Correlation of Pfg377 ortholog gene expression of Plasmodium vivax and mosquito infection.

OBJECTIVE: To determine the expression of Pfg377 ortholog gene in Plasmodium vivax, and examine its correlation with mosquito infection. METHODS: Seventy clinical blood samples positive for P. vivax by microscopy, were used for the mosquito infectivity assay. Infectivity to female Anopheles dirus was determined from oocyst counts. The transcripts of Pfg377 ortholog gene of P. vivax from blood samples infective and non-infective to mosquitoes were examined using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Of 70 P. vivax positive blood samples, 50 (71.4%) samples were mosquito-infective and 20 (28.6%) were not. In infective samples, the expression level of Pfg377 ortholog gene was significantly higher than in the non-infective group (P<0.05). In infective samples, the expression level of Pfg377 ortholog gene at ≥100 copies/ml of blood cut-off point correlated with ≥10 oocysts/mosquito cut-off point of average oocyst numbers and with ≥50% cut-off point of per cent infected mosquitoes (Pearson's chi-square correlation, P=0.014 and P=0.026, respectively). CONCLUSION: The cut-off point of the expression level of Pfg377 ortholog gene could be used to predict the infectiousness of P. vivax gametocytes leading to mosquito infection and parasite transmission in the field.

Multiple enzyme activities of flavivirus proteins.

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.