Rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.     

Genetic structure among Thai populations of Aedes aegypti mosquitoes

Thirty‐one field populations of Aedes aegypti (L.) were compared using isozyme starch gel electrophoresis to characterize genetic variation between populations. Ae. aegypti were collected from seven provinces in Thailand. Thirty‐one isozyme encoding loci, including 19 polymorphic loci, were characterized. Only small levels of genetic differentiation were observed among the 31 district populations in the seven provinces. Isolation by distance among populations from the seven provinces showed no correlation between genetic variation and geographical distance.     

Insecticide susceptible/resistance status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand during 2003-2005

Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide. Almost all Ae. aegypti collected were incipient resistant or resistant to deltamethrin and permethrin, except those from some areas of Songkhla (southern) and Phan district of Chiang Rai (northern) province. Susceptibility to fenitrothion was found in mosquitoes from Bangkok (central), Chonburi (eastern), Chiang Rai, Kanchanaburi (western), and Songkhla, whereas they were resistant in almost all areas of Nakhon Sawan (north central) and Nakhon Ratchasima (northeastern) provinces. Most of Ae. aegypti were susceptible to propoxur except those from Mae Wong, Nakhon Sawan province. Various levels of insecticide resistance and susceptibility in adjacent areas revealed a focal susceptible/resistance profile in the country. It could be noted that almost all of Ae. albopictus were susceptible to the insecticides tested at the same diagnostic doses. In conclusion, resistance to pyrethroids (permethrin and deltamethrin) has developed in Ae. aegypti in most of the collected areas, suggesting that an alternative choice of insecticide or other control measures should be applied.     

Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

Generic delimitation and relationships in Ebenaceae sensu lato: evidence from six plastid DNA regions

Phylogenetic relationships of the pantropical family Ebenaceae s.l. were investigated using plastid DNA sequence data from six regions: atpB, matK, ndhF, trnK intron, trnL intron, and trnL-trnF spacer. Sampling included representatives of all currently recognized genera of Ebenaceae, Diospyros, Euclea, and Lissocarpa, and nearly all taxa that were previously recognized at the generic level, e.g., Cargillia, Gunisanthus, Maba, Macreightia, Royena, and Tetraclis. Our results strongly support monophyly of Ebenaceae s.l. and demonstrate that the previous infrafamilar classifications of the family do not circumscribe monophyletic groups. A new infrafamilial classification based on a phylogenetic approach is proposed here and consists of two subfamilies, Lissocarpoideae and Ebenoideae, and four genera, Lissocarpa, Euclea, Royena, and Diospyros. Relationships and potential synapomorphic characters are discussed and summarized. This study supports a western Gondwanan origin of family and indicates that both vicariant and long-distance dispersal events played an important role in attaining current distributions.     

An in-depth analysis of original antigenic sin in dengue virus infection

The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases.     

Application of activated charcoal and nanocarbon to callus induction and plant regeneration in aromatic rice (Oryza sativa L.)

The investigations of nanotechnology with the application on agricultural products also have been few reported, especially the plant regeneration. The effects of activated charcoal and nanocarbon on the callus induction and plant regeneration of aromatic rice were studied. Activated charcoal was added into the callus induction and regeneration medium. The presence of activated charcoal in the callus induction medium (100–500 mg L^?1), activated charcoal significantly reduced the percentage of the callus induction and biomass accumulation (fresh weight, dry weight and size). Whereas, the regeneration medium supplemented with 100 mg L^?1 of activated charcoal showed the highest percentage of plant regeneration (61.90%) and the ratio of the number of seedlings to the number of regenerated calli (RSR; 3.06) that derived from the callus induction medium (without activated charcoal). Moreover, the induced calli derived from the callus induction medium supplemented with nanocarbon at 5 mg L^?1 showed the highest percentage of callus induction (94.70%), the percentage of green spots (95.83%), the percentage of plant regeneration (60.42%) and the RSR (3.12) when transferred the calli into the regeneration medium (without nanocarbon). After that, nanocarbon was also added into the regeneration medium. The percentage of green spots (96.08%), the percentage of plant regeneration (62.75%) and the RSR (3.16) obtained from the regeneration medium supplemented with 20 mg L^?1 of nanocarbon showed the highest values. This experiment showed that the optimum concentration of activated charcoal and nanocarbon had potential to enhance the callus induction and plant regeneration frequencies in tissue culture medium of aromatic rice.     

Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.