Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BACKGROUND: Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine. RESULTS: We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15-20 µg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4-6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. CONCLUSION: These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Identification of novel VP35 inhibitors: Virtual screening driven new scaffolds.

Ebola virus is a single-stranded, negative-sense RNA virus that causes acute and serious life-threatening illness. In recent years the Ebola virus has spread through several countries in Africa, highlighting the need to develop new treatments for this disease and boosting a new research effort on this subject. However, so far there is no valid treatment for disease created by this pathogen. The Ebola virus Viral Protein 35 (VP35) is a multifunctional protein which is critical for virus replication and infection, and it is considered as a future target for drug development. In this study, we collected 144 VP35 inhibitors which shared the same core scaffold, and a common feature pharmacophore model HypoA was built based on inhibitor-receptor complexes. All 141 compounds were aligned based on the common feature pharmacophore model HypoA (three compounds could not map onto HypoA). The pharmacophore model HypoA was further optimized according to the actual interactions between inhibitors and VP35 protein, resulting in a new pharmacophore model HypoB which was applied for virtual screening. A 3D QSAR model was established by applying the 141 aligned compounds. For the training set, the 3D QSAR model gave a correlation coefficient r2 of 0.897, for the test set, the correlation coefficient r2 was 0.757. Then a virtual screening was carried out, which comprehensively employing the common feature pharmacophore model, 3D QSAR model and docking study, their combination in a hybrid protocol could help to mutually compensate for their limitations and capitalized on their mutual strengths. After the above three virtual screening methods orderly filtering, seven potential inhibitors with novel scaffolds were identified as new VP35 inhibitors. The mapping results of hit compounds onto pharmacophore model and 3D QSAR model, and the molecular interactions of the potential inhibitors with the active site residues have been discussed in detail.

[Time-series analysis on road traffic injury in China].

OBJECTIVE: To establish the predictive models of road traffic injury(RTI) in China, to know the trend of RTI, and to provide the reference data for controlling RTI in China. METHODS: The China RTI data from 1951 to 2003 were collected, and in view of the problem of missing values, the method of intrapolation was adopted. The Box-Jenkins technique was used to analyze and predict the trend of RTI in China. Following the process for stationary time-series analysis, model identification, parameter estimation and model diagnosis, the predictive equation for RTI would be established. RESULTS: A series of predictive equations on RTI were finally established based on ARIMA models. The curve fitting is effective and the predictive data of RTI in 2003 are close to the true statistical data. CONCLUSION: The time-series model thus established proves to be of significant usefulness in RTI prediction.

[Differential expression of genes related to photoperiod-temperature sensitive genic male sterility in wheat, revealed by mRNA differential display using G-box family primer].

mRNA differential display with G-box family primer was used to analyze the differential expression of genes of the photoperiod-temperature sensitive genic male sterile(PTSGMS) line of wheat, BAU3338, between the sterile and fertile conditions. The result indicated that gene expression was significantly different between the two types of condition during the fertility transformation phase. The twelve qualitatively different DNA bands were identified with reverse Northern blot hybridization and five positive clones, HT1-G10, HT1-G3, HT2-G2, HT1-G4 and HT2-G5 were sequenced. The homology search indicated that HT1-G10 was highly homological (96%) to the partial sequences of Triticum aestivum chloroplast genes, rbcL and atpB, HT1-G3 was also homological (88%) to Triticum aestivum histone H2A gene and the other three gene fragments were new sequences in Gen-Bank. The analysis of the candidate gene fragments supplied some effective evidences to reveal the developmental mechanism of PTSGMS.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²7ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.