was added as negative control in these assays. The RNA from tetravalent infections was subjected to four separate, single-serotype real-time RT-PCR assays, with YF-VAX virus serving as the negative control. The specificities of the primer-probe sets were determined as previously described.29 Total RNA for the real-time RT-PCR assay was extracted from 100 µL of the same mosquito suspension used to inoculate Vero cells for plaque titration, using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Five microliters of RNA was combined with 50 pmol of each primer and 7.5 pmol of the FAM-labeled probed in a 50-µL total one-Step TaqMan RT-PCR (PE Applied Biosystems). Reverse transcription of 30 minutes at 50°C was followed by 45 cycles of amplification in an ABI Prism 7700 Sequence Detection System instrument (PE Applied Biosystems) according to manufacturer’s instructions for TaqMan RT-PCR conditions.
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Growth curves. The C6/36 cells (a cell line derived from Ae. albopictus) were obtained from stocks at the Division of Vector-Borne Infectious Diseases (DVBID) of the Centers for Disease Control and Prevention (CDC) (Fort Collins, CO) and grown at 28°C in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 1% each of nonessential amino acids and sodium pyruvate, 1% of 7.5% sodium bicarbonate, and 0.1% gentamicin. Infected C6/36 cells were maintained in 2% FBS medium. Three 25–cm2 flasks of C6/36 cells were infected with each virus at a multiplicity of infection of 0.01 PFU/cell. The pH was adjusted with sodium bicarbonate. Supernatant fluid was removed at 48-hour intervals for 10 days.
Mosquitoes. Field-collected Ae. aegypti eggs from Puerto Rico were provided by the Dengue Branch of DVBID, CDC (San Juan, PR). A laboratory colony was established and maintained at the CDC in Fort Collins as previously described.25 The F4 generations of these mosquitoes were used in this study.
Intrathoracic inoculation of mosquitoes. Mosquitoes were cold anesthetized and inoculated intrathoracically (IT) using a microcapillary needle that had been pulled to a point with a needle puller (Narishige, Tokyo, Japan). Approximately 0.34 µL of virus standardized to 6.0 log10 PFU/mL was injected into each mosquito (2.5 log10 PFU/mosquito; ChimeriVaxTM-DEN-1, 2, 3, 4, mixture, 1.9 log10 PFU of each serotype/mosquito). Inoculated mosquitoes were maintained in cartons at 27°C and a relative humidity of 80% and with 5% sugar water. Three mosquitoes per infection were removed at 48-hour intervals for 10 days; the remainder was collected at 14 days postinoculation. Mosquitoes were frozen at -70°C until assayed.
Oral infection of mosquitoes. Mosquitoes were fed virus-laden artificial blood meals in hanging droplets.30 It was necessary to use fresh virus for blood meals.31 Wild-type DEN viruses were grown in C6/36 cells; because ChimeriVaxTM-DEN viruses did not grow well in C6/36 cells they were cultured in Vero cells. The infection threshold of artificial blood meals in mosquitoes requires a higher virus titer than natural infections.32 Therefore, in the oral infection experiments, the highest virus titer possible was used. Virus supernatant was collected 5–6 days after infection (70% cytopathic effect [CPE]) and clarified by centrifugation at 8,000 rpm at 4°C for 20 minutes. Defibrinated sheep’s blood (Colorado Serum Co., Denver, CO) was washed three times with ice-cold phosphate-buffered saline. Two parts fresh virus (7–8 log10 PFU/ mL) was mixed with two parts defibrinated sheep’s blood and one part 10% sucrose in FBS. Prior to administering the blood meal containing the chimeric tetravalent mixture, the titer of each chimeric virus was determined by Taqman, then mixed in equivalent amounts. The artificial blood meal was quickly heated to 37°C, then offered in hanging droplets to 7–10-day-old mosquitoes that had been starved for 24 hours. Mosquitoes were allowed to feed for 30 min, then were cold anesthetized and sorted. Fully engorged mosquitoes were collected and incubated at 27°C and a relative humidity of 80% with sugar water as previously described.25 Mosquitoes were collected at 48-hour intervals for 10 days and after the 14-day extrinsic incubation period, and were stored at -70°C until they were assayed for infection.
Sequence analysis of the prM and E genes in ChimeriVaxTM - DEN viruses isolated from IT -inoculated mosquitoes.
Genomic sequences of the prM and E regions were obtained from seed viruses and from RNA isolated from single serotype infections of IT-inoculated mosquitoes for each of the ChimeriVaxTM-DEN viruses. Mosquito tissues were triturated in 1 mL of BA-1 diluent and clarified by centrifugation. Because of the low viral titer of the infected mosquitoes, no RT-PCR product could be amplified from these mosquitoes directly. Therefore, 100 µL of mosquito suspension was passed through a 0.2-µm filter (Corning Glass Works, Corning, NY) and used to inoculate a T-25 cm2 flask of Vero cells. Virus supernatant was collected at six days, 
70% CPE, and RNA was extracted from 100 µL of the cell culture supernatant using the QIAamp viral RNA kit. The prM and E genes and surrounding the YF 17D gene region were amplified using the Titan One Tube RT-PCR Kit (Roche Diagnostics, Mannheim, Germany) and primers designed from the ChimeriVax-DEN virus sequence (forward position 413: 5'-ACGCCGTTCCCATGATGTTCTGAC-3'; reverse position 2557: 5'-CTTGTTCAGCCAGTCATCAGAGTC-3'). The resultant 2400-basepair DNA product was purified by gel electrophoresis followed by extraction from the gel using the QIAquick gel extraction kit (Qiagen) and sequenced using CEQ2000 Dye Terminator Cycle Sequencing with Quick Start kit (Beckman Coulter, Fullerton, CA). Sequencing reactions were analyzed on a Beckman Coulter CEQ8000 Genetic Analysis System.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Virus growth in C6/36 cells.
The growth curves of single ChimeriVaxTM-DEN virus infections compared with wt DEN viruses are shown in Figure 2A-C. Growth of each of the ChimeriVaxTM-DEN serotypes in the tetravalent mixture infection is shown in Figure 2D, and, for comparison, in Figure 2A-C as dashed lines. The growth curve of YF-VAX virus is also shown for comparison in Figure 2D. The peak titers of ChimeriVaxTM-DEN-1 and ChimeriVaxTM-DEN-3 were 4.2 and 3.4 log10 RNA/mL, respectively, at 10 days, which were 3.8 and 2.6 logs lower than wt DEN-1 virus and DEN-3 virus peak titers. ChimeriVaxTM-DEN-4 virus grew to 5 log10 RNA/ mL, which was 1 log more than the peak titer of wt DEN-4 virus. The titer and growth rate of each chimeric serotype was similar whether it was a single infection, or part of the tetravalent mix (Figure 2). No interference by one chimeric virus with a faster growth rate over a slower-growing serotype was observed. Peak mean titers in the ChimeriVaxTM-DEN 1, 2, 3, 4 virus infection were 1–2.8 logs lower than the peak mean titer of 6.0 log10 RNA/mL YF-VAX virus (Figure 2D).
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shows the geometric mean ± SD titer of three individual mosquitoes collected at 48-hour intervals following IT inoculation of single ChimeriVaxTM-DEN serotypes compared with wt DEN viruses; replication of each ChimeriVaxTM-DEN serotype IT inoculated into mosquitoes as a tetravalent mixture are included for comparison and shown as dashed lines. The growth of the four chimeric virus serotypes IT inoculated into mosquitoes as a tetravalent mixture is shown and compared with that of YF-VAX virus in Figure 3D. Mean peak titer at 10 days of ChimeriVaxTM-DEN-1 virus in IT-inoculated Ae. aegypti was 1.4 logs lower than the mean peak titer of wt DEN-1 virus. ChimeriVax-DEN3 reached a mean peak titer of 4.0 log10 RNA/mosquito at 10 days; wt DEN-3 virus mean peak titer at 4 days was 4.3 log10 RNA/mosquito. ChimeriVax-DEN-4 grew to higher titer than that of wt DEN-4 virus; the mean peak titer of ChimeriVax-DEN-4 virus was 3.9 log10 RNA/ mosquito at day 8 compared with the wt DEN-4 mean peak titer of 3.2 log10 RNA/mosquito at 10 days. Because of the small sample size, no statistical significance can be attributed to these time course experiments, but they do show the general difference in growth in mosquitoes between the wt DEN and chimeric viruses throughout the 10-day extrinsic incubation period.
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Virus replication and dissemination were also assayed in IT inoculated Ae. aegypti after a 14-day extrinsic incubation period (Table 2). Mosquito heads and bodies were assayed separately. Mosquitoes were defined as infected if viral RNA was detected in the bodies; disseminated infection was defined as the presence of viral RNA in head tissue. As single infections all viruses replicated in mosquitoes at 14 days, and all viruses disseminated to head tissue, except for ChimeriVax-DEN-1, which infected 96% of the mosquitoes at 14 days, and disseminated to head tissue in 88%.
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). Dissemination to head tissue occurred in 100% of the chimeric viruses, except for ChimeriVax-DEN-2 virus. The infection rates and mean titers of ChimeriVax-DEN-2 virus in single infections in a previous study and in the tetravalent mix were equivalent: 56% (1.8 ± 0.7 log10 RNA/ mosquito.25 Dissemination occurred in 36% (mean titer of positive mosquitoes = 1.4 ± 0.6 log10 PFU/mosquito) of mosquitoes inoculated with ChimeriVax-DEN-2 only25 versus 15% (mean titer of positive mosquitoes = 1.3 ± 0.1 log10 RNA/mosquito) of mosquitoes inoculated with the tetravalent mixture.
Oral infection of mosquitoes.
Mosquitoes were fed blood meals containing either wt DEN-1, DEN-3, or DEN-4; single serotypes of ChimeriVaxTMM-DEN-1, ChimeriVaxTMM-DEN-3, or ChimeriVaxTMM-DEN-4; a mixture with equal amounts of the four ChimeriVaxTM-DEN serotypes; or YF-VAX viruses. Virus titers of blood meals were as follows: wt DEN-1 = 7.4 log10 RNA transcripts/mL, ChimeriVaxTM-DEN-1 = 8.1 log10 RNA transcripts/mL, wt DEN-3 = 7.0 log10 RNA transcripts/mL, ChimeriVaxTM-DEN-3 = 8.0 log10 RNA transcripts/mL, wt DEN-4 = 5.8 log10 RNA transcripts/mL, ChimeriVaxTM-DEN-4 = 7.8 log10 RNA transcripts/mL, ChimeriVaxTM-DEN 1, 2, 3, 4 = 7.3 log10 RNA transcripts/ mL of each serotype, and YF-VAX = 7.4 log10 RNA transcripts/mL. In our hands, the peak titer of wt DEN-4 virus (strain 1228) grown in tissue culture was 6 log10 PFU/mL. However, wt DEN-4 virus was shown to competently infect mosquitoes despite the lower titer. Therefore, rather than standardize the titers of all the viruses, which would require dilution of the chimeric DEN viruses, the lower wt DEN-4 virus titer was considered adequate for oral infection experiments. Growth of virus in mosquitoes over a 10-day period following the blood meals is shown in Figure 4. Replication of single ChimeriVaxTM-DEN virus serotypes was compared with wt DEN viruses and the results are shown in Figure 4A-C. Growth curves of each of the chimeric DEN virus serotypes fed to mosquitoes in the tetravalent mixture are shown as shaded dashed lines in Figure 4A-C and together in Figure 4D. At 10 days, none of the mosquitoes fed a blood meal containing ChimeriVaxTM-DEN-3 virus remained infected (Figure 4B); one of three mosquitoes fed blood meals with ChimeriVaxTM-DEN-1 or ChimeriVaxTM-DEN-4 viruses were infected (Figure 4A and C). None of the mosquitoes were infected with any chimeric serotype contained in the tetravalent blood meal at 8 or 10 days following the blood meal (Figure 4D). The oral infection rates were significantly different between wt DEN viruses and ChimeriVaxTM-DEN viruses (P < 0.0001 by Fisher’s exact test for DEN-1, 3, and 4 viruses) (Table 3). Fourteen days post blood feeding 0–9% of the mosquitoes were still infected with one of the chimeric virus serotypes, compared with 10% of mosquitoes infected 14 days following a blood meal containing YF-VAX virus (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aedes aegypti are competent mosquito vectors of YF and DEN viruses. Artificial blood meals containing 5.8–8.0 log10 PFU/mL of wt DEN viruses offered to mosquitoes in hanging droplets infected 59–86% of the mosquitoes. In contrast, 10% of mosquitoes were infected with YF-VAX virus. The oral infection rate of the ChimeriVax-DEN viruses was lower, ranging from 0% to 9% in the tetravalent mixture. Because viremia in nonhuman primates vaccinated with ChimeriVaxTM-DEN 1, 2, 3, and 4 viruses was very low (peak mean titer following primary vaccination = 1.9 log10 PFU/ mL), there is very little probability that mosquitoes would become infected by taking a blood meal on a ChimeriVaxTM-DEN 1, 2, 3, 4 virus vaccinee.27
When introduced directly into C6/36 cells in vitro or into mosquito tissue by IT inoculation of adult Ae. aegypti mosquitoes, the ChimeriVaxTM-DEN 1, 2, 3, 4 viruses were able to infect cells and replicate, although less efficiently than YF-VAX virus. This was not unexpected since YF-VAX virus has been shown to grow in C6/36 mosquito tissue culture and in IT-inoculated Ae. aegypti and Ae. albopictus.24–26 In addition, it has been demonstrated that viruses can replicate in mosquito species that are not natural vectors when introduced via IT inoculation.36
To investigate whether one ChimeriVaxTM-DEN virus serotype replicated more efficiently than the other three virus serotypes in the tetravalent mixture, and possibly inhibited or altered replication of the other ChimeriVaxTM-DEN virus serotypes, mosquitoes were infected with single ChimeriVaxTM-DEN virus serotypes or with a mixture of the four chimeric viruses. When infected as a tetravalent mixture of equal titer, the growth curve of each chimeric virus serotype mirrored that of the single virus serotype infection; no interference by one chimeric virus serotype with a faster growth rate over a slower-growing virus serotype was observed. As with single infections, in the tetravalent mixture ChimeriVaxTM-DEN-2 virus had the lowest peak titer and ChimeriVaxTM-DEN-4 the highest.25
The peak titers of wt DEN-4 virus 1228 strain, from which the ChimeriVaxTM-DEN-4 virus was constructed, were lower than the titers of the other wt DEN viruses and of ChimeriVaxTM-DEN-4 virus in C6/36 cells and IT-inoculated mosquitoes. Moreover, viremia of the parental DEN-4 virus strain 1228 was also lower than the other wt DEN serotypes in nonhuman primate infections.16 Other researchers have observed the natural attenuation of DEN-4 virus in experimental infections of nonhuman primates and in natural infection in humans, and it has been hypothesized that DEN-4 virus infects hosts as secondary infections, through antibody-dependent enhancement of infection.37,38 However, it should be noted that Ae. aegypti were efficiently infected orally with the wt DEN-4 virus (86%) despite the low titer of the blood meal (5.8 log10 RNA/mL). In contrast, only 18% of the mosquitoes became infected following a blood meal containing 7.8 log10 RNA/mL of ChimeriVaxTM-DEN-4 virus (Table 3).
ChimeriVaxTMM viruses have now been evaluated in four mosquito studies. These include ChimeriVaxTM-JE virus in the JE mosquito vector Culex tritaeniorhynchus and the YF vectors Ae. aegypti and Ae. albopictus;24 ChimeriVaxTM-DEN-2 virus in YF and DEN vectors Ae. aegypti and Ae. albopictus;25 and ChimeriVaxTM-WN virus in WN vectors Cx. tritaeniorhynchus, Cx. quinquefasciatus, and Cx. nigripalpus and YF vectors Ae. aegypti and Ae. albopictus.26 Results of the experiments reported here and of the previous ChimeriVaxTM virus-mosquito studies suggest that the ChimeriVaxTM viruses are phenotypically similar to YF-VAX virus. YF-VAX is a live virus that is attenuated in humans and Aedes mosquitoes. Whitman showed in 1939 that YF 17D virus had limited replication activity in mosquito tissue, and was not transmitted by Ae. aegypti mosquitoes.23 Recent studies have confirmed the restricted replication of YF-VAX virus in mosquitoes.24–26 The attenuation of YF-VAX virus in mosquitoes was further supported by results of the experiments reported here.
In general, mosquitoes are not susceptible to oral infection by YF-VAX or ChimeriVaxTM viruses. The titer of virus in the few individual mosquitoes that were infected by either YF-VAX or ChimeriVaxTM virus was very low, usually less than 100 infectious particles, and the virus did not disseminate to head tissue of the mosquitoes. It is probable that a few midgut cells were permissive to infection and limited replication occurred, but that the virus was unable to pass out of the midgut and disseminate to other mosquito tissue.
The ability to replicate in mosquitoes was determined by the YF-VAX portion of the ChimeriVaxTM virus. If the vector mosquito species was not a competent vector of YF, but was a species associated with transmission of the virus contributing the prM and E gene region, the ChimeriVaxTM virus was still unable to replicate in mosquitoes, even when introduced directly by IT inoculation. For example, Culex spp. are important vectors of JE and WN viruses, but are not vectors of YF virus. In a laboratory setting, wt JE and WN viruses efficiently infected the Culex mosquitoes, replicating to high titers. However, YF-VAX, ChimeriVaxTM-JE, and ChimeriVaxTM-WN viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus mosquitoes, even upon introduction via IT inoculation.24,26
The flavivirus envelope protein is involved in cell attachment and entry, and because the prM and E genes of ChimeriVaxTM viruses have been replaced with those of a heterologous flavivirus, the tissue tropism of the chimeric virus might be expected to correspond to the phenotype of the E gene contributing virus.39,40 However, as mentioned earlier in this report, phenotypic characteristics of the ChimeriVaxTM viruses, including the ability to replicate in mosquito tissue, were determined by the YF-VAX portion of the genome in mosquitoes. Mutations in the gene region coding for non-structural proteins are probably responsible for attenuation of YF-VAX, and thus ChimeriVaxTM viruses in mosquitoes.
YF-VAX is a live attenuated vaccine virus that has been in use for more than 60 years, with no reports of natural mosquito infection or transmission. The ChimeriVaxTM viruses have been shown to be similarly attenuated in mosquitoes, and thus vector mosquitoes would be expected to be refractory to infection by these ChimeriVaxTM viruses.
Received August 26, 2003. Accepted for publication September 30, 2003.
Financial support: This work was supported by an Industry Challenge Grant (1 UC 1 AI-49517-01) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by Aventis Pasteur. Barbara W. Johnson was supported by an American Society of Microbiology/National Center for Infectious Diseases postdoctoral research fellowship. Trudy V. Chambers was supported by an Acambis/CDC cooperative agreement.
Authors’ addresses: Barbara W. Johnson, Trudy V. Chambers, Mary B. Crabtree, and Barry R. Miller, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Rampart Road, Foothills Campus, Fort Collins, CO 80521, Telephone: 970-266-3543, Fax: 970-221-6476, E-mail: bfj9{at}cdc.gov. Farshad Guirakhoo and Thomas P. Monath, Acambis, Inc., 38 Sidney Street, Cambridge, MA 02139.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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