7 research outputs found
Anti-CHIKV IgM provides a short period of protection up to day 10 during early phase of infection.
<p>(A) Anti-CHIKV IgM and IgG were measured by ELISA. Samples were categorized by time from disease onset. **<i>P</i><0.01, ***<i>P</i><0.001, Mann-Whitney U test compared to healthy controls. The dotted lines represent the cut-off value. All the samples were within the detection limit except one. (B) The neutralizing titers of IgM, IgG and total antibodies were plotted by days from disease onset. Each point indicates the median of NT<sub>50</sub>. (C) Ratios of NT<sub>50</sub> IgM over NT<sub>50</sub> IgG were calculated and plotted for serum samples by times from disease onset (panel B). A ratio of 1 indicates equal strength of NT<sub>50</sub> IgM over NT<sub>50</sub> IgG. (D) Representative immunofluorescent microscopic images of CHIKV-infected cells incubated with 1:100 diluted serum under different treatment conditions. Objective magnification: 5× from 1 field of view. The serum sample used was from panel B1, collected 4 days post-onset of illness, with IgM and IgG titers of 20480 and 4096, respectively. (E) Panel B1 was divided into groups with either high or low NT<sub>50</sub> IgG. Neutralization of virus infectivity due to IgM or IgG was compared to neutralizing capacity due to total antibodies. Results are expressed as percentage of virus control. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by two-way ANOVA with Bonferroni multiple comparisons test. Data are presented as mean ± SD for individual samples with low NT<sub>50</sub> IgG and mean ± SEM for 12 samples with high NT<sub>50</sub> IgG. (F) Panel B1 was divided into groups with either high or low NT<sub>50</sub> IgG ratio. IgG titers against CHIKV and rE2 were measured. *<i>P</i><0.05, Mann-Whitney U test. The lines in the middle of the boxes indicate medians; the upper and lower boundaries of the box indicate inter-quartile ranges; and the whiskers indicate ranges of values.</p
Anti-CHIKV IgM preferably targets epitopes on E1-E2 glycoproteins.
<p>(A) IgM immunoblotting was performed using approximately 1 μg of recombinant protein (E1 and E2) and 2.5 μg of whole virus antigen under non-reducing conditions at serum dilutions of 1:100 and 1:400 using pooled serum samples from panel B1. Mouse anti-His (α-His) was used as a control. (B) IgM reactivity against rE1 and rE2 in capture ELISA was investigated. The experiments were performed at 1:100 serum dilution. The dotted lines represent the cut-off value. (C) Immunoblotting was performed under non-reducing and reducing conditions at a serum dilution of 1:400 against fusion recombinant E2 and recombinant E1 glycoprotein (rE2-E1-ECSA). (D) Schematic diagram showing the chimeras used in seroneutralization with comparison of infectivity using pooled serum samples from panel B1. Data are presented as means ± SD from 4 independent experiments at a serum dilution of 1:100. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Kruskal-Wallis test.</p
The appearance of CHIKV-specific neutralizing antibodies is associated with a reduction of viremia.
<p>(A) Seroneutralization was performed on sera from panel A which had known viral loads. Experiments were performed in triplicate at 1:100 dilution for detection of neutralizing IgM and IgG. The dotted lines represent the cut-off value determined from the mean—SD values from healthy control sera. (B) Neutralization titers (NT<sub>50</sub>) of total antibodies and previously-determined viral loads [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171989#pone.0171989.ref026" target="_blank">26</a>] of serum samples (panel A) collected at different times of disease onset were plotted. Each point indicates the median of RNA copies/ml targeting E1 and NT<sub>50</sub>, with the inter-quartile range. The dashed line indicates the limit of quantification (1 log<sub>10</sub> RNA copies/reaction or 4.76 log<sub>10</sub> RNA/ml) of the E1 positive-strand PCR assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171989#pone.0171989.ref026" target="_blank">26</a>].</p
Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia
<div><h3>Background</h3><p>Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.</p> <h3>Methods and Findings</h3><p>CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and <em>Aedes</em> mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in <em>Ae. albopictus</em> cells (C6/36). Both CHIKV strains infected <em>Ae. albopictus</em> mosquitoes at a higher rate than <em>Ae. aegypti</em>, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected <em>Ae. aegypti</em> at higher rates.</p> <h3>Conclusions</h3><p>The greater ability of the ECSA strain to replicate in <em>Ae. albopictus</em> may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where <em>Ae. albopictus</em> predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and <em>Aedes</em> species.</p> </div
Comparative infection, replication kinetics, and dissemination of each Malaysian CHIKV strain in <i>Ae. aegypti</i> (⧫, black bars) or <i>Ae. albopictus</i> (⋄, white bars).
<p>The CHIKV isolates MY/06/37348 (Asian) and MY/08/065 (ECSA) were used. Following ingestion with MY/06/37348, infection rates of midguts of mosquitoes (A), replication in midguts as measured by a TCID<sub>50</sub> titration assay with plotted means ± SD of triplicates (B), and dissemination rates in salivary glands of mosquitoes (C) were determined. Following ingestion with MY/08/065, midgut infection (D), midgut replication (E), and salivary gland dissemination (F) were measured. Asterisks indicate significant differences (*p<0.05, **p<0.01). The denominator used to calculate midgut infection rates was the number of mosquitoes sampled, and the denominator for dissemination rates was the number of midguts with detectable virus titre. Denominators are shown (n).</p
Amino acid differences between Malaysian Chikungunya strains of Asian and East/Central/South African genotypes.
<p>Amino acid differences are reported following alignment of sequences obtained in this study from the Bagan Panchor strains MY/06/37348 and MY/06/37350 (Asian genotype), and the Johor strains MY/08/065 and MY/08/068 (ECSA genotype). The first amino acid named is found in the Asian strains, while the second amino acid is found in the ECSA strains. Differences at sites reported to be mosquito adaptation determinants in other alphaviruses are underlined.</p
Comparative replication kinetics of Malaysian CHIKV strains in cells.
<p>The CHIKV isolates MY/06/37348 (Asian, •) and MY/08/065 (ECSA, ○) were used. Replication was measured by virus titration using a TCID<sub>50</sub> assay in (A) Vero, (B) rhabdomyosarcoma, (C) C6/36, and (D) CCL-125 cells. Means ± SD of 3 independent experiments are plotted. Asterisks indicate significant differences (<i>p</i><0.05) at the same time-points.</p