Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species.

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5'dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5'dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2-3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5'dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically.

Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti.

We have constructed an orally infectious Sindbis virus, ME2/5'2J/GFP, that expresses green fluorescent protein (GFP) in the midgut of Aedes aegypti and in other tissues as the virus disseminates. This virus has two unique features that are improvements over the SIN-based expression systems currently used in mosquitoes. First, a subgenomic RNA promoter and GFP coding sequence is located 5'- to the second subgenomic promoter and structural genes of the virus. Second, the E2 glycoprotein gene of TE/5'2J/GFP is replaced with the E2 gene of MRE16 SIN virus. The first feature enhances virus genome stability during virus dissemination from the midgut to other tissues and the second allows efficient virus entry into the midgut epithelial cells and then spread of the virus throughout the mosquito.

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3.

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

Bacillus stearothermophilus cell shape determinant gene, mreC and mreD, and their stimulation of protease production in Bacillus subtilis.

Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19. The cloned fragment stimulated production of a 37-kDa protease in B. subtilis. The nucleotide sequence of the genes and their flanking regions were identical to the B. subtilis cell shape determinant genes mreC and mreD [J. Bacteriol., 176, 6729-6742 (1992); J. Bacteriol., 176, 6717-6728 (1992)]. The mreC and mreD genes in B. subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24 h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli. The protease productions in B. subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes. The extracellular protease was purified, and the molecular mass of the enzyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable at pH 7-10. The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate.