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Chikungunya virus (CHIKV) is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs) are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 108 cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines.

Objectives: Chikungunya virus (CHIKV) is an alphavirus belonging to the Togaviridae family. Alphaviruses cause a chronic non-cytopathic infection in mosquito cells, while they develop a highly cytopathic infection in cells originating from various vertebrates. In this study, we compared the cytopathic effect (CPE) induced by CHIKV in Vero cells and a mosquito cell line, C6/36 cells., Methods: CPE and the virus titers were compared between the CHIKV-infected C6/36 and Vero cells. Apoptosis was measured by TUNEL assay, and the differences between the C6/36 and Vero cells were compared., Results: CHIKV infection induced strong CPE and apoptosis in the Vero cells, but light CPE in the C6/36 cells. The virus titers produced in the C6/36 cells were much higher than those produced in the Vero cells., Conclusions: The reason CHIKV induced strong CPE is that this virus triggers strong apoptosis in Vero cells compared with C6/36 cells. CHIKV established a persistent infection in C6/36 cells after being passaged 20 times. CHIKV infection in mosquito cells was distinct from that in Vero cells. The cell and species specificity of CHIKV-induced cell death implies that the cellular and viral regulators involved in apoptosis may play an important role in determining the outcome of CHIKV infection., (Copyright © 2012 S. Karger AG, Basel.)

Background: Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C)], are recognized by toll-like receptor 3 (TLR3) and induce interferon (IFN)-β in many cell types. Poly (I:C) is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C) elicited IFN-α/β production and natural killer (NK) cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV) infection, the viruses are cleared within 7-10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance., Results: The effects of Poly (I:C) on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C) suppressed cytopathic effects (CPE) induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C) and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C)-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C) treatment of BEAS-2B cells induced the production of IFN-β and increased the expression of anti-viral genes, including IFN-α, IFN-β, MxA, and OAS. Both Poly (I:C) and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells., Conclusions: CHIKV is sensitive to innate immune response induced by Poly (I:C). The inhibition of CHIKV replication by Poly (I:C) may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.

Background: Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial., Results: Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles., Conclusion: The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.

Background: Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance., Methodology/principal Findings: We conducted on a yearly basis a retrospective survey over a ten-year period that included two successive treatment policies, namely quinine during 1990-1994, and chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) as first and second line treatments, respectively, during 1995-1999. Molecular beacon-based genotyping, gene sequencing and microsatellite analysis showed a low prevalence of Pfcrt and Pfdhfr-ts resistance alleles of Southeast Asian origin by the end of 1994 and their effective dissemination within one year of CQ and SP implementation. The Pfcrt resistant allele rose from 9% to 46% prevalence during the first year of CQ reintroduction, i.e., after a mean of 1.66 CQ treatment courses/person/year. The Pfdhfr-ts triple mutant rose from 0% to 20% by end 1996, after a mean of 0.35 SP treatment courses/person in a 16-month period. Both resistance alleles were observed at a younger age than all other alleles. Their spreading was associated with enhanced in vitro resistance and rapidly translated in an increased incidence of clinical malaria episodes during the early post-treatment period., Conclusion/significance: In such a highly endemic setting, selection of drug-resistant parasites took a single year after drug implementation, resulting in a rapid progression of the incidence of clinical malaria during the early post-treatment period. Controlled antimalarial use at the community level did not prevent dissemination of resistance haplotypes. This data pleads against reintroduction of CQ in places where resistant allele frequency has dropped to a very low level after CQ use has been discontinued, unless drastic measures are put in place to prevent selection and spreading of mutants during the post-treatment period.