No abstract

Abstract. 2A⌬30 is a live dengue-4 virus vaccine candidate with a 30-nucleotide deletion in its 3Ј-untranslated region. To assess the transmissibility of 2A⌬30 by mosquitoes, we compared its in vivo replication in mosquitoes with that of its wild type DEN-4 parent. Both the vaccine candidate and wild type virus were equally able to infect the mosquito Toxorhynchites splendens after intrathoracic inoculation. Relative to its wild type parent, 2A⌬30 was slightly restricted in its ability to infect the midgut of Aedes aegypti mosquitoes fed on an artificial blood meal and was even more restricted in its ability to disseminate from the midgut to the salivary glands. Thus, the 30-nucleotide deletion rendered the vaccine candidate more sensitive than its wild type parent to the mosquito midgut escape barrier. Most significantly, 2A⌬30 was not transmitted to 352 Ae. albopictus mosquitoes fed on 10 vaccinees, all of whom were infected with the vaccine candidate.

No abstract

Transformation of rickettsiae is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFPUV) as a fluorometric marker and transformed Rickettsia typhi with anrpoB-GFPUV fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the rpoB-GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV expression, and transformed R. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFPUV) gene.

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin fromR. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negativeProteus mirabilis WPM111 (a HpmA− mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the clonedtlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.