A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

AbstractAn acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host

Elderly patients' knowledge of drug benefit caps and communication with providers about exceeding caps.

ObjectivesMany elderly persons have drug benefits with coverage gaps, such as in Medicare Part D. Because beneficiaries who have such gaps must pay all drug costs, an accurate knowledge of gap thresholds and communication with providers about exceeding caps is important for elderly persons to manage out-of-pocket drug costs.DesignCross-sectional survey.SettingHealth plan.ParticipantsOne thousand three hundred eight health plan members aged 65 and older. The study was a 2002 cross-sectional survey of elderly persons with capped drug benefits in a managed care plan in one state. Participants were sampled so that half reached coverage caps and half did not.MethodsParticipants reported cap levels, communication with providers about exceeding caps, and decreased medication use due to cost.ResultsOf the 1,308 participants (65.4%response rate), 68.6%did not know their correct cap level. Rates were similar in those who exceeded caps (66.2%), reported difficulty paying for medications (63.9%), or decreased medication use (66%). For participants who exceeded caps, 59.1% did not know beforehand that they were close to exceeding caps and 50.2% did not tell providers afterward. In multivariate analyses accounting for demographics and health, the oldest participants (> or =85 vs 65-74) were at greater risk for not knowing cap levels (odds ratio (OR)=2.0, 95% confidence interval (CI)=1.2-3.4) and not telling providers about exceeding caps (OR=2.2, 95% CI=1.1-4.5).ConclusionsElderly patients often did not know correct cap levels and did not tell providers about exceeding caps. Providers, plans, and policymakers should actively assess and improve Medicare beneficiaries' knowledge of Part D coverage gaps