Tick-borne Langat/mosquito-borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: evaluation in rhesus monkeys and in mosquitoes.
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Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all other sequences derived from dengue type 4 virus (DEN4). The live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective E epitopes among themselves and with LGT. Toward that end the chimera, initially recovered in mosquito cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DEN4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-borne encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 10(5) or 10(7) PFU of LGT strain TP21, with 10(5) PFU of DEN4, or with 10(3), 10(5), or 10(7) PFU of the chimera. Each of the monkeys inoculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the level of viremia was at the limit of detection. All monkeys immunized with the chimera or LGT TP21 virus developed a moderate to high level of neutralizing antibodies against LGT TP21 as well as TBEV and were completely protected against subsequent LGT TP21 challenge, whereas monkeys previously immunized with DEN4 virus became viremic when challenged with LGT TP21. These observations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mice subsequently challenged with 100 i.p. 50% lethal doses of the highly virulent TBEV. The issue of transmissibility of the chimera by mosquitoes was addressed by inoculating a nonhematophagous mosquito, Toxorhynchites splendens, intrathoracically with the chimera or its DEN4 or LGT parent. Neither the LGT TP21/DEN4 vaccine candidate nor the wild-type LGT TP21 virus was able to infect this mosquito species, which is highly permissive for dengue viruses. Certain properties of the chimera, notably its attenuation for monkeys, its immunogenicity, and its failure to infect a highly permissive mosquito host, make it a promising vaccine candidate for use in immunization against severe disease caused by many tick-borne flaviviruses. (+info)
Yellow fever in Para State, Amazon region of Brazil, 1998-1999: entomologic and epidemiologic findings.
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Yellow fever (YF) is frequently associated with high severity and death rates in the Amazon region of Brazil. During the rainy seasons of 1998 and 1999, 23 (eight deaths) and 34 (eight deaths) human cases of YF were reported, respectively, in different geographic areas of Para State; most cases were on Marajo Island. Patients were 1 to 46 years of age. Epidemiologic and ecological studies were conducted in Afua and Breves on Marajo Island; captured insects yielded isolates of 4 and 11 YF strains, respectively, from Haemagogus janthinomys pooled mosquitoes. The cases on Marajo Island in 1999 resulted from lack of vaccination near the focus of the disease and intense migration, which brought many nonimmune people to areas where infected vectors were present. We hypothesize that YF virus remains in an area after an outbreak by vertical transmission among Haemagogus mosquitoes. (+info)
Meiotic recombination, cross-reactivity, and persistence in Plasmodium falciparum.
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We incorporate a representation of Plasmodium falciparum recombination within a discrete-event model of malaria transmission. We simulate the introduction of a new parasite genotype into a human population in which another genotype has reached equilibrium prevalence and compare the emergence and persistence of the novel recombinant forms under differing cross-reactivity relationships between the genotypes. Cross-reactivity between the parental (initial and introduced) genotypes reduces the frequency of appearance of recombinants within three years of introduction from 100% to 14%, and delays their appearance by more than a year, on average. Cross-reactivity between parental and recombinant genotypes reduces the frequency of appearance to 36% and increases the probability of recombinant extinction following appearance from 0% to 83%. When a recombinant is cross-reactive with its parental types, its probability of extinction is influenced by cross-reactivity between the parental types in the opposite manner; that is, its probability of extinction after appearance decreases. Frequencies of P. falciparum outcrossing are mediated by frequencies of mixed-genotype infections in the host population, which are in turn mediated by the structure of cross-reactivity between parasite genotypes. The three leading hypotheses about how meiosis relates to oocyst production lead to quantitative, but no qualitative, differences in these results. (+info)
De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase.
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Replication of positive strand flaviviruses is mediated by the viral RNA-dependent RNA polymerases (RdRP). To study replication of dengue virus (DEN), a flavivirus family member, an in vitro RdRP assay was established using cytoplasmic extracts of DEN-infected mosquito cells and viral subgenomic RNA templates containing 5'- and 3'-terminal regions (TRs). Evidence supported that an interaction between the TRs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements is required for RNA synthesis. Two RNA products, a template size and a hairpin, twice that of the template, were formed. To isolate the function of the viral RdRP (NS5) from that of other host or viral factors present in the cytoplasmic extracts, the NS5 protein was expressed and purified from Escherichia coli. In this study, we show that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo initiation. Furthermore, the incubation temperature during initiation, but not elongation phase of RNA synthesis modulates the relative amounts of the hairpin and de novo RNA products. A model is proposed that a specific conformation of the viral polymerase and/or structure at the 3' end of the template RNA is required for de novo initiation. (+info)
A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses.
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A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma. (+info)
Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.
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Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods. (+info)
West Nile virus isolates from mosquitoes in New York and New Jersey, 1999.
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An outbreak of encephalitis due to West Nile (WN) virus occurred in New York City and the surrounding areas during 1999. Mosquitoes were collected as part of a comprehensive surveillance program implemented to monitor the outbreak. More than 32,000 mosquitoes representing 24 species were tested, and 15 WN virus isolates were obtained. Molecular techniques were used to identify the species represented in the WN virus-positive mosquito pools. Most isolates were from pools containing Culex pipiens mosquitoes, but several pools contained two or more Culex species. (+info)
West Nile virus surveillance in Connecticut in 2000: an intense epizootic without high risk for severe human disease.
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In 1999, Connecticut was one of three states in which West Nile (WN) virus actively circulated prior to its recognition. In 2000, prospective surveillance was established, including monitoring bird deaths, testing dead crows, trapping and testing mosquitoes, testing horses and hospitalized humans with neurologic illness, and conducting a human seroprevalence survey. WN virus was first detected in a dead crow found on July 5 in Fairfield County. Ultimately, 1,095 dead crows, 14 mosquito pools, 7 horses, and one mildly symptomatic person were documented with WN virus infection. None of 86 hospitalized persons with neurologic illness (meningitis, encephalitis, Guillain-Barre-like syndrome) and no person in the seroprevalence survey were infected. Spraying in response to positive surveillance findings was minimal. An intense epizootic of WN virus can occur without having an outbreak of severe human disease in the absence of emergency adult mosquito management. (+info)