Identification of a virus-specific and conserved B-cell epitope on NS1 protein of Japanese encephalitis virus
Introduction
Japanese encephalitis (JE), one of the most important mosquito-borne diseases with a mortality rate as high as 20–50%, is widely distributed in most of East and South-east Asia and partly in Oceania (Mackenzie et al., 2001). Up to 50,000 JE cases are reported in Asian countries annually in humans, of which 10,000–15,000 result in fatality (Solomon, 1997, Solomon et al., 2000). A high proportion (nearly 50%) of survivors, especially young children and those greater than 65 years of age, exhibit permanent neurologic and psychiatric sequelae (World Health Organization, 1998). Simultaneously, this disease has accounted for significant economic losses to the pig industry due to the fatal encephalitis and reproductive failure in pregnant sows and hypospermia in boars (Shimizu et al., 1954). To date, there is no specific treatment for JE, a mouse brain-derived formalin-inactivated vaccine, used widely but has many limitations, was approved by World Health Organization (WHO).
JE is caused by Japanese encephalitis virus (JEV). JEV is a member of family Flaviviridae, genus Flavivirus, transmitted mainly by Culex mosquitoes. Except JEV, the Japanese encephalitis virus serocomplex of the family Flaviviridae includes West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). These viruses have a similar ecology; it is very common that two or more of these flaviviruses co-circulate in some regions of the world. Co-existence of these viruses have increased the necessity to distinguish among these viruses serologically, and also underlined the importance of carrying out virus surveillance to track their spread. The initial symptoms of most of the flaviviral infections, however, are similar to each other and high degree of cross-reactivity can be observed in most serodiagnostic assays, which dramatically complicates the specific serological diagnosis for flaviviral diseases. Although the plaque reduction neutralization test (PRNT), virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) are generally conducted to achieve laboratory diagnosis, these methods are time-consuming and not amenable to testing large numbers of specimen. Furthermore, the PRNT needs paired acute- and convalescent-phase serum specimens (Kuno, 2003), and involves manipulation of live virus which requires high level of biosafety laboratory, the method of virus isolation is limited to the period of viremia and RT-PCR assays have the limitation of requiring advanced techniques, equipment, and trained manpower. All of these emphasize the utility of virus-specific epitopes, for the differential diagnosis and epidemiological surveys. The current serologic diagnosis of flavivirus infection is based on detection of antibodies against viral structural proteins, mainly the E protein (Martin et al., 2002), but the E protein contains cross-reactive epitopes (Crill and Chang, 2004, Stiasny et al., 2006), even by applying mutagenesis strategy for the E protein of virus-like particles (VLPs), the cross-reactivity cannot be eliminated completely (Roberson et al., 2007, Chiou et al., 2008). In contrast, the NS1 protein showed more specific reactivity when attempting to differentiate flavivirus, it has been reported that NS1 could induce antibodies without cross-reactivity among flavivirus (Shu et al., 2001, Kitai et al., 2007), and even among different serotypes of dengue virus (DENV) (Huang et al., 2001, Wu et al., 2001, Shu et al., 2004), hence the development of NS1 protein-based specific serological diagnosis is of great interest (Shu et al., 2003, Konishi et al., 2004, Kitai et al., 2007).
The aim of present study is to characterize JEV-specific epitopes without cross-reactivity on NS1 protein to provide important information for the development of specific serological diagnosis of JEV infection, also for further understanding of the antigenic structure of NS1 protein and vaccine design.
Section snippets
Cell lines, virus and serum
Myeloma cell line SP2/0 and Baby hamster kidney (BHK-21) cells were, respectively, cultured in RPMI-1640 (Invitrogen, Scotland, UK) and Dulbecco's modified Eagle's medium (DMEM, Sigma) in humidified 5% CO2 atmosphere at 37 °C. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (PAA, Somerst, UK) and antibiotics (0.1 mg/ml of streptomycin and 100 IU/ml of penicillin). The JEV SA14-14-2 strain (GenBank accession number AF315119) was maintained in our laboratory. The
Production of mAbs
The recombinant JEV NS1 protein was successfully expressed in E. coli BL21 (DE3) after induction with IPTG existed predominantly as inclusion body. The recombinant protein could be recognized by JEV-positive serum from pigs (data not shown). Purified proteins were used to immunize BALB/c mice. After cell fusion and selection, we generated several hundred hybridoma cell lines; among them one strain was selected for its strongest reactivity against native NS1 protein using indirect ELISA and IFA
Discussion
For pathogens of virus infectious diseases, definition of the location of viral protein epitopes and the degree of their conservation is important to understanding the antigenic structure and virus–antibody interactions at a molecular level, and also useful in vaccine design and clinical application. Many investigators have used mAbs, which serves as powerful tool to reveal the properties of proteins, identified linear or mimic epitopes of a variety of infectious agents (Deng et al., 2007,
Acknowledgements
This study was supported by the National Natural Science Foundation of China (30700027) and Commonweal Agriculture Scientific Research Calling Project of China (200803015).
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These authors have contributed equally to this study.