Cloning and expression of envelope protein of Thai genotype I strain KE-093 of Japanese encephalitis virus.

The purpose of this study was to clone and express envelope (E) gene of Japanese encephalitis virus (JEV) genotype I, Thai strain KE-093. The E gene was amplified by PCR and cloned using the expression vector, pET-15b. Analysis of the insert sequence revealed a point mutation, which was corrected by site directed mutagenesis. The envelope 53 kDa protein expression was generated by in vitro coupled transcription translation system. Heterologous expression in Escherichia coli Rosetta 2 strain, but not in E. coli BL21 (DE3) resulted in 2 immunoreactive bands (13 and 53 kDa) using anti-JEV E protein antibodies, and an additional band (35 kDa) using anti-His antibodies, suggesting that E protein antigenicity is located at the carboxy-terminal region. This is the first report of a successful cloning and heterologous expression of an E gene of JEV genotype I. This should prove useful in the application for diagnostics and vaccine development of JEV genotype I strains.

Genetic differences of E gene of a Thai strain of Japanese encephalitis virus that determine small plaque size phenotype but not neurovirulence in suckling mice.

Two small plaque variants of Japanese encephalitis virus (JEV), S4P9 and S9P10, were recovered from the wild type of JEV strain KE-093 using plaque purification in combination with the temperature-shift induction technique. Growth patterns of the S4P9 and S9P10 in BHK-21 cells as well as neurovirulence in suckling mice were similar to that of KE-093. An amino acid substitution, lysine for glutamic acid, was present in envelope protein at residue E-83 in the small plaque variants. This study shows that small plaque phenotype is not always associated with attenuation in vivo.

Characterization of dengue-2 virus binding to surfaces of mammalian and insect cells.

The binding of dengue type 2 (DEN-2) virus to mammalian (LLC-MK(2) and Vero) and mosquito (C6/36 and AP61) cell surfaces was investigated by a virus-binding assay using purified (3)H-labeled DEN-2 virus. The DEN-2 virus binding to all four cell types was specific and saturable, indicating the presence of a single class of receptors (ranging from 3.7 x 10(3) to 3.5 x 10(4) receptors/cell) with a high affinity for DEN-2 virus (K(d) ranging from 98 to 171 pM). Treatment of cell surfaces with certain glycosidases significantly reduced virus binding to mammalian cell lines, but not to the insect cell lines examined. Furthermore, heparin was found to compete with mammalian cell receptors for binding to DEN-2 virus and to inhibit viral infection of mammalian cells, but heparin had no effect on viral binding to or infection of insect cells. These results confirm previous reports suggesting that DEN-2 virus receptors on mammalian cell lines are different from those on insect cell lines.

Genetic variations of human herpesvirus 7 by analysis of glycoproteins B and H, and R2-repeat regions.

Clinical isolates of human herpesvirus 7 (HHV-7) from the saliva of healthy individual were investigated for genetic variations in the regions of two immediate-early (IE) genes, the glycoprotein B (gB) and glycoprotein H (gH) genes, and in R2-repeat. The genomic DNA of 24 isolates from citizens of Thailand, Japan, and the United States was amplified to detect size variations in the IE-1 and IE-2 loci, but none was observed, suggesting that there was no deletion or insertion in these genes, in contrast with an IE gene of human herpesvirus 6 (HHV-6). The sequences of the gB gene from isolates acquired from 5 Japanese and 8 Thai subject were then compared with those of American strains JI and RK with respect to codons that are known to differentiate gB alleles. All the isolates were found to have gB allele C except for the JI strain, which has allele F. Variability was also observed in five specific gH codons, resulting in 6 different groups. The HHV-7 isolates might be classified into two major genetic variants by combining their gB and gH allelic groupings. In the present study, only JI belonged to variant 1, while the rest of the isolates appeared to belong to variant 2. In the R2-repeat region, size heterogeneities were observed among the 24 isolates, due to different repeat numbers (17, 15, 14, 13, or 12 repeats). Therefore, we used the R2-repeat to identify the origins of isolates in a study of HHV-7 transmission, and found HHV-7 to be transmitted within a family from both mothers and fathers to their children.

Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity.

Chitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10 mU of this chitinase, the LD(50) of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10 mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5alpha and sequenced to reveal a single open reading frame of 1,815 bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15 mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.