No abstract

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.

Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.

Abstract. This paper tests the hypothesis that Culicoides (Diptera: Ceratopogonidae) species can be propagated by wind over long distances. Movement patterns of midges were inferred indirectly from patterns of the spread of bluetongue outbreaks between farms (using outbreak data from 1999-2001 for Greece, Bulgaria and Turkey) and then matched to concurrent wind patterns. The general methodology was to determine wind trajectories to and from each outbreak site based on the horizontal and vertical wind components of the European ReAnalysis-40 (ERA-40) dataset from the European centre for medium-range weather forecast (ECMWF). Forward trajectories (downwind or where the windvectors pointed to) and backward trajectories (upwind or where the wind-vectors originated from) were calculated for each outbreak for the period from one week before to one week after it had been recorded. These wind trajectories were then compared with the general outbreak patterns taking into consideration the different serotypes involved. It was found that the wind trajectories could be matched to the temporal distribution of the outbreak cases. Furthermore, the spread of the infected vector via the calculated wind trajectories was corroborated by molecular evidence. The conclusion is that the methodology presented is appropriate for quantifying the risk of spread of infected Culicoides midges by wind and that this approach could form an important component of a regional early-warning system for bluetongue.

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.

Summary 1.The spread of vector-borne diseases into new areas, commonly attributed to environmental change or increased trade and travel, could be exacerbated if novel vector species in newly invaded areas spread infection beyond the range of traditional vectors. 2. By analysing the differential degree of overlap between the environmental envelopes for bluetongue, a devastating livestock disease, and its traditional (Afro-Asian) and potential new (Palearctic) midge vectors, we have implicated the latter in the recent dramatic northward spread of this disease into Europe. 3. The traditional vector of bluetongue virus, the Afro-Asian midge Culicoides imicola , was found to occur in warm (annual mean 12-20 ° C), thermally stable locations that were dry in summer (< 400 mm precipitation). The Palearctic C. obsoletus and C. pulicaris complexes were both found to occur in cooler (down to 7 ° C annual mean), thermally more variable and wetter (up to 700 mm summer precipitation) locations. 4. Of 501 recorded outbreaks from the 1998-2004 bluetongue epidemic in southern Europe, 40% fall outside the climate envelope of C. imicola , but within the species' envelopes of the C. obsoletus and C. pulicaris complexes. 5. The distribution in multivariate environmental space of bluetongue virus is closer to that of the Palaearctic vectors than it is to that of C. imicola . This suggests that Palearctic vectors now play a substantial role in transmission and have facilitated the spread of bluetongue into cooler, wetter regions of Europe. 6. Synthesis and applications . The risk to Northern Europe now depends on how much of the distributions of the widespread, abundant Palearctic midge vectors (the C. obsoletus and C. pulicaris complexes) bluetongue can occupy, perhaps determined by thermal constraints on viral replication. This was highlighted by the sudden appearance in summer 2006 of bluetongue virus at latitudes of more than 50 ° North -approximately 6 ° further North than previous outbreaks in southern Europe. Future surveillance for bluetongue and for related Culicoides -borne pathogens should include studies to record and explain the distributional patterns of all potential Palearctic vector species.

Phenotypes are an important subject of biomedical research for which many repositories have already been created. Most of these databases are either dedicated to a single species or to a single disease of interest. With the advent of technologies to generate phenotypes in a high-throughput manner, not only is the volume of phenotype data growing fast but also the need to organize these data in more useful ways. We have created PhenomicDB (freely available at ), a multi-species genotype/phenotype database, which shows phenotypes associated with their corresponding genes and grouped by gene orthologies across a variety of species. We have enhanced PhenomicDB recently by additionally incorporating quantitative and descriptive RNA interference (RNAi) screening data, by enabling the usage of phenotype ontology terms and by providing information on assays and cell lines. We envision that integration of classical phenotypes with high-throughput data will bring new momentum and insights to our understanding. Modern analysis tools under development may help exploiting this wealth of information to transform it into knowledge and, eventually, into novel therapeutic approaches.

Detection of this highly pathogenic clade in Europe poses a health threat to poultry and humans