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Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.

Because of limited data on dengue virus in Burkina Faso, we conducted 4 consecutive age-stratified longitudinal serologic surveys, ≈6 months apart, among persons 1–55 years of age, during June 2015–March 2017, which included a 2016 outbreak. The seroconversion rate before the serosurvey enrollment was estimated by binomial regression, taking age as the duration of exposure, and assuming constant force of infection (FOI) over age and calendar time. We calculated FOI between consecutive surveys and rate ratios for potentially associated characteristics based on seroconversion using the duration of intervals. Among 2,897 persons at enrollment, 66.3% were IgG-positive, and estimated annual FOI was 5.95%. Of 1,269 enrollees participating in all 4 serosurveys, 438 were IgG-negative at enrollment. The annualized FOI ranged from 10% to 20% (during the 2016 outbreak). Overall, we observed high FOI for dengue. These results could support decision-making about control and preventive measures for dengue.

Objectives: Information on dengue in Africa is limited. To estimate the proportion of dengue-positive cases among febrile patients and describe dengue’s clinical indicators, we conducted passive health facility-based fever surveillance in Mombasa, Kenya. Methods: Non-malarial febrile patients between one and 55 years were enrolled at three health facilities between March 2016 and May 2017. Acute and convalescent blood samples were collected with an interval of 10–21 days. Acute samples were tested with dengue RDT and a selected subset with RT-PCR, and acute/convalescent samples with IgM/IgG ELISA. Results: Among 482 enrollees, 295 (61.2%) were dengue-positive based on laboratory results. The surveillance covered the beginning of a dengue outbreak in April-May 2017, during which 73.9% of enrollees were dengue-positive. By contrast, during the non-outbreak period, 54.6% were dengue-positive. Dengue case status was positively associated with rash, fatigue, headache, retro-orbital pain, nausea/vomiting, nose bleeding, gum bleeding, loss of appetite, myalgia, and arthralgia. Dengue-positive cases in our study had mostly mild disease, with only two requiring observation, and no DHF. Conclusions: The clinical response was generally mild relative to what was observed in SE Asia and the Americas. Given the high level of DENV transmission in Mombasa, more data would be needed to further understand the disease burden and improve case detection for surveillance/monitoring of outbreaks.

BackgroundIn Africa, information on dengue is limited to outbreak reports and focused on some countries with continuing transmission in West and East Africa. To estimate the proportion of dengue-positive cases among febrile patients and identify clinical indicators of dengue cases, we conducted passive facility-based fever surveillance in a catchment area population of 70,000 residents of Lambaréné and its surroundings in Gabon.MethodsNon-malarial febrile patients with current fever or history of fever (≤7 days) between 1 and 55 years of age, were enrolled at Albert Schweitzer Hospital (ASH). Acute (visit 1, day of enrollment) and convalescent blood samples were collected between 10 and 21 days after enrollment. Acute/convalescent samples were tested with IgM/IgG ELISA, and a selected subset of acute samples with RT-PCR.ResultsAmong 682 non-malarial febrile patients enrolled, 119 (17.4%) were identified as dengue-positive (94 dengue-confirmed and 25 dengue-probable cases). Of these dengue-positive cases, 14 were confirmed with PCR, and based on serotyping, two infections were identified to be DENV-2 and two were DENV-3. The majority of our enrolled patients were ConclusionLambaréné is not considered dengue-endemic. However, one in six non-malarial febrile episodes was found to be dengue-positive in the study period. Dengue should be considered more frequently in clinicians' diagnosis among non-malarial febrile patients in Lambaréné. Given the lack of data on dengue in Gabon, additional prospective and longitudinal studies would help to further define the burden and patterns of dengue for improved case detection.

BACKGROUND:In Africa, the magnitude of dengue virus (DENV) transmission is largely unknown. In Burkina Faso, several outbreaks have been reported and data are often based on findings from outbreak investigations. METHODS:To better understand dengue epidemiology and clinical characteristics in Burkina Faso, a fever surveillance study was conducted among patients aged 1-55 years, who presented with non-malarial febrile illness at five primary healthcare facilities in Ouagadougou, Burkina Faso from December 2014 to February 2017, encompassing a 3-month dengue outbreak in September-November 2016. Acute and convalescent blood samples were collected within an interval of 10-21 days between visits. Acute samples were tested with dengue rapid diagnostic tests (RDT) and a selected subset with RT-PCR, and all acute/convalescent samples with IgM/IgG ELISA. RESULTS:Among 2929 non-malarial febrile patients, 740 (25%) were dengue-positive based on RT-PCR and/or IgM/IgG ELISA; 428 out of 777 patients (55%) and 312 out of 2152 (14%) were dengue-positive during outbreak and non-outbreak periods, respectively. There were 11% (316/2929) and 4% (129/2929) patients showing positive for NS1 and IgM, on the RDT, respectively. DENV 2 predominated during the outbreak, whereas DENV 3 predominated before the outbreak. Only 25% of dengue-positive cases were clinically diagnosed with suspected dengue. The odds of requiring observation for ≤3 days (versus routine outpatient care) were 11 times higher among dengue-positive cases than non-dengue cases. In adjusted analyses, dengue-positivity was associated with rash and retro-orbital pain (OR = 2.6 and 7.4, respectively) during the outbreak and with rash and nausea/vomiting (OR = 1.5 and 1.4, respectively) during the non-outbreak period. CONCLUSION:Dengue virus is an important pathogen in Burkina Faso, accounting for a substantial proportion of non-malarial fevers both during and outside outbreak, but is only infrequently suspected by clinicians. Additional longitudinal data would help to further define characteristics of dengue for improved case detection and surveillance.

BACKGROUND:Dengue fever is a rapidly growing public health problem in many parts of the tropics and sub-tropics in the world. While there are existing studies on the economic burden of dengue fever in some of dengue-endemic countries, cost components are often not standardized, making cross-country comparisons challenging. Furthermore, no such studies have been available in Africa. METHODS/PRINCIPAL FINDINGS:A patient-specific survey questionnaire was developed and applied in Burkina Faso, Kenya, and Cambodia in a standardized format. Multiple interviews were carried out in order to capture the entire cost incurred during the period of dengue illness. Both private (patient's out-of-pocket) and public (non-private) expenditure were accessed to understand how the economic burden of dengue is distributed between private and non-private payers. A substantial number of dengue-confirmed patients were identified in all three countries: 414 in Burkina Faso, 149 in Kenya, and 254 in Cambodia. The average cost of illness for dengue fever was $26 (95% CI $23-$29) and $134 (95% CI $119-$152) per inpatient in Burkina Faso and Cambodia, respectively. In the case of outpatients, the average economic burden per episode was $13 (95% CI $23-$29) in Burkina Faso and $23 (95% CI $19-$28) in Kenya. Compared to Cambodia, public contributions were trivial in Burkina Faso and Kenya, reflecting that a majority of medical costs had to be directly borne by patients in the two countries. CONCLUSIONS/SIGNIFICANCE:The cost of illness for dengue fever is significant in the three countries. In particular, the current study sheds light on the potential economic burden of the disease in Burkina Faso and Kenya where existing evidence is sparse in the context of dengue fever, and underscores the need to achieve Universal Health Coverage. Given the availability of the current (CYD-TDV) and second-generation dengue vaccines in the near future, our study outcomes can be used to guide decision makers in setting health policy priorities.

Patterns of colonization and diversification on islands provide valuable insights into evolutionary processes. Due to their unique geographic position and well known history, the Galapagos Islands are an important model system for evolutionary studies. Here we investigate the evolutionary history of a winged grasshopper genus to infer its origin and pattern of colonization in the Galapagos archipelago. The grasshopper genus Sphingonotus has radiated extensively in the Palaearctic and many species are endemic to islands. In the New World, the genus is largely replaced by the genus Trimerotropis. Oddly, in the Caribbean and on the Galapagos archipelago, two species of Sphingonotus are found, which has led to the suggestion that these might be the result of anthropogenic translocations from Europe. Here, we test this hypothesis using mitochondrial and nuclear DNA sequences from a broad sample of Sphingonotini and Trimerotropini species from the Old World and New World. The genetic data show two distinct genetic clusters representing the New World Trimerotropini and the Old World Sphingonotini. However, the Sphingonotus species from Galapagos and the Caribbean split basally within the Old World Sphingonotini lineage. The Galapagos and Caribbean species appear to be related to Old World taxa, but are not the result of recent anthropogenic translocations as revealed by divergence time estimates. Distinct genetic lineages occur on the four investigated Galapagos Islands, with deep splits among them compared to their relatives from the Palaearctic. A scenario of a past wider distribution of Sphingonotus in the New World with subsequent extinction on the mainland and replacement by Trimerotropis might explain the disjunct distribution.

Because of limited data on dengue virus in Burkina Faso, we conducted 4 consecutive age-stratified longitudinal serologic surveys, ≈6 months apart, among persons 1-55 years of age, during June 2015-March 2017, which included a 2016 outbreak. The seroconversion rate before the serosurvey enrollment was estimated by binomial regression, taking age as the duration of exposure, and assuming constant force of infection (FOI) over age and calendar time. We calculated FOI between consecutive surveys and rate ratios for potentially associated characteristics based on seroconversion using the duration of intervals. Among 2,897 persons at enrollment, 66.3% were IgG-positive, and estimated annual FOI was 5.95%. Of 1,269 enrollees participating in all 4 serosurveys, 438 were IgG-negative at enrollment. The annualized FOI ranged from 10% to 20% (during the 2016 outbreak). Overall, we observed high FOI for dengue. These results could support decision-making about control and preventive measures for dengue. [ABSTRACT FROM AUTHOR]

Introduction Dengue is an important and well-documented public health problem in the Asia-Pacific and Latin American regions. However, in Africa, information on disease burden is limited to case reports and reports of sporadic outbreaks, thus hindering the implementation of public health actions for disease control. To gather evidence on the undocumented burden of dengue in Africa, epidemiological studies with standardised methods were launched in three locations in Africa. Methods and analysis In 2014–2017, the Dengue Vaccine Initiative initiated field studies at three sites in Ouagadougou, Burkina Faso; Lambaréné, Gabon and Mombasa, Kenya to obtain comparable incidence data on dengue and assess its burden through standardised hospital-based surveillance and community-based serological methods. Multidisciplinary measurements of the burden of dengue were obtained through field studies that included passive facility-based fever surveillance, cost-of-illness surveys, serological surveys and healthcare utilisation surveys. All three sites conducted case detection using standardised procedures with uniform laboratory assays to diagnose dengue. Healthcare utilisation surveys were conducted to adjust population denominators in incidence calculations for differing healthcare seeking patterns. The fever surveillance data will allow calculation of age-specific incidence rates and comparison of symptomatic presentation between patients with dengue and non-dengue using multivariable logistic regression. Serological surveys assessed changes in immune status of cohorts of approximately 3000 randomly selected residents at each site at 6-month intervals. The age-stratified serosurvey data will allow calculation of seroprevalence and force of infection of dengue. Cost-of-illness evaluations were conducted among patients with acute dengue by Rapid Diagnostic Test. Ethics and dissemination By standardising methods to evaluate dengue burden across several sites in Africa, these studies will generate evidence for dengue burden in Africa and data will be disseminated as publication in peer-review journals in 2018. [ABSTRACT FROM AUTHOR]

Travel-acquired dengue cases have been increasing as the overall global dengue burden has expanded. In Korea, imported dengue cases have been reported since 2000 when it first became a notifiable disease. During the first four months of 2016, three times more dengue cases were reported in Korea than during the same period the previous year. A safe and efficacious vaccine for travelers would be beneficial to prevent dengue disease in individual travelers and potentially decrease the risk of virus spread to non-endemic areas. Here, we summarize the characteristics of dengue vaccines for travelers and review dengue vaccines currently licensed or in clinical development. [ABSTRACT FROM AUTHOR]