Development of persistent HCV genotype 3a infection cell culture model in huh-7 cell

Virology Journal

Sajida Hassan¹  Muhammad Kazim Khan¹  Imran Shahid¹  Sana Gull¹  Muhammad Tahir Sarwar¹  Humera Kausar¹  Waqar Ahmad¹  Bushra Ijaz¹  Sultan Asad¹ 

[1] Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, 53700 Lahore, Pakistan

关键词: HCV Core;    Huh-7 cell line;    siRNA;    HCV models;   

Others  :  1155272
DOI  :  10.1186/1743-422X-9-11

 received in 2011-09-30, accepted in 2012-01-10,  发布年份 2012

【 摘 要 】

Background

Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model.

Methods

We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40^th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells.

Results

HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40^th day, while HCV core protein was detected from the second day up to 40^th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24^th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3^rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA.

Conclusion

Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

【 授权许可】

   
2012 Asad et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Feld JJ, Hoofnagle JH: Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005, 436:967-972.
• [2]Giannini C, Brechot C: Hepatitis C virus biology. Cell Death Differ 2003, 10(Suppl 1):S27-38.
• [3]Idrees M, Riazuddin S: Frequency distribution of hepatitis C virus genotypes in different geographical regions of Pakistan and their possible routes of transmission. BMC Infect Dis 2008, 8:69. BioMed Central Full Text
• [4]Ahmad W, Ijaz B, Javed FT, Jahan S, Shahid I, Khan FM, Hassan S: HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World J Gastroenterol 2010, 16:4321-4328.
• [5]Raja NS, Janjua KA: Epidemiology of hepatitis C virus infection in Pakistan. J Microbiol Immunol Infect 2008, 41:4-8.
• [6]Afdhal NH: The natural history of hepatitis C. Semin Liver Dis 2004, 24(Suppl 2):3-8.
• [7]Mengshol JA, Golden-Mason L, Rosen HR: Mechanisms of Disease: HCV-induced liver injury. Nat Clin Pract Gastroenterol Hepatol 2007, 4:622-634.
• [8]Xie ZC, Riezu-Boj JI, Lasarte JJ, Guillen J, Su JH, Civeira MP, Prieto J: Transmission of hepatitis C virus infection to tree shrews. Virology 1998, 244:513-520.
• [9]Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, Addison WR, Fischer KP, Churchill TA, Lakey JR, et al.: Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001, 7:927-933.
• [10]Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, Koike K: The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 1998, 4:1065-1067.
• [11]Barth H, Robinet E, Liang TJ, Baumert TF: Mouse models for the study of HCV infection and virus-host interactions. J Hepatol 2008, 49:134-142.
• [12]Boonstra A, van der Laan LJ, Vanwolleghem T, Janssen HL: Experimental models for hepatitis C viral infection. Hepatology 2009, 50:1646-1655.
• [13]Kato N, Shimotohno K: Systems to culture hepatitis C virus. Curr Top Microbiol Immunol 2000, 242:261-278.
• [14]Shimizu YK, Iwamoto A, Hijikata M, Purcell RH, Yoshikura H: Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci USA 1992, 89:5477-5481.
• [15]Noguchi M, Hirohashi S: Cell lines from non-neoplastic liver and hepatocellular carcinoma tissue from a single patient. In Vitro Cell Dev Biol Anim 1996, 32:135-137.
• [16]Iacovacci S, Manzin A, Barca S, Sargiacomo M, Serafino A, Valli MB, Macioce G, Hassan HJ, Ponzetto A, Clementi M, et al.: Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro. Hepatology 1997, 26:1328-1337.
• [17]Loriot MA, Bronowicki JP, Lagorce D, Lakehal F, Persico T, Barba G, Mergey M, Vons C, Franco D, Belghiti J, et al.: Permissiveness of human biliary epithelial cells to infection by hepatitis C virus. Hepatology 1999, 29:1587-1595.
• [18]Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, et al.: Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005, 11:791-796.
• [19]Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, Wieland SF, Uprichard SL, Wakita T, Chisari FV: Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005, 102:9294-9299.
• [20]Yi M, Villanueva RA, Thomas DL, Wakita T, Lemon SM: Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 2006, 103:2310-2315.
• [21]el-Awady MK, Tabll AA, el-Abd YS, Bahgat MM, Shoeb HA, Youssef SS, Bader el-Din NG, Redwan el RM, el-Demellawy M, Omran MH, et al.: HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro. World J Gastroenterol 2006, 12:4836-4842.
• [22]Lazaro CA, Chang M, Tang W, Campbell J, Sullivan DG, Gretch DR, Corey L, Coombs RW, Fausto N: Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes. Am J Pathol 2007, 170:478-489.
• [23]Buck M: Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS One 2008, 3:e2660.
• [24]Molina S, Castet V, Pichard-Garcia L, Wychowski C, Meurs E, Pascussi JM, Sureau C, Fabre JM, Sacunha A, Larrey D, et al.: Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent. J Virol 2008, 82:569-574.
• [25]Khaliq S, Jahan S, Ijaz B, Ahmad W, Asad S, Hassan S: Inhibition of hepatitis C virus genotype 3a by siRNAs targeting envelope genes. Arch Virol 2011, 156:433-442.
• [26]Zekri AR, Bahnassy AA, El-Din HM, Salama HM: Consensus siRNA for inhibition of HCV genotype-4 replication. Virol J 2009, 6:13. BioMed Central Full Text
• [27]Bartosch B, Dubuisson J, Cosset FL: Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 2003, 197:633-642.
• [28]Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM: Complete replication of hepatitis C virus in cell culture. Science 2005, 309:623-626.
• [29]Miyamoto M, Kato T, Date T, Mizokami M, Wakita T: Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1). Intervirology 2006, 49:37-43.
• [30]Ji H, Fraser CS, Yu Y, Leary J, Doudna JA: Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc Natl Acad Sci USA 2004, 101:16990-16995.
• [31]Pisarev AV, Shirokikh NE, Hellen CU: Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C R Biol 2005, 328:589-605.
• [32]Rosenfeld AB, Racaniello VR: Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein. J Virol 2005, 79:10126-10137.
• [33]Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P: Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 2005, 309:1577-1581.
• [34]Khaliq S, Jahan S, Ijaz B, Ahmad W, Asad S, Pervaiz A, Samreen B, Khan M, Hassan S: Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs. Virol J 2010, 7:318. BioMed Central Full Text