Surveillance of mosquito vectors in Southern Sweden for Flaviviruses and Sindbis virus.

There are three human pathogenic bird-viruses transmitted by Culex mosquitoes in Europe: the alphavirus Sindbis and the flaviviruses West Nile virus and Usutu virus. Cases of Sindbis fever occur in the north while the flaviviruses are reported from southern Europe. In this study, 7933 Culex pipiens/torrentium mosquitoes from southern Sweden were screened by RTqPCR for these viruses. None of the mosquitoes were positive for viral RNA. The importance of mosquito species composition is discussed as a potential explanation to the lack of detection of mosquito-borne viruses in southern Sweden. However, continued surveillance of mosquitoes for Flaviviruses would be valuable as an early warning for public health awareness.

Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors.

Polyarthritis and rash caused by Sindbis virus (SINV), was first recognised in northern Europe about 50 years ago and is known as Ockelbo disease in Sweden and Pogosta disease in Finland. This mosquito-borne virus occurs mainly in tropical and sub-tropical countries, and in northern Europe it is suggested to cause regularly reoccurring outbreaks. Here a seven-year cycle of SINV outbreaks has been referred to in scientific papers, although the hypothesis is based solely on reported human cases. In the search for a more objective outbreak signal, we evaluated mosquito abundance and SINV prevalence in vector mosquitoes from an endemic area in central Sweden. Vector mosquitoes collected in the River Dalälven floodplains during the years before, during, and after the hypothesised 2002 outbreak year were assayed for virus on cell culture. Obtained isolates were partially sequenced, and the nucleotide sequences analysed using Bayesian maximum clade credibility and median joining network analysis. Only one SINV strain was recovered in 2001, and 4 strains in 2003, while 15 strains were recovered in 2002 with significantly increased infection rates in both the enzootic and the bridge-vectors. In 2002, the Maximum Likelihood Estimated infection rates were 10.0/1000 in the enzootic vectors Culex torrentium/pipiens, and 0.62/1000 in the bridge-vector Aedes cinereus, compared to 4.9/1000 and 0.0/1000 in 2001 and 0.0/1000 and 0.32/1000 in 2003 Sequence analysis showed that all isolates belonged to the SINV genotype I (SINV-I). The genetic analysis revealed local maintenance of four SINV-I clades in the River Dalälven floodplains over the years. Our findings suggest that increased SINV-I prevalence in vector mosquitoes constitutes the most valuable outbreak marker for further scrutinising the hypothesized seven-year cycle of SINV-I outbreaks and the mechanisms behind.

Introduction and Dispersal of Sindbis Virus from Central Africa to Europe.

Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe, as well as in Africa and the Middle East, collected over 58 years. These were analyzed together with 30 additional published whole SINV-I genomes using Bayesian analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southward on two separate occasions in the 1960s and 1970s. Another introduction from central Africa to southern/central Europe seems to have occurred, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where the most divergent SINV-I strains also originated.IMPORTANCE This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular.

Pest occurrence of Aedes rossicus close to the Arctic Circle in northern Sweden.

Major nuisance species are found among the floodwater mosquitoes and snow-pool mosquitoes, with the former being the main reason for mosquito control in most areas. Nuisance species vary with the area, and previous reports from northern areas conclude that the nuisance is most often caused by snow-pool mosquitoes. We investigated the mosquito fauna and abundances of host-seeking females using CDC traps baited with carbon dioxide, in Övertorneå city near the Arctic Circle in northern Sweden, after earlier complaints about massive mosquito nuisance. The abundance of host-seeking female mosquitoes was high in 2014, with a maximum of ∼15,400 individuals per CDC trap night, of which 89% was the floodwater mosquito Aedes rossicus. Surprisingly, the main nuisance species was a floodwater mosquito, occurring at the northernmost location it has ever been recorded in Sweden. Our report is probably the first documentation of such large numbers of Aedes rossicus in any locality and probably the first documentation of a severe floodwater mosquito nuisance near the Arctic Circle. Given the historical data on river discharge in the area, the nuisance is recurrent. We conclude that in northern localities, as well as in more southern localities, production of floodwater mosquitoes is a natural component of the floodplain fauna of rivers with a fluctuating water flow regime. Also, the floodwater mosquitoes Aedes sticticus and Aedes vexans were found north of their formerly known distribution in Sweden.

Do Multi-year Applications of Bacillus thuringiensis subsp. israelensis for Control of Mosquito Larvae Affect the Abundance of B. cereus Group Populations in Riparian Wetland Soils?

Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the Bacillus cereus group (Bcg) and has been used in biocontrol products against nematoceran larvae for several decades. However, knowledge is limited on whether long-term Bti application can affect the structure of indigenous communities of Bcg and the overall abundance of Bti. Using species- and group-specific quantitative PCR assays, we measured the Bcg- and Bti-abundances in riparian wetlands in the River Dalälven floodplains of central Sweden. On five occasions during one vegetative season, soil samples were collected in alder swamps and wet meadows which had been treated with Bti for mosquito larvae control during the preceding 11 years, as well as in untreated control sites and well-drained forests in the same area. The average abundance of Bcg in alder swamps was around three times higher than in wet meadows. Across all sites and habitats, the Bti treatments had no effect on the Bcg-abundance, whereas the Bti-abundance was significantly higher in the treated than in the control sites. However, for individual sampling sites, abundances of Bti and Bcg were not correlated with the number of Bti applications, indicating that added Bti possibly influenced the total population of Bti in the short term but had only a limited effect in the longer term. The findings of this study increase the understanding of the ecology of Bti applications for mosquito control, which can facilitate environmental risk assessment in connection with approval of microbiological control agents.

First report on human-biting Culex pipiens in Sweden.

Culex mosquitoes are vectors of several bird-hosted arboviruses that cause outbreaks in Europe, such as Sindbis virus and West Nile virus. Recently, the human-biting form of Culex pipiens, Cx. pipiens biotype molestus, was found causing big nuisance in a housing cooperative in Gothenburg in southern Sweden, confirmed by molecular identification. This is the first report of human-biting Culex in Scandinavia, signalling increased risk of arbovirus infection in northern Europe.

Temporal Variation in Sindbis Virus Antibody Prevalence in Bird Hosts in an Endemic Area in Sweden.

Sindbis virus (SINV) is a mosquito-borne bird virus that occasionally causes human disease in Fennoscandia, suggested to have cyclic 7-year intervals between outbreaks. Reliable data on human infections in Sweden is however lacking. Here we investigated the SINV antibody prevalence among birds in a Swedish area endemic to SINV to scrutinize if a cyclic variation in antibody prevalence is present in the natural host of SINV. Serum from birds were sampled in the summers of 2002-2004 and 2009 in the floodplains of the River Dalälven in central Sweden, with 2002 and 2009 representing hypothesized years of SINV outbreaks. A total of 963 birds from 52 species (mainly passerines) were tested for the presence of SINV antibodies using a plaque reduction neutralization test. The highest SINV antibody prevalence was found in Turdidae species, specifically Fieldfare, Redwing and Song thrush in which more than 70% of sampled individuals had antibodies to SINV in 2009. The SINV antibody prevalence significantly varied between years with 2% in 2002, 8% in 2003, 14% in 2004 and 37% in 2009. Antibodies were found equally often in hatchlings and in adults and increased from early to late in the season. Clearly, the SINV antibody prevalence was not elevated in the bird hosts in the predicted outbreak year 2002, thus solid evidence of a cyclic occurrence of SINV in Sweden is still lacking.

Genetic and phenotypic variation in central and northern European populations of Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera, Culicidae).

The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape genetics approach (FST , STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations, while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation-by-Distance patterns. Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low-to-moderate FST values on a broader geographical scale indicate a continuous between-population exchange of individuals, including considerable gene flow on the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed data considering population structure in the light of vector control strategies of the mosquito from public health importance.

Trapping biases of Culex torrentium and Culex pipiens revealed by comparison of captures in CDC traps, ovitraps, and gravid traps.

We evaluate three trapping methods for their effectiveness at capturing Culex pipiens and Culex torrentium, both enzootic vectors of bird-associated viruses in Europe. The comparisons, performed in two regions in Sweden, were among CDC traps baited with carbon dioxide, gravid traps, and ovitraps baited with hay infusion. The proportions of the two Culex species in a catch differed between trap types, with CDC traps catching a lower proportion of Cx. torrentium than both gravid traps and ovitraps. Between gravid traps and ovitraps, there was no difference in the proportions of the two species. The results indicate that Cx. torrentium may go undetected or underestimated compared to Cx. pipiens when using carbon dioxide baited CDC traps. The new insight of trap bias presented here adds an important dimension to consider when investigating these vectors of bird-associated viruses in the field.

Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae.

Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.

Culex torrentium Mosquito Role as Major Enzootic Vector Defined by Rate of Sindbis Virus Infection, Sweden, 2009.

We isolated Sindbis virus (SINV) from the enzootic mosquito vectors Culex torrentium, Cx. pipiens, and Culiseta morsitans collected in an area of Sweden where SINV disease is endemic. The infection rate in Cx. torrentium mosquitoes was exceptionally high (36 infections/1,000 mosquitoes), defining Cx. torrentium as the main enzootic vector of SINV in Scandinavia.

Specific detection of the floodwater mosquitoes Aedes sticticus and Aedes vexans DNA in predatory diving beetles.

Floodwater mosquitoes (Diptera: Culicidae) are associated with periodically flooded wet meadows, marshes, and swamps in floodplains of major rivers worldwide, and their larvae are abundant in the shallow parts of flooded areas. The nuisance caused by the blood-seeking adult female mosquitoes motivates mosquito control. Larviciding with Bacillus thuringiensis israelensis is considered the most environmentally safe method. However, some concern has been raised whether aquatic predatory insects could be indirectly affected by this reduction in a potential vital prey. Top predators in the temporary wetlands in the River Dalälven floodplains are diving beetles (Coleoptera: Dytiscidae), and Aedes sticticus and Ae. vexans are the target species for mosquito control. For detailed studies on this aquatic predator-prey system, we developed a polymerase chain reaction (PCR) assay for detection of mosquito DNA in the guts of medium-sized diving beetles. Primers were designed for amplifying short mitochondrial DNA fragments of the cytochrome C oxidase subunit I (COI) gene in Ae. sticticus and Ae. vexans, respectively. Primer specificity was confirmed and half-life detectability of Ae. sticticus DNA in diving beetle guts was derived from a feeding and digestion experiment. The Ae. sticticus DNA within diving beetle guts was detected up to 12 h postfeeding, and half-life detectability was estimated to 5.6 h. In addition, field caught diving beetles were screened for Ae. sticticus and Ae. vexans DNA and in 14% of the diving beetles one or both mosquito species were detected, showing that these mosquito species are utilized as food by the diving beetles.

Investigations on California serogroup orthobunyaviruses in the Tyrols: first description of Tahyna virus in the Alps.

Seroprevalence rates for immunoglobulin G (IgG) antibodies to Tahyna virus (TAHV) and Inkoo virus (INKV) were determined in sera of 1630 blood donors from North, East, and South Tyrol by immunofluorescence assays (IFAs) and confirmatory serum neutralization tests (SNTs). Ten sera (0.6%) reacted positive by TAHV IFA, five of which (0.3%) were confirmed by SNT. Eleven sera (0.7%) reacted positive in the INKV IFA; only one thereof (0.06%) was verified by subsequent SNT. To identify the source of infections, mosquitoes were trapped at 18 sampling sites in the study area, resulting in the collection of 2571 adult mosquitoes: 1254 individuals of the genus Aedes (48.8% of total) including A. albopictus, 640 Culex (24.9%), 303 Coquillettidia (11.8%), 252 Ochlerotatus (9.8%), 49 Anopheles (1.9%), and 73 mosquitoes of the genus Culiseta (2.8%). The mosquitoes were pooled according to species, trapping site, and time, and were tested by RT-PCR for the presence of California serogroup orthobunyavirus nucleic acids. PCR amplification products were obtained in five of 195 pools (2.6%), and all were identified as TAHVs by subsequent sequencing. This represents the first evidence of TAHV circulation and human exposure in the Tyrols and in the alpine region in general. Interestingly, all TAHV sequences were identified in Culex pipiens/torrentium mosquitoes. Whether other California serogroup orthobunyaviruses such as INKV are also circulating in this area is subject of further investigations on larger numbers of mosquitoes.

Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis.

Mosquito-borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito-borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR-based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR-based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.

Outbreaks of tularemia in a boreal forest region depends on mosquito prevalence.

BACKGROUND: We aimed to evaluate the potential association of mosquito prevalence in a boreal forest area with transmission of the bacterial disease tularemia to humans, and model the annual variation of disease using local weather data. METHODS: A prediction model for mosquito abundance was built using weather and mosquito catch data. Then a negative binomial regression model based on the predicted mosquito abundance and local weather data was built to predict annual numbers of humans contracting tularemia in Dalarna County, Sweden. RESULTS: Three hundred seventy humans were diagnosed with tularemia between 1981 and 2007, 94% of them during 7 summer outbreaks. Disease transmission was concentrated along rivers in the area. The predicted mosquito abundance was correlated (0.41, P < .05) with the annual number of human cases. The predicted mosquito peaks consistently preceded the median onset time of human tularemia (temporal correlation, 0.76; P < .05). Our final predictive model included 5 environmental variables and identified 6 of the 7 outbreaks. CONCLUSIONS: This work suggests that a high prevalence of mosquitoes in late summer is a prerequisite for outbreaks of tularemia in a tularemia-endemic boreal forest area of Sweden and that environmental variables can be used as risk indicators.

Geographic distribution and relative abundance of the sibling vector species Culex torrentium and Culex pipiens in Sweden.

Culex torrentium and Culex pipiens are sibling species and potential viral vectors that coexist in Europe. Larvae and females of the two species are morphologically almost identical, and reliable identification can only be done on males. To investigate the distribution and relative abundance of the two species in Sweden, we collected Culex larvae from sites spread over the country, identified them as Culex pipiens/torrentium based on morphology, and identified them to species using a recently developed restriction enzyme method. Cx. torrentium was the dominant species (89%, n=1012) and it occurred in 48 of the 49 sites investigated, and also dominated in most of the study sites. The proportion of Cx. pipiens larvae in relation to Cx. torrentium collected at each site decreased with both increasing latitude and altitude, and the presence of Cx. pipiens decreased with latitude. In addition, Cx. pipiens/torrentium females were sampled with Centres for Disease Control light traps baited with carbon dioxide. The overall country mean was 4.0 Cx. pipiens/torrentium caught per trap night, with decreasing numbers of Cx. pipiens/torrentium caught per trap night with increasing latitude. Thus, the abundance of Cx. pipiens/torrentium decreased, but the proportion Cx. torrentium increased, with increasing latitude. This is the first study that shows the vast dominance of Cx. torrentium over Cx. pipiens in Sweden. The unexpected dominance of Cx. torrentium highlights the importance of distinguishing between the two species in studies of Culex-borne arboviruses in Europe.

Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden.

In Sweden, human cases of tularemia caused by Francisella tularensis holarctica are assumed to be transmitted by mosquitoes, but how mosquito vectors acquire and transmit the bacterium is not clear. To determine how transmission of this bacterium occurs, mosquito larvae were collected in an area where tularemia is endemic, brought to the laboratory, and reared to adults in their original pond water. Screening of adult mosquitoes by real-time PCR demonstrated F. tularensis lpnA sequences in 14 of the 48 mosquito pools tested; lpnA sequences were demonstrated in 6 of 9 identified mosquito species. Further analysis confirmed the presence of F. tularensis holarctica-specific 30-bp deletion region sequences (FtM19inDel) in water from breeding containers and in 3 mosquito species (Aedes sticticus, Ae. vexans, and Ae. punctor) known to take blood from humans. Our results suggest that the mosquitoes that transmit F. tularensis holarctica during tularemia outbreaks acquire the bacterium already as larvae.

Optimization of alpha-ketoamide based p38 inhibitors through modifications to the region that binds to the allosteric site.

We have optimized a novel series of potent p38 MAP kinase inhibitors based on an alpha-ketoamide scaffold through structure based design that due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME, in vivo PK and efficacy studies show these compounds to have drug-like characteristics and have resulted in the nomination of a development candidate which is currently in phase II clinical trials for the oral treatment of inflammatory conditions.