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An ex vivo model of coxsackievirus infection using multilayered human conjunctival epithelial cells

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Abstract

Background

The purpose of this study was to establish an ex vivo model of coxsackievirus infection since there seems to be no suitable disease model currently.

Methods

Human conjunctival epithelial cells (HCECs) were cultured for 2 weeks in a serum-free air–liquid interface system to produce a multilayered structure. The cells were infected with coxsackievirus A24 (CVA24). Histological changes were investigated by staining the cells with H&E and DAPI, and apoptosis was evaluated using the TUNEL technique. Virus replication was measured in HeLa cells infected with viral progeny from multilayered HCECs, after 1 and 3 days, using the TCID50 method.

Results

Cultured HCECs formed multiple layers. The cells showed characteristics of conjunctival epithelial cells and goblet cells, being immunohistochemically positive for CK19 and MUC5AC, respectively. CVA24 replicated readily in cultured multilayered HCECs. A mild cytopathic effect was noted 1 day after viral inoculation. Cell damage was extensive at 3 days. TUNEL imaging confirmed that the cytopathology was attributable to apoptosis. The TCID50 data from HeLa cells indicated that the virus was actively replicating at both 1 and 3 days after inoculation.

Conclusions

This novel infection model may be useful in investigating the pathogenesis of acute hemorrhagic conjunctivitis and the effectiveness of antiviral treatments.

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Acknowledgments

This work was supported by a grant to H. Tchah (no. A08-0593 from the Ministry of Health & Welfare, Republic of Korea). Jooeun Lee and Eun Jung Jun equally contributed to this work. Hee Jin Lee, MD, pathologist, provided advice on review of histological findings.

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Authors and Affiliations

  1. Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, 388-1 Pungnap-2dong, Songpa-gu, Seoul, 138-736, Korea

    Jooeun Lee, Jung Hae Sunwoo, Eun Soon Kim, Jae-hyung Kim, Jae Yong Kim, Myoung Joon Kim & Hungwon Tchah

  2. Department of Microbiology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea

    Eun Jung Jun, Yoo Kyum Kim & Heuiran Lee

  3. Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea

    Eun Jung Jun, Jae Yong Kim, Yoo Kyum Kim, Heuiran Lee & Hungwon Tchah

Authors
  1. Jooeun Lee
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  2. Eun Jung Jun
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  3. Jung Hae Sunwoo
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  10. Hungwon Tchah
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Corresponding author

Correspondence to Hungwon Tchah.

Additional information

This article was presented as a poster at the Association for Research in Vision and Ophthalmology annual meeting, May 2–6, 2010; Fort Lauderdale, Florida. This study was supported by a grant from the Ministry for Health, Welfare and Family Affairs of Korea (A08-0593). No author has a financial or proprietary interest in any material or method mentioned. Jooeun Lee and Eun Jung Jun equally contributed to this work.

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Supplemental Fig. 1

Immunocytochemistry of HeLa cells against VP1. The viral products of HCECs were challenged to HeLa cells and immunocytochemistry with VP1 was performed. Positive staining confirms the replication is by CVA24. MT mocker treated specimen (200×). (DOC 412 kb)

Supplemental Fig. 2

a Sensitivity of primary rabbit-conjunctival epithelial cells toward CVA24 viruses. After infecting CVA24 viruses by MOI 10 each into cells, cytopathologic effect (CPE) was not observed 3 days after the infection. The CPE were examined by light microscopy (LM) and virus protein production (green) was evaluated by fluorescence microscopy (FM) following labeling with primary antibody against VP1 capsid protein. Pictures were taken at an original magnification of 400×. b Adaptation of CVA24 viruses in primary rabbit conjunctival cells. Adaptation for CVA24 viruses in primary rabbit conjunctival cells (a) was performed by the fifth sub-culture. CPE were examined by LM and virus protein production (green) was evaluated by FM following labeling with primary antibody against VP1 capsid protein in primary rabbit conjunctival fibroblast cells. Pictures were taken at an original magnification of 100×. (DOC 969 kb)

Supplemental Fig. 3

Data not for Review 1. Comparison of RNA sequence of wild-type CVA24 with that of adapted CVA24 to rabbit. The RNA sequence of CVA24 showed more than 30% of disparity between the wild-type and the adapted virus. This picture shows part of the RNA sequence from p2 region. (DOC 593 kb)

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Lee, J., Jun, E.J., Sunwoo, J.H. et al. An ex vivo model of coxsackievirus infection using multilayered human conjunctival epithelial cells. Graefes Arch Clin Exp Ophthalmol 249, 1327–1332 (2011). https://doi.org/10.1007/s00417-011-1655-3

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  • DOI: https://doi.org/10.1007/s00417-011-1655-3

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