John P. Kondig

Evidence for Arbovirus Dissemination Conduits from the Mosquito (Diptera: Culicidae) Midgut

Abstract The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.

Identification of ciprofloxacin resistance by SimpleProbe™, High Resolution Melt and Pyrosequencing™ nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro(r)) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe and High Resolution Melt (HRM) dye chemistries and Pyrosequencing genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe and Pyrosequencing successfully identified all known mutants, the HRM assay identified all but those resulting from G<-->C or A<-->T substitutions.

Isolation and genomic characterization of Chaoyang virus strain ROK144 from Aedes vexans nipponii from the Republic of Korea.

During June 2003, mosquito surveillance was conducted at a US Army installation and a US Military training site 2 km south of the demilitarized zone, Republic of Korea. Mosquitoes were collected using Mosquito Magnets™, sorted to species, and assayed for the presence of arboviruses. From the 3,149 mosquitoes that were sorted into 126 pools, one Aedes vexans nipponii pool (out of 73 pools) tested positive for flavivirus RNA by reverse transcription-PCR. After isolation from C6/36 cell culture supernatant, the viral genome was sequenced and found to be 98.9% related to Chaoyang virus, a potential arthropod-specific flavivirus. This report details the first identification of Chaoyang virus in the Republic of Korea and highlights its relationship to other flaviviruses.