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 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).
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