Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background

Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR).

Findings

Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.

Conclusions/Significance

This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

A Modeling Framework to Describe the Transmission of Bluetongue Virus within and between Farms in Great Britain

Background

Recently much attention has been given to developing national-scale micro-simulation models for livestock diseases that can be used to predict spread and assess the impact of control measures. The focus of these models has been on directly transmitted infections with little attention given to vector-borne diseases such as bluetongue, a viral disease of ruminants transmitted by Culicoides biting midges. Yet BT has emerged over the past decade as one of the most important diseases of livestock.

Methodology/Principal Findings

We developed a stochastic, spatially-explicit, farm-level model to describe the spread of bluetongue virus (BTV) within and between farms. Transmission between farms was modeled by a generic kernel, which includes both animal and vector movements. Once a farm acquired infection, the within-farm dynamics were simulated based on the number of cattle and sheep kept on the farm and on local temperatures. Parameter estimates were derived from the published literature and using data from the outbreak of bluetongue in northern Europe in 2006. The model was validated using data on the spread of BTV in Great Britain during 2007. The sensitivity of model predictions to the shape of the transmission kernel was assessed.

Conclusions/Significance

The model is able to replicate the dynamics of BTV in Great Britain. Although uncertainty remains over the precise shape of the transmission kernel and certain aspects of the vector, the modeling approach we develop constitutes an ideal framework in which to incorporate these aspects as more and better data become available. Moreover, the model provides a tool with which to examine scenarios for the spread and control of BTV in Great Britain.     

A Spatial Simulation Model for the Dispersal of the Bluetongue Vector Culicoides brevitarsis in Australia

Background

The spread of Bluetongue virus (BTV) among ruminants is caused by movement of infected host animals or by movement of infected Culicoides midges, the vector of BTV. Biologically plausible models of Culicoides dispersal are necessary for predicting the spread of BTV and are important for planning control and eradication strategies.

Methods

A spatially-explicit simulation model which captures the two underlying population mechanisms, population dynamics and movement, was developed using extensive data from a trapping program for C. brevitarsis on the east coast of Australia. A realistic midge flight sub-model was developed and the annual incursion and population establishment of C. brevitarsis was simulated. Data from the literature was used to parameterise the model.

Results

The model was shown to reproduce the spread of C. brevitarsis southwards along the east Australian coastline in spring, from an endemic population to the north. Such incursions were shown to be reliant on wind-dispersal; Culicoides midge active flight on its own was not capable of achieving known rates of southern spread, nor was re-emergence of southern populations due to overwintering larvae. Data from midge trapping programmes were used to qualitatively validate the resulting simulation model.

Conclusions

The model described in this paper is intended to form the vector component of an extended model that will also include BTV transmission. A model of midge movement and population dynamics has been developed in sufficient detail such that the extended model may be used to evaluate the timing and extent of BTV outbreaks. This extended model could then be used as a platform for addressing the effectiveness of spatially targeted vaccination strategies or animal movement bans as BTV spread mitigation measures, or the impact of climate change on the risk and extent of outbreaks. These questions involving incursive Culicoides spread cannot be simply addressed with non-spatial models.     

High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France

Background

Since 2005, cases of chikungunya (CHIK) were caused by an unusual vector, Aedes albopictus. This mosquito, present in Europe since 1979, has gained importance since its involvement in the first CHIK outbreak in Italy in 2007. The species is capable of transmitting experimentally 26 arboviruses. However, the vectorial status of its temperate populations has remained little investigated. In 2010, autochthonous cases of CHIK and dengue (DEN) were reported in southeastern France. We evaluated the potential of a French population of Ae. albopictus in the transmission of both viruses.

Methodology and Principal Findings

We used two strains of each virus, CHIK and DEN: one strain was isolated from an imported case, and one from an autochthonous case. We used as controls Aedes aegypti from India and Martinique, the source of the imported cases of CHIK and DEN, respectively. We showed that Ae. albopictus from Cagnes-sur-Mer (AL-CSM) was as efficient as the typical tropical vector Ae. aegypti from India to experimentally transmit both CHIK strains isolated from patients in Fréjus, with around 35–67% of mosquitoes delivering up to 14 viral particles at day 3 post-infection (pi). The unexpected finding came from the high efficiency of AL-CSM to transmit both strains of DENV-1 isolated from patients in Nice. Almost 67% of Ae. albopictus AL-CSM which have ensured viral dissemination were able to transmit at day 9 pi when less than 21% of the typical DEN vector Ae. aegypti from Martinique could achieve transmission.

Conclusions/Significance

Temperate Ae. albopictus behaves differently compared to its counterpart from tropical regions, where recurrent epidemic outbreaks occur. Its potential responsibility for outbreaks in Europe should not be minimized.     

The Most Likely Time and Place of Introduction of BTV8 into Belgian Ruminants

Background

In northern Europe, bluetongue (BT) caused by the BT virus (BTV), serotype 8, was first notified in August 2006 and numerous ruminant herds were affected in 2007 and 2008. However, the origin and the time and place of the original introduction have not yet been determined.

Methods and Principal Findings

Four retrospective epidemiological surveys have been performed to enable determination of the initial spatiotemporal occurrence of this emerging disease in southern Belgium: investigations of the first recorded outbreaks near to the disease epicenter; a large anonymous, random postal survey of cattle herds and sheep flocks; a random historical milk tank survey of samples tested with an indirect ELISA and a follow-up survey of non-specific health indicators. The original introduction of BTV into the region probably occurred during spring 2006 near to the National Park of Hautes Fagnes and Eifel when Culicoides become active.

Conclusions/Significance

The determination of the most likely time and place of introduction of BTV8 into a country is of paramount importance to enhance awareness and understanding and, to improve modeling of vector-borne emerging infectious diseases.     

piggybac- and PhiC31-Mediated Genetic Transformation of the Asian Tiger Mosquito,Aedes albopictus (Skuse)

Background

The Asian tiger mosquito, Aedes albopictus (Skuse), is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately.

Methodology/Principal Findings

Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2–3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2–6%.

Conclusions/Significance

Both piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.     

A Wolbachia wMel Transinfection in Aedes albopictus Is Not Detrimental to Host Fitness and Inhibits Chikungunya Virus

Background

Wolbachia inherited intracellular bacteria can manipulate the reproduction of their insect hosts through cytoplasmic incompatibility (CI), and certain strains have also been shown to inhibit the replication or dissemination of viruses. Wolbachia strains also vary in their relative fitness effects on their hosts and this is a particularly important consideration with respect to the potential of newly created transinfections for use in disease control.

Methodology/Principal Findings

In Aedes albopictus mosquitoes transinfected with the wMel strain from Drosophila melanogaster, which we previously reported to be unable to transmit dengue in lab challenges, no significant detrimental effects were observed on egg hatch rate, fecundity, adult longevity or male mating competitiveness. All these parameters influence the population dynamics of Wolbachia, and the data presented are favourable with respect to the aim of taking wMel to high population frequency. Challenge with the chikungunya (CHIKV) virus, for which Ae. albopictus is an important vector, was conducted and the presence of wMel abolished CHIKV dissemination to the saliva.

Conclusions/significance

Taken together, these data suggest that introducing wMel into natural Ae. albopictus populations using bidirectional CI could be an efficient strategy for preventing or reducing the transmission of arboviruses by this species.     

Temperature dependence of the extrinsic incubation period of orbiviruses in Culicoides biting midges

Background

The rate at which viruses replicate and disseminate in competent arthropod vectors is limited by the temperature of their environment, and this can be an important determinant of geographical and seasonal limits to their transmission by arthropods in temperate regions.

Methodology/Principal Findings

Here, we present a novel statistical methodology for estimating the relationship between temperature and the extrinsic incubation period (EIP) and apply it to both published and novel data on virus replication for three internationally important orbiviruses (African horse sickness virus (AHSV), bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV)) in their Culicoides vectors. Our analyses show that there can be differences in vector competence for different orbiviruses in the same vector species and for the same orbivirus in different vector species. Both the rate of virus replication (approximately 0.017-0.021 per degree-day) and the minimum temperature required for replication (11-13°C), however, were generally consistent for different orbiviruses and across different Culicoides vector species. The estimates obtained in the present study suggest that previous publications have underestimated the replication rate and threshold temperature because the statistical methods they used included an implicit assumption that all negative vectors were infected.

Conclusions/Significance

Robust estimates of the temperature dependence of arbovirus replication are essential for building accurate models of transmission and for informing policy decisions about seasonal relaxations to movement restrictions. The methodology developed in this study provides the required robustness and is superior to methods used previously. Importantly, the methods are generic and can readily be applied to other arbovirus-vector systems, as long as the assumptions described in the text are valid.     

Aedes (Stegomyia) albopictus (Skuse): A Potential Vector of Zika Virus in Singapore

Background

Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.

Methodology/Principal Findings

To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80–85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose₅₀ assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.

Conclusions/Significance

The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.     

Diversity and endemism in deglaciated areas: ploidy,relative genome size and niche differentiation in the Galium pusillum complex (Rubiaceae) in Northern and Central Europe

Background and Aims

Plants endemic to areas covered by ice sheets during the last glaciation represent paradigmatic examples of rapid speciation in changing environments, yet very few systems outside the harsh arctic zone have been comprehensively investigated so far. The Galium pusillum aggregate (Rubiaceae) is a challenging species complex that exhibits a marked differentiation in boreal parts of Northern Europe. As a first step towards understanding its evolutionary history in deglaciated regions, this study assesses cytological variation and ecological preferences of the northern endemics and compares the results with corresponding data for species occurring in neighbouring unglaciated parts of Central and Western Europe.

Methods

DNA flow cytometry was used together with confirmatory chromosome counts to determine ploidy levels and relative genome sizes in 1158 individuals from 181 populations. A formalized analysis of habitat preferences was applied to explore niche differentiation among species and ploidy levels.

Key Results

The G. pusillum complex evolved at diploid and tetraploid levels in Northern Europe, in contrast to the high-polyploid evolution of most other northern endemics. A high level of eco-geographic segregation was observed between different species (particularly along gradients of soil pH and competition) which is unusual for plants in deglaciated areas and most probably contributes to maintaining species integrity. Relative monoploid DNA contents of the species from previously glaciated regions were significantly lower than those of their counterparts from mostly unglaciated Central Europe, suggesting independent evolutionary histories.

Conclusions

The aggregate of G. pusillum in Northern Europe represents an exceptional case with a geographically vicariant and ecologically distinct diploid/tetraploid species endemic to formerly glaciated areas. The high level of interspecific differentiation substantially widens our perception of the evolutionary dynamics and speciation rates in the dramatically changing environments of Northern Europe.     

Female-specific flightless (fsRIDL) phenotype for control of Aedes albopictus

Background

Aedes albopictus, the Asian tiger mosquito, is a vector of several arboviruses including dengue and chikungunya, and is also a significant nuisance mosquito. It is one of the most invasive of mosquitoes with a relentlessly increasing geographic distribution. Conventional control methods have so far failed to control Ae. albopictus adequately. Novel genetics-based strategies offer a promising alternative or aid towards efficient control of this mosquito.

Methodology/Principal Findings

We describe here the isolation, characterisation and use of the Ae. albopictus Actin-4 gene to drive a dominant lethal gene in the indirect flight muscles of Ae. albopictus, thus inducing a conditional female-specific late-acting flightless phenotype. We also show that in this context, the Actin-4 regulatory regions from both Ae. albopictus and Ae. aegypti can be used to provide conditional female-specific flightlessness in either species.

Conclusion/Significance

With the disease-transmitting females incapacitated, the female flightless phenotype encompasses a genetic sexing mechanism and would be suitable for controlling Ae. albopictus using a male-only release approach as part of an integrated pest management strategy.     

A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Background

The gene delivery vector for DNA-based therapy should ensure its transfection efficiency and safety for clinical application. The Micro-Linear vector (MiLV) was developed to improve the limitations of traditional vectors such as viral vectors and plasmids.

Methods

The MiLV which contained only the gene expression cassette was amplified by polymerase chain reaction (PCR). Its cytotoxicity, transfection efficiency in vitro and in vivo, duration of expression, pro-inflammatory responses and potential application for Epstein-Barr virus (EBV) positive tumors were evaluated.

Results

Transfection efficiency for exogenous genes transferred by MiLV was at least comparable with or even greater than their corresponding plasmids in eukaryotic cell lines. MiLV elevated the expression and prolonged the duration of genes in vitro and in vivo when compared with that of the plasmid. The in vivo pro-inflammatory response of MiLV group was lower than that of the plasmid group. The MEKK1 gene transferred by MiLV significantly elevated the sensitivity of B95-8 cells and transplanted tumor to the treatment of Ganciclovir (GCV) and sodium butyrate (NaB).

Conclusions

The present study provides a safer, more efficient and stable MiLV gene delivery vector than plasmid. These advantages encourage further development and the preferential use of this novel vector type for clinical gene therapy studies.     

The Spread of Bluetongue Virus Serotype 8 in Great Britain and Its Control by Vaccination

Background

Bluetongue (BT) is a viral disease of ruminants transmitted by Culicoides biting midges and has the ability to spread rapidly over large distances. In the summer of 2006, BTV serotype 8 (BTV-8) emerged for the first time in northern Europe, resulting in over 2000 infected farms by the end of the year. The virus subsequently overwintered and has since spread across much of Europe, causing tens of thousands of livestock deaths. In August 2007, BTV-8 reached Great Britain (GB), threatening the large and valuable livestock industry. A voluntary vaccination scheme was launched in GB in May 2008 and, in contrast with elsewhere in Europe, there were no reported cases in GB during 2008.

Methodology/Principal Findings

Here, we use carefully parameterised mathematical models to investigate the spread of BTV in GB and its control by vaccination. In the absence of vaccination, the model predicted severe outbreaks of BTV, particularly for warmer temperatures. Vaccination was predicted to reduce the severity of epidemics, with the greatest reduction achieved for high levels (95%) of vaccine uptake. However, even at this level of uptake the model predicted some spread of BTV. The sensitivity of the predictions to vaccination parameters (time to full protection in cattle, vaccine efficacy), the shape of the transmission kernel and temperature dependence in the transmission of BTV between farms was assessed.

Conclusions/Significance

A combination of lower temperatures and high levels of vaccine uptake (>80%) in the previously-affected areas are likely to be the major contributing factors in the control achieved in England in 2008. However, low levels of vaccination against BTV-8 or the introduction of other serotypes could result in further, potentially severe outbreaks in future.     

Historical isolation versus recent long-distance connections between Europe and Africa in bifid toadflaxes (Linaria sect. Versicolores)

Background

Due to its complex, dynamic and well-known paleogeography, the Mediterranean region provides an ideal framework to study the colonization history of plant lineages. The genus Linaria has its diversity centre in the Mediterranean region, both in Europe and Africa. The last land connection between both continental plates occurred during the Messinian Salinity Crisis, in the late Miocene (5.96 to 5.33 Ma).

Methodology/Principal Findings

We analyzed the colonization history of Linaria sect. Versicolores (bifid toadflaxes), which includes c. 22 species distributed across the Mediterranean, including Europe and Africa. Two cpDNA regions (rpl32-trnL^UAG and trnK-matK) were sequenced from 66 samples of Linaria. We conducted phylogenetic, dating, biogeographic and phylogeographic analyses to reconstruct colonization patterns in space and time. Four major clades were found: two of them exclusively contain Iberian samples, while the other two include northern African samples together with some European samples. The bifid toadflaxes have been split in African and European clades since the late Miocene, and most lineage and speciation differentiation occurred during the Pliocene and Quaternary. We have strongly inferred four events of post-Messinian colonization following long-distance dispersal from northern Africa to the Iberian Peninsula, Sicily and Greece.

Conclusions/Significance

The current distribution of Linaria sect. Versicolores lineages is explained by both ancient isolation between African and European populations and recent events of long-distance dispersal over sea barriers. This result provides new evidence for the biogeographic complexity of the Mediterranean region.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value?=?sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.

     

Zika Virus in Gabon (Central Africa) – 2007: A New Threat from Aedes albopictus?

Background

Chikungunya and dengue viruses emerged in Gabon in 2007, with large outbreaks primarily affecting the capital Libreville and several northern towns. Both viruses subsequently spread to the south-east of the country, with new outbreaks occurring in 2010. The mosquito species Aedes albopictus, that was known as a secondary vector for both viruses, recently invaded the country and was the primary vector involved in the Gabonese outbreaks. We conducted a retrospective study of human sera and mosquitoes collected in Gabon from 2007 to 2010, in order to identify other circulating arboviruses.

Methodology/Principal Findings

Sample collections, including 4312 sera from patients presenting with painful febrile disease, and 4665 mosquitoes belonging to 9 species, split into 247 pools (including 137 pools of Aedes albopictus), were screened with molecular biology methods. Five human sera and two Aedes albopictus pools, all sampled in an urban setting during the 2007 outbreak, were positive for the flavivirus Zika (ZIKV). The ratio of Aedes albopictus pools positive for ZIKV was similar to that positive for dengue virus during the concomitant dengue outbreak suggesting similar mosquito infection rates and, presumably, underlying a human ZIKV outbreak. ZIKV sequences from the envelope and NS3 genes were amplified from a human serum sample. Phylogenetic analysis placed the Gabonese ZIKV at a basal position in the African lineage, pointing to ancestral genetic diversification and spread.

Conclusions/Significance

We provide the first direct evidence of human ZIKV infections in Gabon, and its first occurrence in the Asian tiger mosquito, Aedes albopictus. These data reveal an unusual natural life cycle for this virus, occurring in an urban environment, and potentially representing a new emerging threat due to this novel association with a highly invasive vector whose geographic range is still expanding across the globe.     

Investigating the effects of topography and clonality on genetic structuring within a large Norwegian population of Arabidopsis lyrata

Background and Aims

The gene flow through pollen or seeds governs the extent of spatial genetic structure in plant populations. Another factor that can contribute to this pattern is clonal growth. The perennial species Arabidopsis lyrata ssp. petraea (Brassicaceae) is a self-incompatible, clonal species found in disjunctive populations in central and northern Europe.

Methods

Fourteen microsatellite markers were employed to study the level of kinship and clonality in a high-altitude mountain valley at Spiterstulen, Norway. The population has a continuous distribution along the banks of the River Visa for about 1·5 km. A total of 17 (10 m × 10 m) squares were laid out in a north–south transect following the river on both sides.

Key Results

It is shown that clonal growth is far more common than previously shown in this species, although the overall size of the genets is small (mean diameter = 6·4 cm). Across the whole population there is no indication of isolation by distance, and spatial genetic structure is only visible on fine spatial scales. In addition, no effect of the river on the spatial distribution of genotypes was found.

Conclusions

Unexpectedly, the data show that populations of small perennials like A. lyrata can behave like panmictic units across relatively large areas at local sites, as opposed to earlier findings in central Europe.     

International Dispersal of Dengue through Air Travel: Importation Risk for Europe

Background

The worldwide distribution of dengue is expanding, in part due to globalized traffic and trade. Aedes albopictus is a competent vector for dengue viruses (DENV) and is now established in numerous regions of Europe. Viremic travellers arriving in Europe from dengue-affected areas of the world can become catalysts of local outbreaks in Europe. Local dengue transmission in Europe is extremely rare, and the last outbreak occurred in 1927–28 in Greece. However, autochthonous transmission was reported from France in September 2010, and from Croatia between August and October 2010.

Methodology

We compiled data on areas affected by dengue in 2010 from web resources and surveillance reports, and collected national dengue importation data. We developed a hierarchical regression model to quantify the relationship between the number of reported dengue cases imported into Europe and the volume of airline travellers arriving from dengue-affected areas internationally.

Principal Findings

In 2010, over 5.8 million airline travellers entered Europe from dengue-affected areas worldwide, of which 703,396 arrived at 36 airports situated in areas where Ae. albopictus has been recorded. The adjusted incidence rate ratio for imported dengue into European countries was 1.09 (95% CI: 1.01–1.17) for every increase of 10,000 travellers; in August, September, and October the rate ratios were 1.70 (95%CI: 1.23–2.35), 1.46 (95%CI: 1.02–2.10), and 1.35 (95%CI: 1.01–1.81), respectively. Two Italian cities where the vector is present received over 50% of all travellers from dengue-affected areas, yet with the continuing vector expansion more cities will be implicated in the future. In fact, 38% more travellers arrived in 2013 into those parts of Europe where Ae. albopictus has recently been introduced, compared to 2010.

Conclusions

The highest risk of dengue importation in 2010 was restricted to three months and can be ranked according to arriving traveller volume from dengue-affected areas into cities where the vector is present. The presence of the vector is a necessary, but not sufficient, prerequisite for DENV onward transmission, which depends on a number of additional factors. However, our empirical model can provide spatio-temporal elements to public health interventions.