The four genetically divergent dengue virus (DENV) types are traditionally classified as serotypes. Antigenic and genetic differences among the DENV types influence disease outcome, vaccine-induced protection, epidemic magnitude, and viral evolution. We characterized antigenic diversity in the DENV types by antigenic maps constructed from neutralizing antibody titers obtained from African green monkeys and after human vaccination and natural infections. Genetically, geographically, and temporally, diverse DENV isolates clustered loosely by type, but we found many are as similar antigenically to a virus of a different type as to some viruses of the same type. Primary infection antisera did not neutralize all viruses of the same DENV type any better than other types did up to two years after infection and did not show improved neutralization to homologous type isolates. That the canonical DENV types are not antigenically homogenous has implications for vaccination and research on the dynamics of immunity, disease, and the evolution of DENV.

In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.

BackgroundSingle amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar.Methodology/Principal FindingsWe determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study.Conclusions/SignificancePyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3′ and 5′ ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6–36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

In 2001, Myanmar (Burma) had its largest outbreak of dengue—15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses.

SummaryWe calculated pathogen-specific attributable fractions of acute watery diarrhea in children from 16 countries using quantitative polymerase chain reaction testing for a broad range of enteropathogens. Rotavirus remained the leading etiology, despite a clear impact of rotavirus vaccine introduction.

SummaryBackgroundIn southeast Asia, antibiotic prescription in febrile patients attending primary care is common, and a probable contributor to the high burden of antimicrobial resistance. The objective of this trial was to explore whether C-reactive protein (CRP) testing at point of care could rationalise antibiotic prescription in primary care, comparing two proposed thresholds to classify CRP concentrations as low or high to guide antibiotic treatment.MethodsWe did a multicentre, open-label, randomised, controlled trial in participants aged at least 1 year with a documented fever or a chief complaint of fever (regardless of previous antibiotic intake and comorbidities other than malignancies) recruited from six public primary care units in Thailand and three primary care clinics and one outpatient department in Myanmar. Individuals were randomly assigned using a computer-based randomisation system at a ratio of 1:1:1 to either the control group or one of two CRP testing groups, which used thresholds of 20 mg/L (group A) or 40 mg/L CRP (group B) to guide antibiotic prescription. Health-care providers were masked to allocation between the two intervention groups but not to the control group. The primary outcome was the prescription of any antibiotic from day 0 to day 5 and the proportion of patients who were prescribed an antibiotic when CRP concentrations were above and below the 20 mg/L or 40 mg/L thresholds. The primary outcome was analysed in the intention-to-treat and per-protocol populations. The trial is registered with ClinicalTrials.gov, number NCT02758821, and is now completed.FindingsBetween June 8, 2016, and Aug 25, 2017, we recruited 2410 patients, of whom 803 patients were randomly assigned to CRP group A, 800 to CRP group B, and 807 to the control group. 598 patients in CRP group A, 593 in CRP group B, and 767 in the control group had follow-up data for both day 5 and day 14 and had been prescribed antibiotics (or not) in accordance with test results (per-protocol population). During the trial, 318 (39%) of 807 patients in the control group were prescribed an antibiotic by day 5, compared with 290 (36%) of 803 patients in CRP group A and 275 (34%) of 800 in CRP group B. The adjusted odds ratio (aOR) of 0·80 (95% CI 0·65–0·98) and risk difference of −5·0 percentage points (95% CI −9·7 to −0·3) between group B and the control group were significant, although lower than anticipated, whereas the reduction in prescribing in group A compared with the control group was not significant (aOR 0·86 [0·70–1·06]; risk difference −3·3 percentage points [–8·0 to 1·4]). Patients with high CRP concentrations in both intervention groups were more likely to be prescribed an antibiotic than in the control group (CRP ≥20 mg/L: group A vs control group, p<0·0001; CRP ≥40 mg/L: group B vs control group, p<0·0001), and those with low CRP concentrations were more likely to have an antibiotic withheld (CRP <20 mg/L: group A vs control group, p<0·0001; CRP <40 mg/L: group B vs control group, p<0·0001). 24 serious adverse eve...