- Short report
- Open Access
Development and characterization of the replicon system of Japanese encephalitis live vaccine virus SA14-14-2
- Shi-Hua Li†1,
- Xiao-Feng Li†1,
- Hui Zhao1,
- Yong-Qiang Deng1,
- Xue-Dong Yu1,
- Shun-Ya Zhu1,
- Tao Jiang1,
- Qing Ye1,
- E-De Qin1 and
- Cheng-Feng Qin1Email author
© Li et al.; licensee BioMed Central Ltd. 2013
- Received: 10 October 2012
- Accepted: 22 February 2013
- Published: 26 February 2013
Viral self-replicating sub-genomic replicons represent a powerful tool for studying viral genome replication, antiviral screening and chimeric vaccine development. Many kinds of flavivirus replicons have been developed with broad applications.
The replicon system of JEV live vaccine strain SA14-14-2 was successfully developed in this study. Two kinds of replicons that express enhanced green fluorescent protein (EGFP) and Renilla luciferase (R.luc) were constructed under the control of SP6 promoter, respectively. Robust EGFP and R.luc signals could be detected in the replicon-transfected BHK-21 cells. Furthermore, the potential effects of selected amino acids in the C-terminal of envelope protein on replication were characterized using the replicon system.
Our results provide a useful platform not only for the study of JEV replication, but also for antiviral screening and chimeric vaccine development.
- Japanese encephalitis virus (JEV)
Japanese encephalitis is now recognized as the leading cause of viral encephalitis in Asian countries, including China, Japan, Korea, the Philippines, Thailand, and India [1, 2]. Clinical Japanese encephalitis is a severe disease with a high case fatality rate. World Health Organization (WHO) estimates that approximately 50,000 cases of Japanese encephalitis occur each year, resulting in about 10,000 deaths and 15,000 cases of neurological or psychiatric sequelae [3, 4]. Japanese encephalitis virus (JEV) is transmitted in an enzootic cycle between Culex species mosquitoes and vertebrates, primarily birds with pigs serving as amplifying hosts. In recent years, JEV has begun to spread to other geographic areas such as Pakistan and Australia [5, 6]. The geographic expansion and high fatality rates have drawn increasing attention from the international public health community .
Vaccination has been recognized as the most reliable and economic measure for protection against Japanese encephalitis. Currently, three kinds of vaccines are available: inactivated vaccine produced in mouse-brain or cell culture and live attenuated vaccine produced on primary hamster kidney (PHK) cells . The live vaccine (SA14-14-2) was initially licensed in 1989 in mainland China, and now exported to most JEV-endemic countries, including India, Sri Lanka, Nepal, Thailand and South Korea under the recommendation of WHO . Large scale immunizations in more than 300 million children have well demonstrated its excellent safety and efficacy profile. Very recently, a novel chimeric JEV live vaccine based on the genetic background of Yellow fever virus (YFV) 17D strain was licensed in Australia and is under active consideration for license in Thailand .
JEV belongs to the Flavivirus genus in the family Flaviviridae together with YFV, dengue virus (DENV), West Nile virus (WNV), Murray Valley encephalitis virus (MVEV) and tick-borne encephalitis virus (TBEV). The genome of JEV is a positive-sense single-stranded RNA molecule comprising 10, 976 nucleotides with a long open reading frame coding for three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins. The RNA genome has a type I cap structure at its 5′-end and lacks the poly (A) tail at its 3′-end .
A viral replicon is a self-replicating sub-genomic viral RNA originated from viral genome, which contains viral non-structural genes that are critical for viral genome replication with structural proteins deleted or replaced by foreign genes. This non-infectious replicon provides a valuable platform to study the function and structure of viral genome RNA, express foreign proteins and develop novel vaccines. In recent years, many flavivirus replicons have been developed, including Kunjin virus , Tick-borne encephalitis virus , DENV [14–17], Yellow fever virus [18, 19], and West Nile virus [20–23].
The reverse genetic system of JEV is greatly hampered due to the toxicity of JEV cDNA in bacteria. Despite extensive efforts for many years [24–27], a genetically stable full-length infectious cDNA clone of JEV was not obtained until the bacterial artificial chromosome (BAC) was used as a vector in 2003. Then, several JEV replicons using the BAC vector were constructed based on a Korean JEV strain K87P39 to express the foreign proteins . In this work, we described the preption of a sub-genomic replicon derived from JEV attenuated strain SA14-14-2, as well as, a series of replicons with Enhanced green fluorescent protein (EGFP) and Renilla luciferase (R.luc) reporter genes were constructed and characterized, respectively. These replicons should be useful for studying many aspects of JEV replication, expressing foreign proteins and developing new vaccines.
To further adapt the potential application of JEV replicon system, a series of JEV replicons expressing the enhanced green fluorescent protein (EGFP) reporter gene (Figure 1) were constructed. A DNA fragment encoding 2A protease of foot-and-mouth disease virus (FMDV-2A) in fusion with the downstream region of the EGFP encoding sequence was amplified from pEGFP-N1 vector (Promega) using primer set F-KAS-EGFP and J-2A-E3-R. The resulting fusion PCR fragment was digested by Kas I and Bsp EI and ligated into the pJE3Rep, yielding the JEV reporter-replicon pJ/EGFP2A/E3Rep. Another two JEV reporter-replicons retaining the C-terminal 25 and 71 amino acids residues of E protein, pJ/EGFP2A/E25Rep and pJ/EGFP2A/E71Rep, respectively, were constructed. All the molecular constructs were prepared by using standard molecular biology techniques, and confirmed by restriction digest analysis and DNA sequencing.
Previously, we have generated the infectious clone of JEV and constructed mutant JEV by using reverse genetic technology . In this study, we have constructed a series of JEV replicons based on the JEV live vaccine strain SA14-14-2, and all these replicons are functionally active to replicate and express the desired foreign reporter genes. These replicons constructed herein are under the control of SP6 promoter. Previously, some DNA-based JEV replicons that under the control of CMV promoter have been developed and adapted for pseudo infectious particles [35, 36]. These JEV replicons not only help to understand the molecular mechanism of viral replication, but also provide a powerful tool for foreign proteins expression, chimeric vaccine and single-round virus like particles (VLP) based vaccine development. The live vaccine virus SA14-14-2 has been widely used in the most JEV endemic countries owing to its highly efficiency, and very few adverse effects [37–39]. Another flavivirus live vaccine strain, YFV 17D, has been widely used as genetic backbone for chimeric flavivirus vaccine development [40–45]. The potential applications of JEV SA14-14-2 in vaccine development are of high significance and deserve further investigation [11, 46, 47]. Currently, we are working with these JEV replicons to generate a series of chimeric flaviviruses vaccine candidates.
We thank Chengdu Institute of Biological Products for providing the work seed of JEV live vaccine SA14-14-2. This work was supported in part by the National Science and Technology Major Project of China (2013ZX10004-805), National Basic Research Project of China (2012CB518904) and National Natural Science Foundation of China (81101243 and 31270974).
- Kumar R, Tripathi P, Singh S, Bannerji G: Clinical features in children hospitalized during the 2005 epidemic of Japanese encephalitis in uttar pradesh, India. Clin Infect Dis 2006, 43: 123-131. 10.1086/505121PubMedView ArticleGoogle Scholar
- Murgod UA, Muthane UB, Ravi V, Radhesh S, Desai A: Persistent movement disorders following Japanese encephalitis. Neurology 2001, 57: 2313-2315. 10.1212/WNL.57.12.2313PubMedView ArticleGoogle Scholar
- Misra UK, Kalita J: Overview: Japanese encephalitis. Prog Neurobiol 2010, 91: 108-120. 10.1016/j.pneurobio.2010.01.008PubMedView ArticleGoogle Scholar
- Erlanger TE, Weiss S, Keiser J, Utzinger J, Wiedenmayer K: Past, present, and future of Japanese encephalitis. Emerg Infect Dis 2009, 15: 1-7. 10.3201/eid1501.080311PubMedPubMed CentralView ArticleGoogle Scholar
- Endy TP, Nisalak A: Japanese encephalitis virus: ecology and epidemiology. Curr Top Microbiol Immunol 2002, 267: 11-48. 10.1007/978-3-642-59403-8_2PubMedGoogle Scholar
- Mackenzie JS, Johansen CA, Ritchie SA, van den Hurk AF, Hall RA: Japanese encephalitis as an emerging virus: the emergence and spread of Japanese encephalitis virus in Australasia. Curr Top Microbiol Immunol 2002, 267: 49-73. 10.1007/978-3-642-59403-8_3PubMedGoogle Scholar
- Yun SI, Choi YJ, Song BH, Lee YM: 3′ cis-acting elements that contribute to the competence and efficiency of Japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions. J Virol 2009, 83: 7909-7930. 10.1128/JVI.02541-08PubMedPubMed CentralView ArticleGoogle Scholar
- Oya A, Kurane I: Japanese encephalitis for a reference to international travelers. J Travel Med 2007, 14: 259-268. 10.1111/j.1708-8305.2007.00134.xPubMedView ArticleGoogle Scholar
- Halstead SB, Thomas SJ: New Japanese encephalitis vaccines: alternatives to production in mouse brain. Expert Rev Vaccines 2011, 10: 355-364. 10.1586/erv.11.7PubMedView ArticleGoogle Scholar
- Fischer M, Lindsey N, Staples JE, Hills S: Japanese encephalitis vaccines: recommendations of the advisory committee on immunization practices (ACIP). MMWR Recomm Rep 2010, 59: 1-27.Google Scholar
- Yun SI, Choi YJ, Yu XF, Song JY, Shin YH, Ju YR, Kim SY, Lee YM: Engineering the Japanese encephalitis virus RNA genome for the expression of foreign genes of various sizes: implications for packaging capacity and RNA replication efficiency. J Neurovirol 2007, 13: 522-535. 10.1080/13550280701684651PubMedView ArticleGoogle Scholar
- Khromykh AA, Westaway EG: Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 1997, 71: 1497-1505.PubMedPubMed CentralGoogle Scholar
- Gehrke R, Ecker M, Aberle SW, Allison SL, Heinz FX, Mandl CW: Incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line. J Virol 2003, 77: 8924-8933. 10.1128/JVI.77.16.8924-8933.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Mosimann AL, de Borba L, Bordignon J, Mason PW, dos Santos CN: Construction and characterization of a stable subgenomic replicon system of a Brazilian dengue virus type 3 strain (BR DEN3 290–02). J Virol Methods 2010, 163: 147-152. 10.1016/j.jviromet.2009.09.004PubMedView ArticleGoogle Scholar
- Suzuki R, de Borba L, Santos CN Dd, Mason PW: Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1. Virology 2007, 362: 374-383. 10.1016/j.virol.2006.11.026PubMedPubMed CentralView ArticleGoogle Scholar
- Ng CY, Gu F, Phong WY, Chen YL, Lim SP, Davidson A, Vasudevan SG: Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing. Antiviral Res 2007, 76: 222-231. 10.1016/j.antiviral.2007.06.007PubMedView ArticleGoogle Scholar
- Pang X, Zhang M, Dayton AI: Development of Dengue virus type 2 replicons capable of prolonged expression in host cells. BMC Microbiol 2001, 1: 18. 10.1186/1471-2180-1-18PubMedPubMed CentralView ArticleGoogle Scholar
- Corver J, Lenches E, Smith K, Robison RA, Sando T, Strauss EG, Strauss JH: Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization. J Virol 2003, 77: 2265-2270. 10.1128/JVI.77.3.2265-2270.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Jones CT, Patkar CG, Kuhn RJ: Construction and applications of yellow fever virus replicons. Virology 2005, 331: 247-259. 10.1016/j.virol.2004.10.034PubMedView ArticleGoogle Scholar
- Rossi SL, Zhao Q, O’Donnell VK, Mason PW: Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 2005, 331: 457-470. 10.1016/j.virol.2004.10.046PubMedView ArticleGoogle Scholar
- Shi PY, Tilgner M, Lo MK: Construction and characterization of subgenomic replicons of New York strain of west nile virus. Virology 2002, 296: 219-233. 10.1006/viro.2002.1453PubMedView ArticleGoogle Scholar
- Yamshchikov VF, Wengler G, Perelygin AA, Brinton MA, Compans RW: An infectious clone of the west nile flavivirus. Virology 2001, 281: 294-304. 10.1006/viro.2000.0795PubMedView ArticleGoogle Scholar
- Moritoh K, Maeda A, Nishino T, Sasaki N, Agui T: Development and application of west nile virus subgenomic replicon RNA expressing secreted alkaline phosphatase. J Vet Med Sci 2011, 73: 683-686. 10.1292/jvms.10-0495PubMedView ArticleGoogle Scholar
- Mishin VP, Cominelli F, Yamshchikov VF: A ‘minimal’ approach in design of flavivirus infectious DNA. Virus Res 2001, 81: 113-123. 10.1016/S0168-1702(01)00371-9PubMedView ArticleGoogle Scholar
- Sumiyoshi H, Hoke CH, Trent DW: Infectious Japanese encephalitis virus RNA can be synthesized from in vitro -ligated cDNA templates. J Virol 1992, 66: 5425-5431.PubMedPubMed CentralGoogle Scholar
- Sumiyoshi H, Tignor GH, Shope RE: Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA. J Infect Dis 1995, 171: 1144-1151. 10.1093/infdis/171.5.1144PubMedView ArticleGoogle Scholar
- Zhang F, Huang Q, Ma W, Jiang S, Fan Y, Zhang H: Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector. J Virol Methods 2001, 96: 171-182. 10.1016/S0166-0934(01)00331-7PubMedView ArticleGoogle Scholar
- Yoshii K, Holbrook MR: Sub-genomic replicon and virus-like particles of Omsk hemorrhagic fever virus. Arch Virol 2009, 154: 573-580. 10.1007/s00705-009-0345-5PubMedView ArticleGoogle Scholar
- Alvarez DE, Lodeiro MF, Luduena SJ, Pietrasanta LI, Gamarnik AV: Long-range RNA-RNA interactions circularize the dengue virus genome. J Virol 2005, 79: 6631-6643. 10.1128/JVI.79.11.6631-6643.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Bredenbeek PJ, Kooi EA, Lindenbach B, Huijkman N, Rice CM, Spaan WJ: A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication. J Gen Virol 2003, 84: 1261-1268. 10.1099/vir.0.18860-0PubMedView ArticleGoogle Scholar
- Khromykh AA, Meka H, Guyatt KJ, Westaway EG: Essential role of cyclization sequences in flavivirus RNA replication. J Virol 2001, 75: 6719-6728. 10.1128/JVI.75.14.6719-6728.2001PubMedPubMed CentralView ArticleGoogle Scholar
- Cao F, Li XF, Yu XD, Deng YQ, Jiang T, Zhu QY, Qin ED, Qin CF: A DNA-based west nile virus replicon elicits humoral and cellular immune responses in mice. J Virol Methods 2011, 178: 87-93. 10.1016/j.jviromet.2011.08.018PubMedView ArticleGoogle Scholar
- Patkar CG, Larsen M, Owston M, Smith JL, Kuhn RJ: Identification of inhibitors of yellow fever virus replication using a replicon-based high-throughput assay. Antimicrob Agents Chemother 2009, 53: 4103-4114. 10.1128/AAC.00074-09PubMedPubMed CentralView ArticleGoogle Scholar
- Ye Q, Li XF, Zhao H, Li SH, Deng YQ, Cao RY, Song KY, Wang HJ, Hua RH, Yu YX: A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1’ formation and contributes to attenuation. J Gen Virol 2012, 93: 1959-1964. 10.1099/vir.0.043844-0PubMedView ArticleGoogle Scholar
- Huang Y, Liu S, Yang P, Wang C, Du Y, Yu W, Sun Z: Replicon-based Japanese encephalitis virus vaccines elicit immune response in mice. J Virol Methods 2012, 179: 217-225. 10.1016/j.jviromet.2011.11.002PubMedView ArticleGoogle Scholar
- Huang Y, Liu S, Yang P, Wang C, Du Y, Sun Z, Yu W: Influence of Japanese enciphalitis virus capsid protein on the self-replicate ability of JEV replicon vectors. Sheng Wu Gong Cheng Xue Bao 2010, 26: 1088-1094.PubMedGoogle Scholar
- Hennessy S, Liu Z, Tsai TF, Strom BL, Wan CM, Liu HL, Wu TX, Yu HJ, Liu QM, Karabatsos N: Effectiveness of live-attenuated Japanese encephalitis vaccine (SA14-14-2): a case–control study. Lancet 1996, 347: 1583-1586. 10.1016/S0140-6736(96)91075-2PubMedView ArticleGoogle Scholar
- Tandan JB, Ohrr H, Sohn YM, Yoksan S, Ji M, Nam CM, Halstead SB: Single dose of SA 14-14-2 vaccine provides long-term protection against Japanese encephalitis: a case–control study in Nepalese children 5 years after immunization. Vaccine 2007, 25: 5041-5045. 10.1016/j.vaccine.2007.04.052PubMedView ArticleGoogle Scholar
- Ohrr H, Tandan JB, Sohn YM, Shin SH, Pradhan DP, Halstead SB: Effect of single dose of SA 14-14-2 vaccine 1 year after immunisation in Nepalese children with Japanese encephalitis: a case–control study. Lancet 2005, 366: 1375-1378. 10.1016/S0140-6736(05)67567-8PubMedView ArticleGoogle Scholar
- Franco D, Li W, Qing F, Stoyanov CT, Moran T, Rice CM, Ho DD: Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development. Vaccine 2010, 28: 5676-5685. 10.1016/j.vaccine.2010.06.052PubMedView ArticleGoogle Scholar
- McGee CE, Lewis MG, Claire MS, Wagner W, Lang J, Guy B, Tsetsarkin K, Higgs S, Decelle T: Recombinant chimeric virus with wild-type dengue 4 virus premembrane and envelope and virulent yellow fever virus Asibi backbone sequences is dramatically attenuated in nonhuman primates. J Infect Dis 2008, 197: 693-697. 10.1086/527329PubMedView ArticleGoogle Scholar
- Chambers TJ, Liang Y, Droll DA, Schlesinger JJ, Davidson AD, Wright PJ, Jiang X: Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model. J Virol 2003, 77: 3655-3668. 10.1128/JVI.77.6.3655-3668.2003PubMedPubMed CentralView ArticleGoogle Scholar
- van Der Most RG, Murali-Krishna K, Ahmed R, Strauss JH: Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response. J Virol 2000, 74: 8094-8101. 10.1128/JVI.74.17.8094-8101.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree M, Arroyo J, Georgakopoulos K, Catalan J, Monath TP: Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000, 74: 5477-5485. 10.1128/JVI.74.12.5477-5485.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Guirakhoo F, Zhang ZX, Chambers TJ, Delagrave S, Arroyo J, Barrett AD, Monath TP: Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis. Virology 1999, 257: 363-372. 10.1006/viro.1999.9695PubMedView ArticleGoogle Scholar
- Chambers TJ, Droll DA, Jiang X, Wold WS, Nickells JA: JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: biological properties in the mouse model of neuroinvasive disease. Virology 2007, 366: 51-61. 10.1016/j.virol.2007.04.016PubMedPubMed CentralView ArticleGoogle Scholar
- Yun SI, Song BH, Koo Y, Jeon I, Byun SJ, Park JH, Joo YS, Kim SY, Lee YM: Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles. Virus Res 2009, 144: 298-305. 10.1016/j.virusres.2009.04.014PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.