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      Characterization of a Chikungunya virus strain isolated from banked patients’ sera

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          Abstract

          Background

          Chikungunya virus (CHIKV) is a prevalent mosquito-borne pathogen that is emerging in many parts of the globe causing significant human morbidity. Here, we report the isolation and characterization of an infectious CHIKV from banked serum specimens of suspected patients from the 2009 epidemic in Thailand.

          Methods

          Standard plaque assay was used for CHIKV isolation from the banked serum specimens. Isolated CHIKV was identified base on E1 structural gene sequence. Growth kinetic, infectivity, cell viability and cytokine gene expression throughout CHIKV infection in a permissive cell line, 293T cells, was performed using several approaches, including standard plaque assay, immunofluorescence assay, classical MTT assay, and quantitative real-time PCR. Two tailed Student’s t test was used for evaluation statistically significance between the mean values of the groups.

          Results

          Based on the E1 structural gene sequence and phylogenetic analysis, we identified the virus as the CHIK/SBY8/10 isolate from Indonesia. Assessment of the growth kinetics, cytopathic effects as well as its ability to induce cellular immune responses suggested that the currently isolated CHIK/SBY8/10 virus is relatively more virulent than a known CHIKV vaccine strain, which also induces more dramatic proinflammatory responses.

          Conclusions

          Our studies further add to the infectivity of a less-studied yet infectious CHIKV isolate as well as underscored the importance and utility of 293T cells as an excellent cell culture model for studying viral growth, CHIKV-induced inflammatory cellular responses and cell death. Together, these studies provide novel information on the CHIKV biology, infectivity and virus-cell interaction, which would help develop novel interventions against the infection.

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          Most cited references25

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          Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography.

          Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV, here we report the crystal structures of the precursor p62-E1 heterodimer and of the mature E3-E2-E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2-E1 heterodimer at acidic pH (ref. 3), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.
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            Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses.

            A Igarashi (1978)
            Twenty clones were isolated from cultured Aedes albopictus (Singh) cells in the presence of anti-Chikungunya (CHIK) virus serum. Each clone was tested for its yields of Dengue (DEN) viruses, types 1, 2, 3 and 4, and also CHIK virus. Clone C6 showed the highest yield of each virus tested. Forty-three clones obtained by recloning C6 in the presence of anti-DEN sera showed almost the same virus yields as C6. One of the clones, C6/36, showed mild to extensive cytopathic effects several days after virus infection, in contrast to the original uncloned (SAAR) cells. Fluorescent antibody staining revealed that the amount of virus antigen accumulated in the cytoplasm was almost the same in every cell in the case of clone C6/36, while it was highly heterogeneous for uncloned SAAR cells. Growth curves of the viruses indicated that clone C6/36 gave a significantly higher yield for each virus than uncloned SAAR cells up to 7 days after infection. Virus sensitivity of the C6/36 clone did not change by growing the cells with the medium used for uncloned SAAR cells, nor did the virus sensitivity of uncloned cells increase in medium used for clone C6/36. However, the C6/36 clone became resistant to CHIK virus, but not to DEN or Sindbis viruses, after incubation with the medium used for another A. albopictus cell line (SAAK). The transfer of the specific resistance to CHIK may be mediated by some latent virus related to CHIK.
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              Persistent Arthralgia Induced by Chikungunya Virus Infection is Associated with Interleukin-6 and Granulocyte Macrophage Colony-Stimulating Factor

              Background. Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients. Methods. A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated. Results. When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)–6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor. Conclusions. The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.
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                Author and article information

                Contributors
                kunbc14@gmail.com
                sarunyouchusri@hotmail.com
                stefan.fernandez.mil@mail.mil
                wilaiwan58@hotmail.com
                janguita@cicbiogune.es
                upal@umd.edu
                kpromnares@gmail.com , kamoltip.p@psu.ac.th
                Journal
                Virol J
                Virol. J
                Virology Journal
                BioMed Central (London )
                1743-422X
                2 September 2016
                2 September 2016
                2016
                : 13
                : 1
                : 150
                Affiliations
                [1 ]Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hatyai, Songkhla 90112 Thailand
                [2 ]Division of Infectious Disease, Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University, Hatyai, Songkhla 90112 Thailand
                [3 ]Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand
                [4 ]CIC bioGUNE, 48160 Derio, Bizkaia Spain
                [5 ]Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Bizkaia Spain
                [6 ]Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742 USA
                Article
                606
                10.1186/s12985-016-0606-3
                5009685
                26728778
                d2d4ffc7-e415-4701-bd42-6f8b0b9b18a8
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 15 June 2016
                : 24 August 2016
                Funding
                Funded by: Prince of Songkla University Budget (Beginner Research Scholarship)
                Award ID: SCI550151S
                Award Recipient :
                Funded by: Prince of Songkla University National Budget
                Award ID: SCI570067S
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Microbiology & Virology
                chikungunya virus,293t cells,chik/sby8/10 isolate,chikv 181/clone 25,gene expression,cytokines

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