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BackgroundMost regions in Indonesia experience annual dengue epidemics. However, the province of East Nusa Tenggara has consistently reported low incidence. We conducted a dengue molecular epidemiology study in Kupang, the capital of the province.MethodsDengue patients were recruited from May 2016 to September 2017. Dengue virus (DENV) screening was performed using NS1 and immunoglobulin G (IgG)/IgM detection. Serotype was determined using reverse transcription polymerase chain reaction and the envelope genes were sequenced to infer the genetic identity and phylogeny.ResultsFrom 119 patients, dengue was confirmed in 62 (52%). Compared with official data, underreporting of dengue incidence was observed. The majority (36%) of patients were children ConclusionsThe low dengue incidence was most likely caused by sustained local circulation of endemic viruses. This study provides information on the epidemiology of dengue in a low-endemicity setting that should help future mitigation and disease management.

Background Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. Methods We evaluated a method designed to address this challenge, using the ‘Primal Scheme’ multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. Results The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested ($$ \overline{x} $$x¯ = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17–99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69–99.92%). However, coding-region coverage was reduced at this depth ($$ \overline{x} $$x¯ = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. Conclusion The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available.

Transient computing enables application execution to be performed despite power outages. Although it handles the non-deterministic nature of energy harvesting (EH), sensor systems envisioned by the IoT seek more cost- and volume-effective solutions, which are better tailored to application requirements. Additionally, a major drawback of transient computing, keeping track of time, hinders its widespread adoption in the IoT. To overcome these challenges, this paper proposes a control flow for sensor systems by combining two state-of-the-art transient computing schemes in an energy-aware manner, underpinned by a strategy for timekeeping. It enables application execution to be reliably performed even under the most severe EH conditions, with an improved cost and volume efficiency, i.e., smaller energy storage. Benefiting from the combination of the two schemes, dynamic adjustment of system performance is achieved, while the time is accurately tracked. To illustrate the applicability of this flow to actual sensor systems, two case studies: a bicycle trip computer and a step counter, are presented. Empirical results reveal that, even with a tiny amount of energy harvested ( $\simeq$ tens of $\mu \text{J}$ ), our proposed approach can meet application requirements with smaller storage, i.e., 40% and 66% reduction in required capacitance for the presented case studies.

Background The introduction of rotavirus A vaccination across the developing world has not proved to be as efficacious as first hoped. One cause of vaccine failure may be infection by zoonotic rotaviruses that are very variable antigenically from the vaccine strain. However, there is a lack of genomic information about the circulating rotavirus A strains in farm animals in the developing world that may be a source of infection for humans. We therefore screened farms close to Accra, Ghana for animals sub-clinically infected with rotavirus A and then sequenced the virus found in one of these samples. Results 6.1% of clinically normal cows and pigs tested were found to be Rotavirus A virus antigen positive in the faeces. A subset of these (33.3%) were also positive for virus RNA. The most consistently positive pig sample was taken forward for metagenomic sequencing. This gave full sequence for all open reading frames except segment 5 (NSP1), which is missing a single base at the 5′ end. The virus infecting this pig had genome constellation G5-P[7]-I5-R1-C1-M1-A8-N1-T7-E1-H1, a known porcine genotype constellation. Conclusions Farm animals carry rotavirus A infection sub-clinically at low frequency. Although the rotavirus A genotype discovered here has a pig-like genome constellation, a number of the segments most closely resembled those isolated from humans in suspected cases of zoonotic transmission. Therefore, such viruses may be a source of variable gene segments for re-assortment with other viruses to cause vaccine breakdown. It is recommended that further human and pig strains are characterized in West Africa, to better understand this dynamic.

Funder: US-CDC, Background: Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. Methodology/Principal Findings: Four hundred and seventy nine acutely febrile patients presenting between September 2017–2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4–3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. Conclusions/Significance: Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia.

Modern embedded systems need to cater for several needs depending upon the application domain in which they are deployed. For example, mixed-critically needs to be considered for real-time and safety-critical systems and energy for battery-operated systems. At the same time, many of these systems demand for their reliability and security as well. With electronic systems being used for increasingly varying type of applications, novel challenges have emerged. For example, with the use of embedded systems in increasingly complex applications that execute tasks with varying priorities, mixed-criticality systems present unique challenges to designing reliable systems. The large design space involved in implementing cross-layer reliability in heterogeneous systems, particularly for mixed-critical systems, poses new research problems. Further, malicious security attacks on these systems pose additional extraordinary challenges in the system design. In this paper, we cover both the industry and academia perspectives of the challenges posed by these emergent aspects of system design towards designing high-performance, energy-efficient, reliable and/or secure embedded systems. We also provide our views on paths forward.

As apex predators, pinnipeds are considered to be useful bioindicators of marine and coastal environments. Endemic to a small archipelago in the South Pacific, the Juan Fernandez fur seal (JFFS) is one of the less‐studied members of the pinniped family Otariidae. This study aimed to characterize the fecal microbiome of the JFFS for the first time, to establish a baseline for future studies of host–microbial–environment interactions and monitoring programs. During two consecutive reproductive seasons, 57 fecal samples were collected from seven different JFFS colonies within the Juan Fernandez Archipelago, Chile. Bacterial composition and abundance were characterized by sequencing the V4 region of the 16S rRNA gene. The overall microbiome composition was dominated by five phyla: Firmicutes (40% ±24), Fusobacteria (30% ±17), Bacteroidetes (22% ±10), Proteobacteria (6% ±4), and Actinobacteria (2% ±3). Alpha diversity was higher in Tierras Blancas. However, location was not found to be a dominant driver of microbial composition. Interestingly, the strongest signal in the data was a negative association between the genera Peptoclostridium and Fusobacterium, which explained 29.7% of the total microbial composition variability between samples. The genus Peptoclostridium has not been reported in other pinniped studies, and its role here is unclear, with interpretation challenging due to a lack of information regarding microbiome functionality in marine mammals. As a first insight into the JFFS fecal microbiome, these results contribute towards our understanding of the natural microbial diversity and composition in free‐ranging pinnipeds., Two distinct microbial patterns were identified in Juan Fernandez fur seal fecal samples. We suspect that sex, age, and especially diet are most likely to be influencing the observed patterns.

The Gambian epauletted fruit bat, Epomophorus gambianus, is widely distributed across sub-Saharan Africa. Its assembled and annotated mitochondrial genome (GenBank accession no. KT963027) is 16,702 bases in length, containing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions: the control region (D-loop) and the origin of light-strand replication (OL). The average base composition is 32.2% A; 27.6% C; 14% G; and 26.1% T. The mitogenome presented a structural composition greatly conserved between members of the Pteropodidae family.

Energy harvesting wireless sensor nodes are sensitive to spatial and temporal fluctuations in energy availability. This issue is especially prevalent in batteryless systems, where devices are directly connected to power sources with little or no buffering. The strong coupling of energy supply and demand introduces a new dimension to the problem of designing robust networked sensing systems. We propose a modeling framework for this class of batteryless systems with an emphasis on the interactions between energy and function. The tool models energy harvesters, power management circuitry, energy storage, microcontrollers, sensors, radio modules, environmental models, and is fully extensible. The microcontroller model is based on cycle-accurate instruction set simulators from Fused, with various peripheral extensions to enable board-level functionality, such as SPI, DMA, hardware multiplier etc. The tool enables virtual prototyping of self-powered wireless sensor nodes, but is especially useful for studying intermittent operation and developing application specific software, hardware, or combined solutions. The simulator is capable of executing real workloads under realistic conditions and this is demonstrated through a case study where the same compiled binary is executed on a virtual prototype and its corresponding physical wireless sensor system to yield matching digital traces and current profiles

The people of Indonesia have been afflicted by dengue, a mosquito-borne viral disease, for over 5 decades. The country is the world's largest archipelago with diverse geographic, climatic, and demographic conditions that may impact the dynamics of disease transmissions. A dengue epidemiology study was launched by us to compare and understand the dynamics of dengue and other arboviral diseases in three cities representing western, central, and eastern Indonesia, namely, Batam, Banjarmasin, and Ambon, respectively. A total of 732 febrile patients were recruited with dengue-like illness during September 2017–2019 and an analysis of their demographic, clinical, and virological features was performed. The seasonal patterns of dengue-like illness were found to be different in the three regions. Among all patients, 271 (37.0%) were virologically confirmed dengue, while 152 (20.8%) patients were diagnosed with probable dengue, giving a total number of 423 (57.8%) dengue patients. Patients' age and clinical manifestations also differed between cities. Mostly, mild dengue fever was observed in Batam, while more severe cases were prominent in Ambon. While all dengue virus (DENV) serotypes were detected, distinct serotypes dominated in different locations: DENV-1 in Batam and Ambon, and DENV-3 in Banjarmasin. We also assessed the diagnostic features in the study sites, which revealed different patterns of diagnostic agreements, particularly in Ambon. To detect the possibility of infection with other arboviruses, further testing on 461 DENV RT-PCR-negative samples was performed using pan-flavivirus and -alphavirus RT-PCRs; however, only one chikungunya infection was detected in Ambon. A diverse dengue epidemiology in western, central, and eastern Indonesia was observed, which is likely to be influenced by local geographic, climatic, and demographic conditions, as well as differences in the quality of healthcare providers and facilities. Our study adds a new understanding on dengue epidemiology in Indonesia.

Dengue virus (DENV) sequencing is a vital tool for surveillance and epidemiology studies. However, the current methods employed for sequencing DENV are expensive, laborious and technically demanding, often due to intra- and inter-serotype variability. Therefore, on-site DENV sequencing is not feasible in many of the areas where DENV is endemic. Surveillance in these areas can only be performed by shipping samples to well-equipped central laboratories for sequencing. However, long periods of inadequate storage and unreliable shipping conditions mean that such samples can arrive degraded, rendering sequence recovery difficult. We therefore aimed to develop an approach that is simple, portable and effective, to be used for on-site DENV sequencing in limited resource settings.To achieve this, we first used the ‘Primal Scheme’ primer design tool to develop a simple and robust protocol for generating multiple short amplicons, covering the complete coding-region of DENV isolates. We then paired this method with the Nanopore MinION, a portable and affordable sequencing device, well-suited to minimal resource settings.The multiplex PCR method produced full-coding-region coverage of all DENV samples tested with no optimisation required, and Nanopore sequencing of the short amplicons generated consensus sequences with high accuracy (99.52 - 99.92 %). Phylogenetic analysis of the consensus sequences generated using the new method showed that they formed monophyletic clusters with those produced by the current, long-amplicon, Illumina method, thus demonstrating that the two approaches are comparable. The multiplex method’s simplicity and portability compared to the current DENV sequencing approach make it well-suited for use in resource-limited, DENV-endemic regions. Deployment of the method in these regions would increase the capacity for DENV surveillance and has the potential to provide vital resolution for future DENV epidemiology studies.

Background: Long-term immunosuppression following solid organ transplantation is associated with increased morbidity and mortality. Viral infections as a result of immunosuppression are a major cause of prolonged hospitalization and poor prognostic outcome post-transplantation. Additionally, changes in the viral populations inhabiting these patients (the virome) may affect the immune response, with implications for graft survival. Methods: We performed metagenomic sequencing of RNA and DNA extracted from paired plasma samples taken from patients pre- and post-renal transplant. This untargeted approach was used to detect changes in the virome following immunosuppression and to determine viral targets for further characterisation. Correlations between viral presence, viral load and clinical outcome were established to determine the relevance of these viruses to patient outcomes. Findings: Anellovirus load was found to be a strong predictor of immunosuppression-related infectious complications, especially viral reactivation events, in the first year post-transplant. Anellovirus load was increased in patients who had been treated for acute graft rejection 1-2 months prior to sampling, revealing additional immunosuppressive measures can have a prolonged negative impact on a patient. Interpretation: Currently no assays exist for predicting infection risk in immunosuppressed patients, meaning that potentially life-threatening infections must be dealt with retrospectively. Our observations support the possibility of a viral marker being used to monitor immunosuppressive protocols, pre-emptively minimizing risk of severe infections including viral re-activation events, reducing hospitalization and improving long-term allotransplant success rates. Funding Statement: This work was funded by Wellcome Programme Grant number WT091501/Z/10/Z, which provided funding for sequencing, reagents and computing infrastructure. MRC programme grant number MR/L019027/1 provided funding for collection of the samples. MRC CASE Studentship MR/K017071/1 provided funding for reagents and the PhD studentship of SS, of which this study formed a part. LW is funded by a Wellcome Clinical Training Fellowship and ECJ received funding from an MRC Clinical Research Fellowship, Sackler Studentship and Evelyn Trust funding. Declaration of Interests: SS reports an MRC CASE studentship in collaboration with GlaxoSmithKline, outside of the submitted work. GSK did not influence any aspect of this study including data collection, analysis, writing of the manuscript or decision to submit for publication. All other authors have nothing to disclose. Ethics Approval Statement: All samples were anonymized and obtained with informed written consent under the approval of the Cambridge Local Research Ethical Committee (reference 08/H0308/176).

Feline morbillivirus (FeMV) is associated with the presence of tubulo-interstitial nephritis (TIN) in cats, however the seroprevalence of FeMV in the UK and the association between the presence of FeMV and renal azotemia is unknown HYPOTHESIS/OBJECTIVES: To identify whether paramyxoviruses are present in urine samples of geriatric cats and to develop an assay to assess FeMV seroprevalence. To investigate the relationship between both urinary paramyxovirus (including FeMV) excretion and FeMV seroprevalence and azotemic chronic kidney disease (CKD). Seventy-nine cats (40 for FeMV detection; 72 for seroprevalence). Retrospective cross-sectional, case control study. Viral RNA was extracted from urine for RT-PCR. PCR products were sequenced for virus identification and comparison. The FeMV N protein gene was cloned and partially purified for use as an antigen to screen cat sera for anti-FeMV antibodies by Western Blot. Feline morbillivirus RNA from five distinct morbilliviruses were identified. Detection was not significantly different between azotemic CKD (1/16) and nonazotemic groups (4/24; P = .36). Three distinct, non-FeMV paramyxoviruses were present in the nonazotemic group but their absence from the azotemic group was not statistically significant (P = .15). 6/14 (43%) azotemic cats and 40/55 (73%) nonazotemic cats were seropositive (P = .06). Feline morbillivirus was detected in cats in the UK for the First time. However, there was no association between virus prevalence or seropositivity and azotemic CKD. These data do not support the hypothesis that FeMV infection is associated with the development of azotemic CKD in cats in the UK. [Abstract copyright: © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.]

Marks (1973) and Gur & Hilgard (1975) have reported success in predicting performance in visual-memory tasks from scores in a questionnare of self-rated vividness of imagery, i.e. the Vividness of Visual Imagery Questionnaire (VVIQ; Marks, 1973). These findings were disconfirmed in two experiments in which the VVIQ was used and vivid pictures were presented in memory tasks. (Editor/RK)

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.

Background: Pregnant women with heart disease are at increased risk of complications. At our centre, management algorithms for this population have been implemented since 2001. However, the impact of standardized care on outcomes has not been evaluated. Methods: We prospectively recorded baseline

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

During pregnancy, hormonally mediated changes in blood volume, red cell mass, and heart rate result in a 50% increase in intravascular volume and cardiac output, peaking during the second trimester and remaining constant through the remainder of the pregnancy (1). Gestational hormones, circulating prostaglandins, and the low-resistance vascular bed in the placenta result in concomitant decreases in peripheral vascular resistance and blood pressure. During labor and delivery, pain and uterine contractions result in additional increases in cardiac output and blood pressure. Immediately following delivery, relief of caval compression and autotransfusion from the emptied and contracted uterus produce a further increase in cardiac output. The hemodynamic changes of pregnancy persist for at least several days postpartum and may not fully resolve until the sixth postpartum month.

The authenticity of a posthypnotically released emotion was examined to see whether this emotion also could modify the reaction times (RTs) of subjects. The simulant design proposed by Orne (1979) was utilized, with 10 highly hypnotizable subjects and 10 non-hypnotizable simulators participating in the experiment. The subjects received a post-hypnotic suggestion to the effect that every presentation of a light stimulus would release a feeling of discomfort and that a second light stimulus would be neutral. They were informed that they could terminate the stimulation by pressing a RT switch and received a suggestion of amnesia for the posthypnotic suggestion. The simulators skilfully imitated the objective behavior of the hypnotic subjects; their physiological reactions increased and their RTs were shorter when exposed to the aversive stimulation. The main differences occurred in the subjective reports. The hypnotic subjects reported that the emotion was released involuntarily and found relief in escaping from it, while the simulators reported no corresponding experiences, saying that they had deliberately manipulated the objective behavior.