Chikungunya virus was isolated in Thailand, 2010

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008–2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-014-1105-5) contains supplementary material, which is available to authorized users

Variation at position 350 in the Chikungunya virus 6K-E1 protein determines the sensitivity of detection in a rapid E1-antigen test

Abstract Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay

DENV serotypes, viremia titer, and anti-DENV antibody isotypes in DENV-infected patients.

a<p>ID, patient identification.</p>b<p>Dengue disease was determined by examining the clinical symptoms of patients according to the WHO criteria.</p>c<p>DENV serotype was determined by RT-PCR with universal and serotype-specific primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Yenchitsomanus1" target="_blank">[31]</a>.</p>d<p>Viremia titers were determined in a focus-forming immunoassay in semi-adherent K562 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Kurosu1" target="_blank">[30]</a>.</p>e<p>IgM in patient serum was detected by Dengue Virus IgM Capture DxSelect. An index value of >1.00 was interpreted as positive (POS) and an index value of <1.00 was interpreted as negative (NEG).</p>f<p>IgG in patient serum was detected by Dengue Virus IgG DxSelect. An index value of >1.00 was interpreted as positive (POS) and an index value of <1.00 was interpreted as negative (NEG).</p>g<p>Cases with an IgM/IgG index ratio of ≤1.2 were diagnosed as secondary infections <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Shu1" target="_blank">[32]</a>.</p>h<p>Patient’s blood specimen was used for huMAb preparation as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Guy1" target="_blank">[24]</a>. ND, not detectable.</p

Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients

<div><p>Background</p><p>The majority of dengue patients infected with any serotype of dengue virus (DENV) are asymptomatic, but the remainder may develop a wide spectrum of clinical symptoms, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). Severe cases occur more often in patients who experience a secondary infection with a different virus serotype. A phenomenon called antibody-dependent enhancement (ADE) has been proposed to explain the onset of these severe cases, but the exact mechanism of ADE remains unclear.</p><p>Methodology/Principal Finding</p><p>Virus neutralization and ADE assays were performed using ultracentrifugation supernatants of acute-phase sera from patients with secondary infections or human monoclonal antibodies (HuMAbs) as anti-DENV antibodies. Virus sources included infectious serum-derived viruses from the ultracentrifugation precipitates, laboratory-culture adapted DENV, or recombinant DENVs derived from patient sera. In contrast to the high levels of ADE observed with laboratory virus strains, low ADE was observed with autologous patient-derived viruses, when patient sera were used to provide the antibody component in the ADE assays. Similar results were obtained using samples from DF and DHF patients. Recombinant-viruses derived from DHF patients showed only minor differences in neutralization and ADE activity in the presence of HuMAbs or plasma derived from the same DHF patient.</p><p>Conclusion/Significance</p><p>Serum or plasma taken from patients during the acute phase of a secondary infection showed high levels of ADE, but no neutralization activity, when assayed in the presence of laboratory-adapted virus strains. By contrast, serum or plasma from the same patient showed high levels of neutralization activity but failed to induce significant ADE when the assays were performed with autologous virus. These results demonstrate the significance of the virus source when measuring ADE. They also suggest that repeated passage of DENV in cell culture has endowed it with the capacity to induce high levels of ADE.</p></div

Low ADE when using serum and viruses from DENV-patients.

<p>Serum samples (DENV-1, DENV-2, and DENV-3) from HTD dengue patients with DF and DHF symptoms were ultracentrifuged to precipitate DENV virions which were used in assays without any subsequent passage in cells. The supernatant fractions were heat-inactivated at 56°C for 30 minutes and then serially diluted 10-fold. The dilutions were mixed for 30 minutes at 37°C with the precipitated virions from autologous plasma at an MOI of 0.02. The virus-antibody complexes were added to K562 cells and incubated for 2 hours at 37°C before the addition of maintenance medium supplemented with 2% FBS. The cells were then incubated for a further 3 days. Supernatants were harvested for virus titration by focus-forming immunoassay in Vero cells, and the results are expressed as FFU/ml. The mean ± SD of triplicate experiment is shown. ‘No Ab’ means virus infection in the absence of plasma. The ‘No Ab’ value was used as a baseline for calculating virus infection enhancement.</p

Replication kinetics of recombinant DENVs.

<p>DENV-2 16681 or individual recombinant DENVs were used to infect C6/36 (A) and Vero cells (B) at an MOI of 0.001, or K562 cells (C) at an MOI of 0.1. After 2 hours of incubation, the supernatants were removed and cells were washed twice with plain medium before the addition of maintenance medium supplemented with 2% FBS. For infected C6/36 and Vero cells, the supernatants were harvested daily. For infected K562 cells, both the culture medium and infected cells were harvested and centrifuged. Virus titers in the supernatants were determined in focus-forming assays in Vero cells. Results are expressed as mean ± SD of triplicate experiment (*<i>p<0.05</i> and **<i>p<0.01</i>, unpaired two-tailed Student’s t-test, n = 3 per point). Statistically significant differences between data points are indicated by # (# <i>p<0.05</i>, ## <i>p<0.01</i>).</p

List of primers used in the study.

<p>List of primers used in the study.</p