Discovery of DNA Viruses in Wild-Caught Mosquitoes Using Small RNA High throughput Sequencing

BACKGROUND: Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18-30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/- strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5'- and 3' -untranslated regions where no transcripts were expected. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors

Dissemination of bloodmeal acquired Rickettsia felis in cat fleas, Ctenocephalides felis

BACKGROUND: Cat fleas, Ctenocephalides felis, are known biological vectors for Rickettsia felis. Rickettsial transmission can be vertical via transovarial transmission within a flea population, as well as horizontal between fleas through a bloodmeal. The previously undescribed infection kinetics of bloodmeal-acquired R. felis in cat fleas provides insight into the R. felis-flea interaction. FINDINGS: In the present study, dissemination of R. felis in previously uninfected cat fleas fed an R. felis-infected bloodmeal was investigated. At weekly intervals for 28 days, rickettsial propagation, accumulation, and dissemination in gut epithelial cells, specifically in the hindgut and the specialized cells in the neck region of midgut, were observed on paraffin sections of infected cat fleas by immunofluorescence assay (IFA) and confirmed by PCR detection of R. felis 17-kDa antigen gene. IFA results demonstrate ingested rickettsiae in vacuoles during early infection of the gut; lysosomal activity, indicated by lysosome marker staining of freshly-dissected gut, suggests the presence of phagolysosome-associated vacuoles. Subsequent to infection in the gut, rickettsiae spread to the hemocoel and other tissues including reproductive organs. Densely-packed rickettsiae forming mycetome-like structures were observed in the abdomen of infected male cat fleas during late infection. Ultrastructural analysis by transmission electron microscopy (TEM) confirmed the presence and infection characteristics of Rickettsia including rickettsial destruction in the phagolysosome, rickettsial division, and accumulation in the flea gut. CONCLUSIONS: This study intimately profiles R. felis dissemination in cat fleas and further illuminates the mechanisms of rickettsial transmission in nature