Metagenomic arbovirus detection using MinION nanopore sequencing.

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.

Improving public health intervention for mosquito-borne disease: the value of geovisualization using source of infection and LandScan data.

Epidemiological studies use georeferenced health data to identify disease clusters but the accuracy of this georeferencing is obfuscated by incorrectly assigning the source of infection and by aggregating case data to larger geographical areas. Often, place of residence (residence) is used as a proxy for the source of infection (source) which may not be accurate. Using a 21-year dataset from South Australia of human infections with the mosquito-borne Ross River virus, we found that 37% of cases were believed to have been acquired away from home. We constructed two risk maps using age-standardized morbidity ratios (SMRs) calculated using residence and patient-reported source. Both maps confirm significant inter-suburb variation in SMRs. Areas frequently named as the source (but not residence) and the highest-risk suburbs both tend to be tourist locations with vector mosquito habitat, and camping or outdoor recreational opportunities. We suggest the highest-risk suburbs as places to focus on for disease control measures. We also use a novel application of ambient population data (LandScan) to improve the interpretation of these risk maps and propose how this approach can aid in implementing disease abatement measures on a smaller scale than for which disease data are available.

Arbovirus models to provide practical management tools for mosquito control and disease prevention in the Northern Territory, Australia.

Ross River virus (RRV) causes the most common human arbovirus disease in Australia. Although the disease is nonfatal, the associated arthritis and postinfection fatigue can be debilitating for many months, impacting on workforce participation. We sought to create an early-warning system to notify of approaching RRV disease outbreak conditions for major townships in the Northern Territory. By applying a logistic regression model to meteorologic factors, including rainfall, a postestimation analysis of sensitivity and specificity can create rainfall cut-points. These rainfall cut-points indicate the rainfall level above which previous epidemic conditions have occurred. Furthermore, rainfall cut-points indirectly adjust for vertebrate host data from the agile wallaby (Macropus agilis) as the life cycle of the agile wallaby is intricately meshed with the wet season. Once generated, cut-points can thus be used prospectively to allow timely implementation of larval survey and control measures and public health warnings to preemptively reduce RRV disease incidence. Cut-points are location specific and have the capacity to replace previously used models, which require data management and input, and rarely provide timely notification for vector control requirements and public health warnings. These methods can be adapted for use elsewhere.

Image segmentation for automatic particle identification in electron micrographs based on hidden Markov random field models and expectation maximization.

Three-dimensional reconstruction of large macromolecules like viruses at resolutions below 10 A requires a large set of projection images. Several automatic and semi-automatic particle detection algorithms have been developed along the years. Here we present a general technique designed to automatically identify the projection images of particles. The method is based on Markov random field modelling of the projected images and involves a pre-processing of electron micrographs followed by image segmentation and post-processing. The image is modelled as a coupling of two fields--a Markovian and a non-Markovian. The Markovian field represents the segmented image. The micrograph is the non-Markovian field. The image segmentation step involves an estimation of coupling parameters and the maximum á posteriori estimate of the realization of the Markovian field i.e, segmented image. Unlike most current methods, no bootstrapping with an initial selection of particles is required.

Natural history of Ross River virus-induced epidemic polyarthritis.

OBJECTIVE: To describe the natural history, treatment and cost of Ross River virus-induced epidemic polyarthritis (RRV disease). DESIGN: Questionnaire-based longitudinal prospective study. PARTICIPANTS AND SETTING: Patients in the greater Brisbane area, Queensland, diagnosed with RRV disease by their general practitioners based on clinical symptoms and paired serological tests between November 1997 and April 1999. MAIN OUTCOME MEASURES: Scores on two validated quality-of-life questionnaires (Clinical Health Assessment Questionnaire and Medical Outcomes Study Short Form 36) were obtained soon after diagnosis and one, two, three, six and 12 months thereafter. Scores were compared between patients diagnosed with RRV disease alone and those with RRV disease plus other conditions. RESULTS: 67 patients were enrolled. Most patients with RRV disease alone had severe acute symptoms, but followed a consistent path to recovery within three to six months. Other conditions, often chronic rheumatic diseases or depression, were identified in half the cohort; their quality-of-life scores suggested stable chronic illness between six and 12 months after diagnosis. Non-steroidal anti-inflammatory drugs (NSAIDs) were taken by 58% of patients (average use, 7.6 weeks; range, 2-22 weeks). Time off work averaged 1.9 days, and direct cost to the community was estimated as 1018 Australian dollars per patient. CONCLUSIONS: Symptom duration and frequency of long-term symptoms may have been overestimated by previous studies of RRV disease. Disease persisting six to 12 months after RRV diagnosis was largely attributable to other conditions, highlighting the need to seek other diagnoses in RRV patients with persistent symptoms.

Assessment of the potential of dogs and cats as urban reservoirs of Ross River and Barmah Forest viruses.

OBJECTIVE: To determine whether dogs and cats are potential reservoirs of Ross River (RR) and Barmah Forest (BF) viruses METHOD: Young seronegative female dogs and cats were experimentally exposed to the viruses using Ochlerotatus vigilax (Skuse) mosquitoes. RESULTS: Only one of the 10 dogs and one of the 10 cats exposed to RR developed neutralising antibody. None of the animals developed detectable viraemia or clinical signs. One dog and three cats exposed to BF developed neutralising antibody. In addition, a serological survey of sera obtained from domestic dogs and cats residing in the Brisbane region indicated that 23.7% and 1.3% of dogs, and 14% and 2% of cats, had neutralising antibodies to RR and BF respectively. CONCLUSIONS: Although dogs and cats are exposed naturally to these viruses, and can become infected, they are unlikely to be important urban reservoirs of either virus.

Health impact assessments of malaria and Ross River virus infection in the Southern Highlands Province of Papua New Guinea.

Malaria at an elevation of 1050 metres is common and highly endemic in the Tagari Valley in the Southern Highlands of Papua New Guinea. Health impact assessments showed that the risks of malaria and epidemic polyarthritis at a gasfield development project in this area were high. Baseline malariometric surveys were conducted in four villages in June and August 1990 and two follow-up surveys (May and December 1991) were made in the village of Nogolitogo near the gasfield pioneer base camp. A total of 941 blood smears were examined. Average malaria prevalence rates decreased with altitude from 56% (at 1050 m) to 9% (at 1700 m) for children 1-9 years of age and from 45% (at 1050 m) to 8% (at 1550 m) for those aged 10 years or more. The spleen rate for children less than 10 years old did not vary significantly with altitude, but average enlarged spleen for all ages decreased with altitude. Mean packed cell volume increased with altitude. Plasmodium falciparum was the most common malaria parasite found and Anopheles punctulatus the predominant vector. Ross River arbovirus (RRV) antibody prevalence was 59%. These results indicate frequent or constant transmission of malaria and pathogenic arboviruses. Entomological and epidemiological data suggested that the vulnerability of the valley community, the receptivity of the environment and the health hazards from malaria and RRV were high. Nonimmune Papua New Guineans and expatriate employees face high health hazards; therefore effective preventive measures are required to mitigate epidemics and avoid the likely heightened transmission of malaria and arboviruses caused by the development project.

Ross River virus infection on the North Coast of New South Wales.

A retrospective follow-up survey was undertaken of residents of the North Coast of New South Wales infected with Ross River virus in 1992. The aims of the study were to describe the epidemiology and acute symptomatology of Ross River virus infection, its natural history during the first 12 months of infection, and its effects on those infected. Questionnaires were distributed to both cases and their medical practitioners. Of 129 people infected, aged between six and 85 years, 81 (63 per cent) were male and 48 (37 per cent) were female. The peak age-specific incidence was in the age group 50 to 59 years. The most common symptoms were arthralgia (95 per cent) and tiredness (91 per cent). Over 60 per cent took time off work. At 12 months follow-up, over 50 per cent reported persistent arthralgia, 35 per cent reported persistent tiredness and 15 per cent were still unable to carry out their normal activities. The median duration of symptoms was in the range 7 to 12 months, and of incapacity was in the range five weeks to three months. There were some differences from previous reports of Ross River virus outbreaks, in the incidence of major symptoms and the duration of illness and incapacity. These are likely to be at least partly due to inconsistent measurement methods. In this study, there were systematic differences between medical practitioners' and patients' estimates of periods of incapacity. Previous estimates of the direct economic costs and indirect human costs of infection based on data obtained from medical practitioners, although alarming, are almost certainly underestimates.

Human sentinels for arbovirus surveillance and regional risk classification in South Australia.

OBJECTIVES: To assess the use of human sentinels to monitor arbovirus activity in South Australia and to use age-specific seroprevalence data from the same sentinels to classify regions according to risk from Ross River virus (RRV). METHODS: Between 1 January 1992 and 15 August 1992, 4776 serum samples were obtained from Red Cross blood donors in the State. All sera were tested for the presence of total antibody (IgA, IgG and IgM) by indirect enzyme immunoassay. To test for recent infection, positive sera were further tested for IgA and IgM specific antibody to Ross River virus with a view to initiating public health interventions if necessary. The age, sex and postcode of residence of each donor were also recorded for seroepidemiological studies. RESULTS: Of the 4776 sera, 2952 were tested to the end of May (the end of the arbovirus season in South Australia). There was evidence of RRV infection in 248 sera (8.4%) but none had serological markers consistent with recent infection. The arbovirus season was considered non-epidemic, and no reactive public health interventions were introduced. Analysis of age-specific seroprevalence by post-code in the full bank of 4776 sera indicated that the Riverland is endemic for RRV, the Murray Mallee and Upper South East epidemic for RRV, and the remaining regions of the State at variable risk, depending on their proximity to these regions. CONCLUSIONS: The ongoing use of human sentinels is a new public health surveillance system for this disease. In the first year of operation of this surveillance system, a suboptimal number of sera was collected from some areas, and sera were tested only for RRV. However, the number of sera tested could easily be increased, as could the range of arboviruses covered by the testing.