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Sun J.,CAS Shanghai Institutes for Biological Sciences | Yu Y.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Deubel V.,CAS Shanghai Institutes for Biological Sciences
Microbes and Infection | Year: 2012

Japanese encephalitis virus (JEV) is a flavivirus with a complex life cycle involving mosquito vectors that mainly target birds and pigs, and causes severe encephalitis in children in Asia. Neurotropic flaviviruses of the JEV serogroup have a particular characteristic of expressing a unique nonstructural NS1' protein, which is a prolongation of NS1 at the C terminus by 52 amino acids derived from a pseudoknot-driven-1 translation frameshift. Protein NS1' is associated with virus neuro-invasiveness. In this study, the need of the pseudoknot structure for NS1' synthesis was confirmed. By using a specific antibody against the prolonged peptide, NS1' was found to be absent from the JEV SA14-14-2 vaccine strain, resulting from a single nucleotide silent mutation in the pseudoknot. A partial cleavage of NS1' at a specific site of its C-terminal appendix recognized by caspases and inhibited by caspase inhibitors suggests a unique feature of intracellular NS1'. © 2012 Institut Pasteur. Source

Yu Y.,Chinese National Institute for the Control of Pharmaceutical and Biological Products
Vaccine | Year: 2010

A novel Japanese encephalitis (JE) attenuated live vaccine virus SA14-14-2 was licensed for commercial application in China in 1989. Since then this vaccine has been widely used in China and other countries in Asia, and no vaccine associated encephalitis case was reported. The neurovirulence of the SA14-14-2 was tested in JE susceptible laboratory animals, such as mice, monkeys, hamsters and athymic nude mice. The results showed that the attenuated virus strain was avirulent to these animals by intracerebral inoculation (i.c.) or intraperitoneal inoculation (i.p.). Studies on the neuroattenuation stability revealed that no reversion after 17 times tissue culture passages or one i.c. sucking mice passage. Mosquito infection studies indicated that after one mosquito intrathoracical passage, the progeny viruses in the infected mosquitoes were unable to cause sucking mice or weanling mice disease. Molecules characteristics' studies of the SA14-14-2 virus strain showed that there are 57-61 nucleotide changes and 24-31 amino acid substitutions, eight substitutions in the E protein gene are the critical amino acid related to the virus attenuation. The E gene sequence studies have showed that the 8 critical amino acids were not changed after 22 passages in tissue cultures or one passage in mosquitoes. Comparison of the full-length sequence to the parental SA14 virus has revealed that after 22 passages in the tissue cultures, only 8 nucleotides changed leading to 4 amino acid substitutions. However they were not the reverse mutation and none of the 8 critical residues changed. The homology of the nucleotide and amino acid between the virus of passage 22 and the primary seed virus in Genbank was 99.93% and 99.88% respectively. The above results demonstrated that the SA14-14-2 virus is highly attenuated for the various JE susceptible animals. The attenuated phenotypes and the genetic characteristics of the SA14-14-2 strain are highly stable after multiple in vitro passages or mosquitoes infection. Therefore the safety of the live JE vaccine is due to a high degree of neuroattenuation and a number of stable phenotypically and genetically characteristics, suggesting that reversion to neurovirulence of the vaccine strain would be highly unlikely. © 2010 Elsevier Ltd. All rights reserved. Source

Maddison L.A.,Vanderbilt University | Lu J.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Chen W.,Vanderbilt University
Methods in Cell Biology | Year: 2011

While several mutagenesis methods have been successfully applied in zebrafish, these mutations do not allow tissue- or temporal-specific functional analysis. We have developed a strategy that will allow tissue- or temporal-specific disruption of genes in zebrafish. This strategy combines gene-trap mutagenesis and FlEx modules containing target sites for site-specific recombinases. The gene-trap cassette is highly mutagenic in one orientation and nonmutagenic in the opposite orientation, with different fluorescent proteins as indicators of the orientation. The inclusion of the FlEx modules allows two rounds of stable inversion mediated by the Cre and Flp recombinases. This gene-trap cassette can be easily delivered via transposons. Through large-scale community-wide efforts, broad genome coverage can be obtained. This should allow investigation of cell/tissue-specific gene function of a wide range of genes. © 2011 Elsevier Inc. Source

Wang L.,Chinese National Institute for the Control of Pharmaceutical and Biological Products
Chinese Journal of Biologicals | Year: 2010

Objective: To evaluate the adaptivity of CVS-11 strain as the challenge virus in the rapid fluorescent focus inhibition test (RFFTT) for determination of neutralizing antibody against rabies virus on the basis of sequencing the complete genome of CVS-11 strain and comparative research on important antigenic sites among CVS-11 and rabies vaccine strains used in China. Methods: Eight overlapped gene fragments were amplified by RT-PCR in order to cover the complete genome and then were cloned into pGEM-T Easy vector respectively. Each gene fragment was sequenced on both strands, based on which sequences were analyzed with DNAStar software, and the results were compared with those of rabies vaccine virus strains CTN-1, aG, FluryLEP, PM and PV for analysis of homology and important antigenic sites. Results: The full-length of genome of CVS-11 strain was 11 927 bp. The structural gene arrangement of CVS-11 strain was quite similar to those of other known rabies virus strains except the long non-coding sequences of G mRNA and M mRNA. The homologies of CVS-11 strain to the current rabies vaccine virus strains in China were 84.3% - 99.5%. Based on N gene phylogenetic tree, we found the highest homology of PM strain to CVS-11 strain, while CTN-1 and aG strains were more independent relative to CVS-11 strain than the other vaccine strains. Comparative analysis of the important antigenic sites of G protein also indicated a certain amino acid variation between CVS-11 strain and rabies vaccine virus strains. Conclusion: CVS-11 strain is highly homologous to the current rabies vaccine virus strains in China. However, the variation of antigenic sites of G proteins between CVS-11 strain and various rabies vaccine virus strains may influence the evaluation of immune effects of various rabies vaccines at different degrees. Source

Wang S.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Nie J.,Chinese National Institute for the Control of Pharmaceutical and Biological Products | Wang Y.,Chinese National Institute for the Control of Pharmaceutical and Biological Products
Virus Research | Year: 2011

Subtype C is the most prevalent HIV-1 subtype globally and a common circulating recombinant form virus, CRF_BC, is predominant in China. It is unclear whether subtype C-based vaccines are capable of neutralizing CRF_BC viruses or can be evaluated using a neutralization assay based on CRF_BC pseudoviruses. Phylogenetic analysis of full-length gp160 nucleotide sequences showed that 37 functional env clones formed two unique clusters. The 30 clones belonged to 07_BC and seven clones 08_BC. Increased length in gp120 was found in 07_BC clones when compared to subtype C and 08_BC clones. The subtype BC clones had more PNLG sites on gp160, and 08_BC had more PNLG sites in the V5 region than subtype C. However, the number of PNLG sites in the C4 region of 07_BC was less than subtype C. The neutralization properties of Env-pseudovirus were analyzed against HIV-positive plasma panels belonging to three subtypes: BC, B and AE. Subtype BC pseudoviruses had higher sensitivities to all the three plasma panels than subtype C viruses. CRF07_BC clones were the most sensitive to subtype BC antibodies, regardless of whether the CRF07_plasma panel or CRF08_BC were used. Heatmap analyses of inhibition ratios showed that clones in this study were antigenically diverse despite being genetic similarity. Thus, genetic and neutralization properties of HIV CRF07/08_BC Env isolates predominating in China show different properties than those of subtype C. These findings are likely to provide useful information for the design and evaluation of HIV-1 neutralizing antibody-based vaccine development in China. © 2010 Elsevier B.V. Source

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