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Several studies in dengue on cloned or cell culture passaged in wild and attenuated virus have been performed, but each report described different sites of nucleotide and amino acid changes possibly associated with virulence. The aim of this study was to investigate protein expression of dengue type 2 virus infected C6/36 cells through two-dimensional gel electrophoresis followed by N-terminal Edman degradation sequencing to identify target proteins.
Two type of samples; dengue type 2 virus-infected C6/36 cells and uninfected C6/36 cells were prepared. Immobilized non-linear pH gradient strips, pH 3-10 were used in isoelectric focusing, and 10% homogeneous gels were used in Sodium dodecyl sulphate - Polyacylamide gel electrophoresis. The target protein spots were subsequently transferred onto polyvinyldifluoride membrane by western blotting and visualized by coomassie brilliant blue for N-terminal sequencing purpose.
This study revealed four target protein spots in dengue type 2 virus-infected C6/36 cells that were not present in uninfected C6/36 cells. The N-terminal sequencing result was D-S-G-C-V-V-S-W-K-N-K which was identical to nonstructural glycoprotein NS1 (Swissprot-database) associated with the replication process of flaviviruses.
Nonstructural NS1 might be used as a diagnostic marker and/or as a parameter to evaluate the effect of antiviral agents for dengue type 2 virus infection/replication.
Cisneros A, Diaz-Bdillo A, Cruz-Martinez G, Tovar R, Amirez-Palacios LR, Jimenez-Rojaz F, et al. Dengue 2 genotypes in the state of Oaxaca, Mexico. Arch Virol 2006; 151:113-25.
World Health Organization. Epidemic and pandemic alert and response impact of dengue. World Health Organization, Geneva, Switzerland 2005. Available at: http:/www.who.int/csr/disease/dengue/impact/en/index.html. Accessed 18 April, 2006.
Gubler D. Dengue virus and dengue hemorrhagic fever. J Clin Microbiol Rev 1998; 11: 480-96.
McBride WJH, Ohmann HB. Dengue viral infections; pathogenesis and epidemiology. Microbes Infect 2000; 2:1041-50.
Dussart P, Labeau B, Lagathu G, Louis P, Nunes MRT, Rodrigues SG, et al. Evaluation of an Enzyme Immunoassay for detection of dengue virus NS1 antigen in human serum. Clin and Vac Immunol 2006; pp.1185-9.
Guzman MG, Kourl G. Dengue: an update. Lancet Infect Dis 2002; 2:33-42.
Kalayanarooj S, Vaughn DW, Nimmannitya S, Green S, Suntayakorn S, Kunentrasai N, et al. Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 1997; 176:313-21.
Hommel D, Talarmin A, Deubel V, Reynes JM, Drouet MT, Sarthou JL, et al. Dengue encephalitis in French Guiana. Res Virol. 1998; 149:235-8.
Murgue B, Deparis X, Chungue E, Cassar O, Roche C. Dengue: an evaluation of dengue severity in French Polynesia based on an analysis of 403 laboratory confirmed cases. Trop Med Int Health 1999; 4:765-73.
Pan American Health Organization. Dengue hemorrhagic fever in Venezuela. Epidemiol Bull 1990; 11: 7-9.
Calvert AE, Huang CYH, Kinney RM, Roehrig T. Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge. J Gen Virol 2006; 87:339-46.
Wengler G. The carboxy-terminal part of NS3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 1991; 184: 707-15.
Lindenbach BD, Rice CM. trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 1997; 71:9608-17.
Roehrig JT. Antigenic structure of flavivirus protein. Adv Virus Res 2003; 59:141-75.
Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD. High level expression of recombinant dengue viral NS1 protein and its potential use as a diagnostic antigen. J Med Virol 2001; 65:553-60.
Anderson NG, Anderson NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996; 17: 443-53.
Edman P. A method for the determination of the amino acid sequence of peptides. Arch Biochem Biophys 1949; 22: 475-83.
Corthals GL, Wasinger VC, Hochstrasser DF, Sanchez JC. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000; 21: 1104-15.
Berkelman T, Stenstedt T. 2-D Electrophoresis Principles and Methods, 1998.
Shuib AS, Chua CT, Hashim OH. Sera of IgA Nephropathy Patients contain a Heterogeneous Population of Relatively Cationic Alpha-heavy chains. Nephron 1998; 78:290-5.
Wengler G. The carboxy-terminal part of NS3 protein of the west Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 1991; 184: 707-15.
Anderson L, Seilhamer J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 1997; 18: 533-7.
Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. J Clin Microbiol 2000; 38:1053-7.
Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD. High level expression of recombinant dengue viral NS1 protein and its potential use as a diagnostic antogen. J Med Virol 2001; 65: 553-60.