THE REPLICATION EFFICIENCY OF DENGUE VIRUS SEROTYPE 1 ISOLATED FROM PATIENTS WITH DENGUE FEVER IN HUMAN HEPATOCYTE CELL LINES
DOI:
https://doi.org/10.21010/Ajid%20v19i2.4Keywords:
Huh-7, HepG2, Vero, DENV-1, Efficiency, ReplicationAbstract
ABSTRACT
Background. The Efficiency of viral replication in cells depends on the capability of supporting virus replication by the cells. We characterized the effectiveness of Dengue Virus Serotype 1 (DENV-1) replication in various cell lines and various multiplicity of infection (MOI) starting from 2 FFU/cell up to 0,3125 FFU/cell.
Material and Methods. We used HepG2 and Huh-7 human hepatocyte cell lines and in addition, we also used nonhuman kidney cells (Vero cells). DENV-1 strain IDS 11/2010 was isolated from DF patients and previously propagated in Huh7 and Vero cells as DENV-1-adapted Huh-7 and DENV-1-adapted Vero cells, respectively. Huh7 cells, Hep G2 cells, and Vero cells were infected with DENV-1 at various MOI and incubated for 48 hours at 370C with 5% CO2. DENV-infected cells were determined by indirect immuno-peroxidase staining using 3,3'-Diaminobenzidine (DAB). DENV-1 infected cells as foci were counted under inverted light microscopy and were used to determine the virus titer.
Result. The virus was adapted to Huh-7 and Vero cells, with results showing that Vero cells exhibited the highest replication efficiency, evidenced by significant viral titers. Among human hepatocyte cell lines, DENV-1 demonstrated greater replication in Huh-7 cells than in HepG2 cells. Notably, no foci formation was observed in HepG2 cells after 48 hours of infection.
Conclusion. These findings underscore the suitability of Vero and Huh-7 cells as optimal environments for DENV-1 replication, offering valuable insights for enhancing laboratory diagnostics and advancing antiviral strategies and vaccine development against DENV-1.
Keywords: Huh-7, HepG2, Vero, DENV-1, Efficiency, Replication
References
Angelina, M., Hanafi, M., Suyatna, F. D., Ratnasari, S. and Dewi, B. E. (2017). Antiviral effect of sub fraction Cassia alata leaves extract to dengue virus Serotype-2 strain new guinea C in human cell line Huh-7 it-1. IOP Conference Series: Earth and Environmental Science, IOP Publication, 012004.
Balsitis, S. J., Coloma, J., Castro, G., Alava, A., Flores, D., Mckerrow, J. H. (2009). Tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining. The American journal of tropical medicine and hygiene, 80, 416-424.
Bolívar-Marin, S., Bosch, I. and Narváez, C. F. (2022). Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease. Journal of Tropical Medicine, 2022, 2177183.
Cabrera‐Hernandez, A., Thepparit, C., Suksanpaisan, L. and Smith, D. R. (2007). Dengue virus entry into liver (HepG2) cells is independent of hsp90 and hsp70. Journal of medical virology, 79, 386-392.
Carpp, L. N., Fong, Y., Bonaparte, M., Moodie, Z., Juraska, M., Huang, Y. (2020). Microneutralization assay titer correlates analysis in two phase 3 trials of the CYD-TDV tetravalent dengue vaccine in Asia and Latin America. PLoS One, 15, e0234236.
Chan, J. F., Yip, C. C., Tsang, J. O., Tee, K. M., Cai, J. P., Chik, K. K. (2016). Differential cell line susceptibility to the emerging Zika virus: implications for disease pathogenesis, non-vector-borne human transmission and animal reservoirs. Emerg Microbes Infect, 5, e93.
Charretier, C., Saulnier, A., Benair, L., Armanet, C., Bassard, I., Daulon, S. (2018). Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process. J Virol Methods, 252, 57-64.
Chen, Y., Maguire, T., Hileman, R. E., Fromm, J. R., Esko, J. D., Linhardt, R. J. (1997). Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nature medicine, 3, 866-871.
Chin, J., Chu, J. and Ng, M. (2007). The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry. Microbes and Infection, 9, 1-6.
Clark, K. B., Hsiao, H. M., Bassit, L., Crowe, J. E., Jr., Schinazi, R. F., Perng, G. C. (2016). Characterization of dengue virus 2 growth in megakaryocyte-erythrocyte progenitor cells. Virology, 493, 162-72.
Coombe, D. and Kett, W. (2005). Heparan sulfate-protein interactions: therapeutic potential through structure-function insights. Cellular and Molecular Life Sciences CMLS, 62, 410-424.
Couvelard, A., Marianneau, P., Bedel, C., Drouet, M.-T., Vachon, F., Hénin, D. (1999). Report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis. Human pathology, 30, 1106-1110.
Dewi, B. E., Angelina, M., Meilawati, L., Hartati, S., Dewijanti, I. D., Santi, M. R. (2018). Antiviral Effect of Pterocarpus indicus Willd Leaves Extract Against Replication of Dengue Virus (DENV) In vitro. Journal of Tropical Life Science, 8.
Dewi, B. E., Takasaki, T. and Kurane, I. (2008). Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin. Journal of General Virology, 89, 642-652.
Diamond, M. S., Edgil, D., Roberts, T. G., Lu, B. and Harris, E. (2000). Infection of human cells by dengue virus is modulated by different cell types and viral strains. Journal of virology, 74, 7814-7823.
Durbin, A. P., Vargas, M. J., Wanionek, K., Hammond, S. N., Gordon, A., Rocha, C. (2008). Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever. Virology, 376, 429-435.
Fang, S., Wu, Y., Wu, N., Zhang, J. and An, J. (2013). [Retracted] Recent Advances in DENV Receptors. The Scientific World Journal, 2013, 684690.
Gutiérrez-Barbosa, H., Castañeda, N. Y. and Castellanos, J. E. (2020). Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells. Braz J Infect Dis, 24, 13-24.
Haryanto, S., Yohan, B., Santoso, M. S., Hayati, R. F., Denis, D., Udjung, G. (2019). Clinical features and virological confirmation of perinatal dengue infection in Jambi, Indonesia: A case report. Int J Infect Dis, 86, 197-200.
Huerre, M. R., Trong Lan, N., Marianneau, P., Bac Hue, N., Khun, H., Thanh Hung, N. (2001). Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Archiv, 438, 107-115.
Jácome, F. C., Caldas, G. C., Rasinhas, A. D. C., De Almeida, A. L. T., De Souza, D. D. C., Paulino, A. C. (2021). Immunocompetent mice infected by two lineages of dengue virus type 2: observations on the pathology of the lung, heart and skeletal muscle. Microorganisms, 9, 2536.
Jindadamrongwech, S. and Smith, D. R. (2004). Virus Overlay Protein Binding Assay (VOPBA) reveals serotype specific heterogeneity of dengue virus binding proteins on HepG2 human liver cells. Intervirology, 47, 370-373.
Kongmanas, K., Punyadee, N., Wasuworawong, K., Songjaeng, A., Prommool, T., Pewkliang, Y. (2020). Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy. PLoS Negl Trop Dis, 14, e0008835.
López-Medina, E., Biswal, S., Saez-Llorens, X., Borja-Tabora, C., Bravo, L., Sirivichayakul, C. (2022). Efficacy of a dengue vaccine candidate (TAK-003) in healthy children and adolescents 2 years after vaccination. The Journal of infectious diseases, 225, 1521-1532.
Lu, D., Liu, J., Zhang, Y., Liu, F., Zeng, L., Peng, R. (2018.) Discovery and optimization of phthalazinone derivatives as a new class of potent dengue virus inhibitors. Eur J Med Chem, 145, 328-337.
Marianneau, P., Mégret, F., Olivier, R., Morens, D. M. and Deubel, V. (1996). Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs. Journal of general virology, 77, 2547-2554.
Ng, Y. L., Mok, C. K. and Chu, J. J. H. (2022). Cytopathic Effect (CPE )-Based Drug Screening Assay for SARS-CoV-2. Methods Mol Biol, 2452, 379-391.
Obi, J., Gutiérrez-Barbosa, H., Chua, J., and Deredge, D. (2021). Current trends and limitations in dengue antiviral research. Trop Med Infect Dis 6: 180.
Panda, K., Alagarasu, K., Patil, P., Agrawal, M., More, A., Kumar, N. V. (2021). In vitro Antiviral Activity of α-Mangostin against Dengue Virus Serotype-2 (DENV-2). Molecules, 26.
Rabelo, K., Trugilho, M. R. O., Costa, S. M., Pereira, B. A. S., Moreira, O. C., Ferreira, A. T. S. (2017)). The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach. J Proteomics, 152, 339-354.
Sakoonwatanyoo, P., Boonsanay, V. and Smith, D. R. (2006). Growth and production of the dengue virus in C6/36 cells and identification of a laminin-binding protein as a candidate serotype 3 and 4 receptor protein. Intervirology, 49, 161-172.
Sasmono, R. T., Wahid, I., Trimarsanto, H., Yohan, B., Wahyuni, S., Hertanto, M. (2015). Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia. Infection, Genetics and Evolution, 32, 165-177.
Schneider-Schaulies, J. R., Martin, M. J., Logan, J. S., Firsching, R., Ter Meulen, V., and Diamond, L. E. (2000). CD46 transgene expression in pig peripheral blood mononuclear cells does not alter their susceptibility to measles virus or their capacity to downregulate endogenous and transgenic CD46. Journal of General Virology, 81, 1431-1438.
Scroggs, S. L. P., Gass, J. T., Chinnasamy, R., Widen, S. G., Azar, S. R., Rossi, S. L. (2021). Evolution of resistance to fluoroquinolones by dengue virus serotype 4 provides insight into mechanism of action and consequences for viral fitness. Virology, 552, 94-106.
Shue, B., Chiramel, A. I., Cerikan, B., To, T. H., Frölich, S., Pederson, S. M. (2021). Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication. J Virol, 95, e0059621.
Suttitheptumrong, A., Khunchai, S., Panaampon, J., Yasamut, U., Morchang, A., Puttikhunt, C. (2013). Compound A, a dissociated glucocorticoid receptor modulator, reduces dengue virus-induced cytokine secretion and dengue virus production. Biochem Biophys Res Commun, 436, 283-8.
Thepparit, C., Khakpoor, A., Khongwichit, S., Wikan, N., Fongsaran, C., Chingsuwanrote, P. (2013). Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death. BMC Res Notes, 6, 372.
Thepparit, C., Phoolcharoen, W., Suksanpaisan, L. and Smith, D. R. (2004). Internalization and propagation of the dengue virus in human hepatoma (HepG2) cells. Intervirology, 47, 78-86.
Tsypysheva, I. P., Lai, H. C., Kiu, Y. T., Koval'skaya, A. V., Tsypyshev, D. O., Huang, S. H. (2021). Synthesis and antiviral evaluation of cytisine derivatives against dengue virus types 1 and 2. Bioorg Med Chem Lett, 54, 128437.
Vasilakis, N., Deardorff, E. R., Kenney, J. L., Rossi, S. L., Hanley, K. A. and Weaver, S. C. (2009). Mosquitoes put the brake on arbovirus evolution: experimental evolution reveals slower mutation accumulation in mosquito than vertebrate cells. PLoS Pathog, 5, e1000467.
Wang, W. H., Urbina, A. N., Chang, M. R., Assavalapsakul, W., Lu, P. L., Chen, Y. H., (2020). Dengue hemorrhagic fever - A systemic literature review of current perspectives on pathogenesis, prevention and control. J Microbiol Immunol Infect, 53, 963-978.
Zandi, K., Teoh, B.-T., Sam, S.-S., Wong, P.-F., Mustafa, M. R. and Abubakar, S. (2012). Novel antiviral activity of baicalein against dengue virus. BMC complementary and alternative medicine, 12, 1-9.
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