Search

Abstract Background Clinical cases of leishmaniasis caused by Leishmania (Mundinia) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania (Mundinia) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. Methods We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021–2023. Midge samples were molecularly screened for Leishmania using SSU rRNA-qPCR and ITS1-PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb-PCRs and a nanopore-based metabarcoding approach. Results A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera (Remmia, Trithecoides, Avaritia, Hoffmania, and Meijerehelea) and two species groups (Shortti and Calvipalpis) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA-qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma, C. guttifer, C. orientalis, C. mahasarakhamense, C (Trithecoides) spp., C. innoxius, C. shortti, C. arakawae, C. sumatrae, C. actoni, and C. fulvus, with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1-PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. Conclusions This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides. Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission. Graphical Abstract

Abstract Background Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females. Methods We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search. Results In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns. Conclusions In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors. Graphical Abstract

Abstract Background Mosquitoes are important vectors of pathogens. They are usually collected with CO2-baited traps and subsequently identified by morphology. This procedure is very time-consuming. Automatic counting traps could facilitate timely evaluation of the local risk for mosquito-borne pathogen transmission or decision-making on vector control measures, but the counting accuracy of such devices has rarely been validated in the field. Methods The Biogents (BG)-Counter 2 automatically counts mosquitoes by discriminating the size of captured objects directly in the field and transmits the data to a cloud server. To assess the accuracy of this counting device, 27 traps were placed at 19 sampling sites across Germany and used in daily, weekly or bimonthly intervals from April until October 2021. The BG-Counter 2 was attached to a CO2-trap (BG-Pro trap = CO2-Pro) and the same trap was converted to also attract gravid mosquitoes (upside-down BG-Pro trap with a water container beneath = CO2-Pro-gravid). All captured mosquitoes were identified by morphology. The number of females (unfed and gravid), mosquito diversity and the number of identified specimens in relation to the counting data of the BG-Counter were compared between the two trapping devices to evaluate sampling success and counting accuracy. Results In total 26,714 mosquitoes were collected during 854 trap days. The CO2-Pro-gravid trap captured significantly more mosquitoes per trap day for all specimens, gravid females and non-gravid females, while there was no difference in the mosquito diversity. The linear model with the captured mosquitoes as a response and the counted specimens as a predictor explained only a small degree of the variation within the data (R 2 = 0.16), but per individual trap the value could reach up to 0.62 (mean R 2 = 0.23). The counting accuracy for the daily samples had a significant positive correlation with sample size, resulting in higher accuracy for the CO2-Pro-gravid trap and higher accuracy for sites and sampling months with high mosquito abundance. Conclusions While the accuracy of the BG-Counter 2 is quite low, the device is able to depict mosquito phenology and provide information about local population dynamics. Graphical Abstract

Abstract Background Batai virus (BATV) is a zoonotic arbovirus of veterinary importance. A high seroprevalence in cows, sheep and goats and infection in different mosquito species has been observed in Central Europe. Therefore, we studied indigenous as well as exotic species of the genera Culex and Aedes for BATV vector competence at different fluctuating temperature profiles. Methods Field caught Culex pipiens biotype pipiens, Culex torrentium, Aedes albopictus and Aedes japonicus japonicus from Germany and Aedes aegypti laboratory colony were infected with BATV strain 53.3 using artificial blood meals. Engorged mosquitoes were kept under four (Culex species) or three (Aedes species) fluctuating temperature profiles (18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C) at a humidity of 70% and a dark/light rhythm of 12:12 for 14 days. Transmission was measured by testing the saliva obtained by forced salivation assay for viable BATV particles. Infection rates were analysed by testing whole mosquitoes for BATV RNA by quantitative reverse transcription PCR. Results No transmission was detected for Ae. aegypti, Ae. albopictus or Ae. japonicus japonicus. Infection was observed for Cx. p. pipiens, but only in the three conditions with the highest temperatures (21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C). In Cx. torrentium infection was measured at all tested temperatures with higher infection rates compared with Cx. p. pipiens. Transmission was only detected for Cx. torrentium exclusively at the highest temperature of 27 ± 5 °C. Conclusions Within the tested mosquito species, only Cx. torrentium seems to be able to transmit BATV if the climatic conditions are feasible. Graphical Abstract

Abstract Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019–2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray–Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

The Usutu Virus (USUV) is a mosquito-borne flavivirus originated in Africa. The virus circulates in Germany since 2010. It is primarily transmitted and maintained in the natural cycle by Culex mosquitoes and primarily affects birds, particularly Eurasian blackbird (Turdus merula), leading to significant mortality. Several studies have reported a high co-infection rate of European birds with both USUV and haemosporidians. Haemosporidians are blood parasites which maintain an enzootic life cycle with birds via different arthropod vectors. This study conducted screenings of birds from Germany received through a citizen's science project for both, USUV and haemosporidians between 2016 and 2021. The prevalence of USUV reached its peak in 2018, when it was first detected throughout most parts of Germany rather than being limited to localised hotspots. Subsequently, USUV prevalence consistently declined. On the other hand, the prevalence of haemosporidians initially declined between 2016 and 2019, but experienced a subsequent increase in the following years, exhibiting a more or less inverse pattern compared to the prevalence of USUV. In 2020, a statistically significant positive association between both pathogens was found, which was also detected across all years combined, indicating if at all a weak relationship between these pathogens.

ABSTRACTTwo Wolbachia strains, wMel and wAlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains wMel and wAlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the wMel strain exhibited several significant differences compared with those infected with the wAlbB strain. Specifically, wMel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia-based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia-infected Aedes aegypti: wMel and wAlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that wMel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on mosquito fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations.

Abstract Background Mosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported. Methods A mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed. Results A strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status. Conclusions Taken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses. Graphical Abstract

Abstract The spatial distribution of mosquito species in the course of globalization and climate warming is highly dynamic. Different studies have demonstrated the spread and establishment of thermophilic mosquito species, potentially increasing the prevalence of ‘nuisance’ mosquitoes and the local transmission of pathogens. Here we report the first recorded sampling of Anopheles hyrcanus in Wrocław, southwest Poland. This is the most northern detection of this species to date in Europe. Future spread and population development of this potential vector of malaria parasites, viruses or zoonotic helminths, such as Dirofilaria spp., must be monitored carefully. Potential factors underlying the spread of this species are discussed. Graphical Abstract

Abstract Background The impact of the arbovirus vector Aedes aegypti is of major concern for global public health as the viruses that it transmits affect millions of people each year worldwide. Originating in Africa, Ae. aegypti has now spread throughout much of the world. While the genetic makeup of Ae. aegypti in the New World has been extensively studied, there is limited knowledge on its genetic diversity in Africa, particularly at a microgeographical level. Methods We investigated mitochondrial cytochrome oxidase I of four Ae. aegypti populations from Benin and employed wing morphometric analyses as a cost-effective and reliable tool to explore population structure. Our sampling encompassed various areas of Benin, from the southern to the northern borders of the country, and included urban, semi-urban, and sylvatic sites. Results We observed a notable level of genetic diversity (haplotype diversity of 0.8333) and nucleotide diversity (0.00421986), and identified seven distinct haplotypes. Sylvatic and semi-urban sites exhibited a greater number of haplotypes compared to urban sites. Utilizing 18 wing landmarks, we calculated the centroid size, which revealed significant variation among the three landscape types. However, principal component analysis, employed to assess wing shape variation, did not demonstrate significant differences between populations based on landscape type. Conclusions Our findings indicate substantial genetic and morphological diversity among Ae. aegypti populations in Benin, and provide insight into important biological characteristics of these populations with respect to their potential to transmit viruses. To the best of our knowledge, this is the first study undertaken in Africa to integrate genetics with morphology to analyse the population structure of the major arbovirus vector Ae. aegypti. Graphical Abstract

The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations.

Background: Endemic and imported vector- and rodent-borne infectious agents can be linked to high morbidity and mortality. Therefore, vector- and rodent-borne human diseases and the effects of climate change are important public health issues. Methods: For this review, the relevant literature was identified and evaluated according to the thematic aspects and supplemented with an analysis of surveillance data for Germany. Results: Factors such as increasing temperatures, changing precipitation patterns, and human behaviour may influence the epidemiology of vector- and rodent-borne infectious diseases in Germany. Conclusions: The effects of climatic changes on the spread of vector- and rodent-borne infectious diseases need to be further studied in detail and considered in the context of climate adaptation measures.

In 2021, three encephalitis cases due to the Borna disease virus 1 (BoDV-1) were diagnosed in the north and east of Germany. The patients were from the states of Thuringia, Saxony-Anhalt, and Lower Saxony. All were residents of known endemic areas for animal Borna disease but without prior diagnosed human cases. Except for one recently detected case in the state of Brandenburg, all >30 notified cases had occurred in, or were linked to, the southern state of Bavaria. Of the three detected cases described here, two infections were acute, while one infection was diagnosed retrospectively from archived brain autopsy tissue samples. One of the acute cases survived, but is permanently disabled. The cases were diagnosed by various techniques (serology, molecular assays, and immunohistology) following a validated testing scheme and adhering to a proposed case definition. Two cases were classified as confirmed BoDV-1 encephalitis, while one case was a probable infection with positive serology and typical brain magnetic resonance imaging, but without molecular confirmation. Of the three cases, one full virus genome sequence could be recovered. Our report highlights the need for awareness of a BoDV-1 etiology in cryptic encephalitis cases in all areas with known animal Borna disease endemicity in Europe, including virus-endemic regions in Austria, Liechtenstein, and Switzerland. BoDV-1 should be actively tested for in acute encephalitis cases with residence or rural exposure history in known Borna disease-endemic areas.

Culex pipiens s.s./Culex torrentium belong to the most widespread mosquito taxa in Europe and are the main vectors of Sindbis, West Nile and Usutu virus. The adult overwintering females can act as reservoir for these arthropod-borne viruses (arboviruses), thus contributing to their local persistence when transmission cycles are interrupted during the winter. However, the main overwintering sites of Cx. torrentium are unknown.In a study from 2017, 3455 Cx. pipiens s.s./Cx. torrentium specimens were collected from abandoned animal burrows in Poznan, Poland. These specimens were retrospectively identified to species-level with a PCR assay, which revealed Cx. torrentium as dominant species (> 60%). Motivated by these results, we conducted a field study from February to July 2022 to systematically analyse the overwintering site patterns of Cx. pipiens s.s./Cx. torrentium. Mosquitoes were sampled using pipe traps in abandoned animal burrows (n = 20) and with aspirators in nearby anthropogenic overwintering sites (n = 23). All Cx. pipiens s.s./Cx. torrentium were screened for Flaviviridae RNA.In total, 4710 mosquitoes of five different taxa were collected from anthropogenic sites. 3977 of them were identified as Cx. p. pipiens/Cx. torrentium (Cx. p. pipiens: 85%, Cx torrentium: 1%, pools with both species: 14%). In contrast, only Cx. p. pipiens/Cx. torrentium (1688 specimens) were collected from animal burrows dominated by Cx. torrentium (52%), followed by pools with both species (40%) and Cx. p pipiens (8%). A single pool of 10 Cx. torrentium specimens collected from an animal burrow was positive for Usutu virus.The detection of Usutu virus demonstrates that Cx. torrentium can act as winter reservoir for arboviruses. Abandoned animal burrows may by the primary overwintering site for the species and should be considered in future surveillance programmes, when sampling overwintering mosquitoes.

Snowshoe hare virus (SSHV) is a zoonotic arthropod-borne virus (arbovirus) circulating in colder areas of the Northern Hemisphere. SSHV is maintained in an enzootic cycle between small mammals and mosquitoes, assumably of the genera Aedes and Culiseta. Symptoms of SSHV human infection can range from asymptomatic to severe neuroinvasive disease. Studies on SSHV transmission are limited, and there is no information available on whether mosquitoes of the genus Culex are able to transmit SSHV. Therefore, we investigated six mosquito species via salivation assay for their vector competence. We demonstrated that SSHV can be transmitted by the abundant European Culex species Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, and Cx. torrentium with low transmission efficiency between 3.33% and 6.67%. Additionally, the invasive species Ae. albopictus can also transmit SSHV with a low transmission efficiency of 3.33%. Our results suggest that local transmission of SSHV after introduction to Europe seems to be possible from a vector perspective.

Abstract Background The invasive species Aedes albopictus, commonly known as the Asian tiger mosquito, has undergone extreme range expansion by means of steady introductions as blind passengers in vehicles traveling from the Mediterranean to south-west Germany. The more than 25 established populations in the State of Baden-Württemberg, Palatine and Hesse (south-west Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as a vector of arboviruses, including dengue, chikungunya and Zika viruses. In Germany, Ae. albopictus control programs are implemented by local communities under the auspices of health departments and regulatory offices. Methods The control strategy comprised three pillars: (i) community participation (CP) based on the elimination of breeding sites or improved environmental sanitation, using fizzy tablets based on Bacillus thuringiensis israelensis (fizzy Bti tablets; Culinex® Tab plus); (ii) door-to-door (DtD) control by trained staff through the application of high doses of a water-dispersible Bti granular formulation (Vectobac® WG) aimed at achieving a long-lasting killing effect; and (iii) implementation of the sterile insect technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to initiating large-scale city-wide treatments on a routine basis, the efficacy of the three elements was evaluated in laboratory and semi-field trials. Special emphasis was given to the mass release of sterile Ae. albopictus males. Results More than 60% of the local residents actively participated in the first pillar (CP) of the large-scale control program. The most effective element of the program was found to be the DtD intervention, including the application of Vectobac® WG (3000 ITU/mg) to potential breeding sites (10 g per rainwater container, maximum of 200 l = maximum of approx. 150,000 ITU/l, and 2.5 g per container

Abstract Background Pneumococcal conjugate vaccines (PCVs) effectively prevent pneumococcal disease, but the global impact of pneumococcal vaccination is hampered by its cost. The evaluation of reduced dose schedules of PCV includes measurement of effects on immunogenicity and carriage acquisition compared to standard schedules. The relevance and feasibility of trials of reduced dose schedules is greatest in middle- and low-income countries, such as The Gambia, where the introduction of PCV resulted in good disease control but where transmission of vaccine-type pneumococci persists. We designed a large cluster-randomised field trial of an alternative reduced dose schedule of PCV compared to the standard schedule, the PVS trial. We will also conduct a sub-study to evaluate the individual-level effect of the two schedules on carriage acquisition, immunogenicity, and co-administration of PCV with yellow fever vaccine, the PVS-AcqImm trial. Methods PVS-AcqImm is a prospective, cluster-randomised trial of one dose of PCV scheduled at age 6 weeks with a booster dose at age 9 months (i.e. alternative ‘1+1’ schedule) compared to three primary doses scheduled at 6, 10, and 14 weeks of age (i.e. standard ‘3+0’ schedule). Sub-groups within the alternative schedule group will receive yellow fever vaccine separately or co-administered with PCV at 9 months of age. The primary endpoints are (a) rate of nasopharyngeal vaccine-type pneumococcal acquisition from 9 to 14 months of age, (b) geometric mean concentration of vaccine-type pneumococcal IgG at 18 months of age, and (c) proportions with yellow fever neutralising antibody titre ≥8 four weeks after administration of yellow fever vaccine. Participants and field staff will not be masked to group allocation while the measurement of laboratory endpoints will be masked. Approximately equal numbers of participants will be resident in each of 28 geographic clusters (14 clusters in alternative and standard schedule groups); 784 enrolled for acquisition measurements and 336 for immunogenicity measurements. Discussion Analysis will account for potential non-independence of measurements by cluster and so interpretation of effects will be at the individual level (i.e. a population of individuals). PVS-AcqImm will evaluate whether acquisition of vaccine-type pneumococci is reduced by the alternative compared to the standard schedule, which is required if the alternative schedule is to be effective. Likewise, evidence of superior immune response at 18 months of age and safety of PCV co-administration with yellow fever vaccine will support decision-making regarding the use of the alternative 1+1 schedule. Acquisition and immunogenicity outcomes will be essential for the interpretation of the results of the large field trial comparing the two schedules. Trial registration International Standard Randomised Controlled Trial Number 72821613 .

Abstract The genus Ocotea (Lauraceae) includes about 450 species, of which about 90% are Neotropical, while the rest is from Macaronesia, Africa and Madagascar. In this study we present the first complete chloroplast genome sequences of seven Ocotea species, six Neotropical and one from Macaronesia. Genome sizes range from 152,630 (O. porosa) to 152,685 bp (O. aciphylla). All seven plastomes contain a total of 131 (114 unique) genes, among which 87 (80 unique) encode proteins. The order of genes (if present) is the same in all Lauraceae examined so far. Two hypervariable loci were found in the LSC region (psbA-trnH, ycf2), three in the SSC region (ycf1, ndhH, trnL(UAG)-ndhF). The pairwise cp genomic alignment between the taxa showed that the LSC and SSC regions are more variable compared to the IR regions. The protein coding regions comprise 25,503–25,520 codons in the Ocotea plastomes examined. The most frequent amino acids encoded in the plastomes were leucine, isoleucine, and serine. SSRs were found to be more frequent in the two dioecious Neotropical Ocotea species than in the four bisexual species and the gynodioecious species examined (87 vs. 75–84 SSRs). A preliminary phylogenetic analysis based on 69 complete plastomes of Lauraceae species shows the seven Ocotea species as sister group to Cinnamomum sensu lato. Sequence divergence among the Ocotea species appears to be much lower than among species of the most closely related, likewise species-rich genera Cinnamomum, Lindera and Litsea.

ABSTRACT Bats host a large variety of viruses, including some that may infect other vertebrates and humans. Research on bat-borne viruses attracted significant attention in recent years mainly due to epizootics caused by viruses having bats as hosts. The characterization of the viral communities of bats was then prioritized, but despite increasing efforts, there are large disparities in the geographical ranges covered and the methodologies employed around the world. As a result, large gaps remain in our current understanding of bat viromes and their role in disease emergence. This is particularly true for megadiverse regions in Latin America. This review aims to summarize the current understanding about bat viruses that inhabit Brazilian biomes, one of the most bat species-rich and diverse regions of the globe. Taking into account all known bat-associated viral families studied in Brazilian biomes, we found that almost half of all bat species (86/181 species) were not investigated for viruses at all. Moreover, only a small fraction of viral lineages or families have been studied more in depth, usually employing targeted methods with limited power to characterize a broad virus diversity. Additionally, these studies relied on limited spatiotemporal sampling and small sample sizes. Therefore, our current understanding of bat viral communities in the Brazilian biomes is limited and biased at different levels, limiting zoonotic risk assessments of bat-borne viruses. Considering these limitations, we propose strategies to bridge the existing gaps in the near future. IMPORTANCE Bat-borne viruses have attracted much attention due to zoonotic outbreaks with large consequences to humans. Because of that, virus characterization in bats has been prioritized in tropical regions of the globe. However, bat-virus research in Latin America and particularly in Brazil, which are among the most bat species-rich regions of the world, are highly biased toward zoonotic viruses and known bat reservoir species. These results have direct implication for virus studies in general but also for new zoonotic virus and spillover events characterization. The limited knowledge we currently have about the virome of Brazilian bats drastically limits any broad assessment of zoonotic viruses they carry and calls for coordinated and large-scale studies to fill this crucial knowledge gap.

Abstract Background In Europe, Aedes albopictus is an important vector of chikungunya virus and Dirofilaria nematodes and has been involved in local autochthonous circulation of dengue and Zika viruses. Due to the ongoing spread, targeted field surveillance at potential points of entry of invasive Aedes mosquitoes was initiated by the Republic of Moldova in 2020 as part of the transboundary “Invasive Aedes Mosquitoes COST-Action project.” Methods In 2020, ovitraps were positioned at each of three locations: the border crossing to Romania in Leuşeni (Hancesti region), Chişinӑu International Airport and Chişinӑu Botanical Garden. Results A total of 188 Aedes spp. eggs were collected at the Chişinӑu International Airport between August and September 2020. Twenty-three adults reared in the laboratory were identified morphologically as Ae. albopictus (Skuse, 1895), and 12 selected specimens were confirmed by molecular barcoding of the cytochrome oxidase subunit I gene region. In addition, one adult Ae. albopictus female at the same site was caught with a manual aspirator. Conclusions This is the first documented report of Ae. albopictus in the Republic of Moldova. The presence of immature and adult stages indicates the local reproduction of the species in the country. Therefore, it is crucial to extend and strengthen surveillance of the invasive Aedes mosquitoes to prevent Ae. albopictus and other exotic mosquito species from becoming established in the Republic of Moldova. Graphical abstract

Abstract Background For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. Methods In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. Results A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. Conclusion The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory. Graphical Abstract

Abstract Background The true burden and geographical distribution of human Borna disease virus 1 (BoDV-1) encephalitis is unknown. All detected cases so far have been recorded in Bavaria, southern Germany. Case presentation A retrospective laboratory and epidemiological investigation of a 2017 case of fatal encephalitis in a farmer in Brandenburg, northeast Germany, demonstrated BoDV-1 as causative agent by polymerase chain reaction, immunohistochemistry and in situ hybridization. Next-generation sequencing showed that the virus belonged to a cluster not known to be endemic in Brandenburg. The investigation was triggered by a recent outbreak of animal Borna disease in the region. Multiple possible exposures were identified. The next-of-kin were seronegative. Conclusions The investigation highlights clinical awareness for human BoDV-1 encephalitis which should be extended to all areas endemic for animal Borna disease. All previously diagnosed human cases had occurred > 350 km further south. Further testing of shrews and livestock with Borna disease may show whether this BoDV-1 cluster is additionally endemic in the northwest of Brandenburg.

Human bornavirus encephalitis is a severe and often fatal infection caused by variegated squirrel bornavirus 1 (VSBV-1) and Borna disease virus 1 (BoDV-1). We conducted a prospective study of bornavirus etiology of encephalitis cases in Germany during 2018–2020 by using a serologic testing scheme applied along proposed graded case definitions for VSBV-1, BoDV-1, and unspecified bornavirus encephalitis. Of 103 encephalitis cases of unknown etiology, 4 bornavirus infections were detected serologically. One chronic case was caused by VSBV-1 after occupational-related contact of a person with exotic squirrels, and 3 acute cases were caused by BoDV-1 in virus-endemic areas. All 4 case-patients died. Bornavirus etiology could be confirmed by molecular methods. Serologic testing for these cases was virus specific, discriminatory, and a practical diagnostic option for living patients if no brain tissue samples are available. This testing should be guided by clinical and epidemiologic suspicions, such as residence in virus-endemic areas and animal exposure.

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background Usutu virus (USUV) is an emerging neurotropic arthropod-borne virus recently involved in massive die offs of wild birds predominantly reported in Europe. Although primarily asymptomatic or presenting mild clinical signs, humans infected by USUV can develop neuroinvasive pathologies (including encephalitis and meningoencephalitis). Similar to other flaviviruses, such as West Nile virus, USUV is capable of reaching the central nervous system. However, the neuropathogenesis of USUV is still poorly understood, and the virulence of the specific USUV lineages is currently unknown. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages circulating at the same time and in the same location. Methods The aim of this work was to determine the neurovirulence of isolates from the six main lineages circulating in Europe using mouse model and several neuronal cell lines (neurons, microglia, pericytes, brain endothelial cells, astrocytes, and in vitro Blood-Brain Barrier model). Results Our results indicate that all strains are neurotropic but have different virulence profiles. The Europe 2 strain, previously described as being involved in several clinical cases, induced the shortest survival time and highest mortality in vivo and appeared to be more virulent and persistent in microglial, astrocytes, and brain endothelial cells, while also inducing an atypical cytopathic effect. Moreover, an amino acid substitution (D3425E) was specifically identified in the RNA-dependent RNA polymerase domain of the NS5 protein of this lineage. Conclusions Altogether, these data show a broad neurotropism for USUV in the central nervous system with lineage-dependent virulence. Our results will help to better understand the biological and epidemiological diversity of USUV infection.

The variegated squirrel bornavirus 1 (VSBV-1) is a recently discovered emerging viral pathogen which causes severe and eventually fatal encephalitis in humans after contact to exotic squirrels in private holdings and zoological gardens. Understanding the VSBV-1 epidemiology is crucial to develop, implement, and maintain surveillance strategies for the detection and control of animal and human infections. Based on a newly detected human encephalitis case in a zoological garden, epidemiological squirrel trade investigations and molecular phylogeny analyses of VSBV-1 with temporal and spatial resolution were conducted. Phylogenetic analyses indicated a recent emergence of VSBV-1 in European squirrel holdings and several animal–animal and animal–human spill-over infections. Virus phylogeny linked to squirrel trade analysis showed the introduction of a common ancestor of the known current VSBV-1 isolates into captive exotic squirrels in Germany, most likely by Prevost’s squirrels (Callosciurus prevostii). The links of the animal trade between private breeders and zoos, the likely introduction pathway of VSBV-1 into Germany, and the role of a primary animal distributor were elucidated. In addition, a seroprevalence study was performed among zoo animal caretakers from VSBV-1 affected zoos. No seropositive healthy zoo animal caretakers were found, underlining a probable high-case fatality rate of human VSBV-1 infections. This study illustrates the network and health consequences of uncontrolled wild pet trading as well as the benefits of molecular epidemiology for elucidation and future prevention of infection chains by zoonotic viruses. To respond to emerging zoonotic diseases rapidly, improved regulation and control strategies are urgently needed.

Abstract Background Vector-borne diseases (VBD) are of growing global importance. Sand flies are potential vectors for phleboviruses (family Phenuiviridae) including Toscana virus (TOSV), Sicilian virus, Sandfly fever, Naples virus, and Leishmania parasites in Europe. To date, only two phlebotomine species have been recorded for Germany: Phlebotomus perniciosus and Phlebotomus mascittii. This study updates the distribution and abundance of the two occurring species. Methods An entomological field study was carried out during 2015–2018 to assess the abundance of sand flies in Southwest Germany within the federal states Baden-Wuerttemberg (BW) and Rhineland-Palatinate (RLP). A total of 176 collection sites were examined using CDC light traps. Results A total of 149 individuals of P. mascittii were collected. During 2015–2018, P. mascittii was found at all sites known positive from previous studies and was detected at 15 additional sites previously unknown for the presence of sand flies. Although the environment has changed considerably in 30 years, no significant difference in sand fly dynamics and distribution was found. Phlebotomus perniciosus has only been trapped once since 2001. Conclusions This study showed that sand flies occur in different areas in Southern Germany where they had not been recorded previously. Therefore, it can be assumed that they are more widespread than expected. In addition, sand flies could be found over several years at the same trapping sites, indicating population stability. This supports the need for continued surveillance of possible vector populations and urgent clarification of the vector competence of P. mascittii.

Abstract Background Culicoides biting midges are potential vectors of different pathogens. However, especially for eastern Europe, there is a lack of knowledge on the host-feeding patterns of this vector group. Therefore, this study aimed to identify Culicoides spp. and their vertebrate hosts collected in a wetland ecosystem. Methods Culicoides spp. were collected weekly from May to August 2017, using Biogents traps with UV light at four sites in the Danube Delta Biosphere Reserve, Romania. Vectors and hosts were identified with a DNA barcoding approach. The mitochondrial cytochrome c oxidase subunit 1 was used to identify Culicoides spp., while vertebrate hosts were determined targeting cytochrome b or 16S rRNA gene fragments. A maximum likelihood phylogenetic tree was constructed to verify the biting midge identity against other conspecific Palaearctic Culicoides species. A set of unfed midges was used for morphological confirmation of species identification using slide-mounted wings. Results Barcoding allowed the species identification and detection of corresponding hosts for 1040 (82.3%) of the 1264 analysed specimens. Eight Culicoides spp. were identified with Culicoides griseidorsum, Culicoides puncticollis and Culicoides submaritimus as new species records for Romania. For 39 specimens no similar sequences were found in GenBank. This group of unknown Culicoides showed a divergence of 15.6–16.3% from the closest identified species and clustered in a monophyletic clade, i.e. a novel species or a species without reference sequences in molecular libraries. For all Culicoides spp., nine mammalian and 24 avian species were detected as hosts. With the exception of C. riethi (n = 12), at least one avian host was detected for all Culicoides spp., but this host group only dominated for Culicoides kibunensis and the unknown Culicoides sp.. The most common host group were mammals (n = 993, 87.6% of all identified blood sources) dominated by cattle (n = 817, 70.6%). Conclusions Most Culicoides spp. showed a broad host-feeding pattern making them potential bridge vectors. At the same time, new records of biting midge species for Romania, as well as a potentially unknown Culicoides species, highlight the lack of knowledge regarding the biting midge species and their genetic diversity in eastern Europe.

The West Nile Virus (WNV) and Sindbis virus (SINV) are avian-hosted mosquito-borne zoonotic viruses that co-circulate in some geographical areas and share vector species such as Culex pipiens and Culex torrentium. These are widespread in Europe, including northern parts and Finland, where SINV is endemic, but WNV is currently not. As WNV is spreading northwards in Europe, we wanted to assess the experimental vector competence of Finnish Culex pipiens and Culex torrentium mosquitoes to WNV and SINV in different temperature profiles. Both mosquito species were found susceptible to both viruses and got infected via infectious blood meal at a mean temperature of 18 °C. WNV-positive saliva was detected at a mean temperature of 24 °C, whereas SINV-positive saliva was detected already at a mean temperature of 18 °C. Cx. torrentium was found to be a more efficient vector for WNV and SINV over Cx. pipiens. Overall, the results were in line with the previous studies performed with more southern vector populations. The current climate does not seem optimal for WNV circulation in Finland, but temporary summertime transmission could occur in the future if all other essential factors are in place. More field data would be needed for monitoring and understanding the northward spreading of WNV in Europe.

Transmission of arthropod-borne viruses (arboviruses) are an emerging global health threat in the last few decades. One important arbovirus family is the Togaviridae, including the species Sindbis virus within the genus Alphavirus. Sindbis virus (SINV) is transmitted by mosquitoes, but available data about the role of different mosquito species as potent vectors for SINV are scarce. Therefore, we investigated seven mosquito species, collected from the field in Germany (Ae. koreicus, Ae. geniculatus, Ae. sticticus, Cx. torrentium, Cx. pipiens biotype pipiens) as well as lab strains (Ae. albopictus, Cx. pipiens biotype molestus, Cx. quinquefasciatus), for their vector competence for SINV. Analysis was performed via salivation assay and saliva was titrated to calculate the amount of infectious virus particles per saliva sample. All Culex and Aedes species were able to transmit SINV. Transmission could be detected at all four investigated temperature profiles (of 18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C or 27 ± 5 °C), and no temperature dependency could be observed. The concentration of infectious virus particles per saliva sample was in the same range for all species, which may suggest that all investigated mosquito species are able to transmit SINV in Germany.

It is evident that all the countries surrounding Ghana have experienced epidemics of key arboviruses of medical importance, such as the recent dengue fever epidemic in Burkina Faso. Therefore, Ghana is considered a ripe zone for epidemics of arboviruses, mainly dengue. Surprisingly, Ghana never experienced the propounded deadly dengue epidemic. Indeed, it is mysterious because the mosquito vectors capable of transmitting the dengue virus, such as Aedes aegypti, were identified in Ghana through entomological investigations. Additionally, cases may be missed, as the diagnostic and surveillance capacities of the country are weak. Therefore, we review the arbovirus situation and outline probable reasons for the epidemic mystery in the country. Most of the recorded cases of arbovirus infections were usually investigated via serology by detecting IgM and IgG immunoglobulins in clinical samples, which is indicative of prior exposure but not an active case. This led to the identification of yellow fever virus and dengue virus as the main circulating arboviruses among the Ghanaian population. However, major yellow fever epidemics were reported for over a decade. It is important to note that the reviewed arboviruses were not frequently detected in the vectors. The data highlight the necessity of strengthening the diagnostics and the need for continuous arbovirus and vector surveillance to provide an early warning system for future arbovirus epidemics.

Abstract Background The analysis of large mosquito samples is expensive and time-consuming, delaying the efficient timing of vector control measurements. Processing a fraction of a sample using a subsampling method can significantly reduce the processing effort. However, a comprehensive evaluation of the reliability of different subsampling methods is missing. Methods A total of 23 large mosquito samples (397–4713 specimens per sample) were compared in order to evaluate five subsampling methods for the estimation of the number of specimens and species: area, volume, weight, selection of 200 random specimens and analyses with an image processing software. Each sample was distributed over a grid paper (21.0 × 29.7 cm; 25 grid cells of 4.2 × 5.9 cm) with 200 randomly distributed points. After taking pictures, mosquito specimens closest to each of the 200 points on the paper were selected. All mosquitoes per grid cell were identified by morphology and transferred to scaled tubes to estimate the volume. Finally, the fresh and dry weights were determined. Results The estimated number of specimens and species did not differ between the area-, volume- and weight-based method. Subsampling 20% of the sample gave an error rate of approximately 12% for the number of specimens, 6% for the proportion of the most abundant species and between 6–40% for the number of species per sample. The error for the estimated number of specimens using the picture processing software ImageJ gave a similar error rate when analyzing 15–20% of the total sample. By using 200 randomly selected specimens it was possible to give a precise estimation of the proportion of the most abundant species (r = 0.97, P

Variegated squirrel bornavirus 1 (VSBV-1) is a zoonotic virus that causes fatal encephalitis in humans who are infected after contact with exotic squirrels. We analyzed the brain lesions and the immune responses in all 4 known human cases that showed panencephalitis. Inflammatory infiltrates in areas positive for VSBV-1 RNA and antigen consisted of CD4+ and CD8+ T cells, with perivascular B-cell accumulation. Strong microglial response and bizarre astroglial expansion were present. Areas of malacia contained neutrophils and foamy microglia and macrophages. Immunopathologic examination during infection showed cleavage of caspase 3 in brain cells adjacent to CD8+ cells and widespread p53 expression, hallmarks of apoptosis. Cerebrospinal fluid analyses over time demonstrated increasing protein concentrations and cell counts, paralleled by pathologic lactate elevations in all patients. The most severe cerebrospinal fluid and histologic changes occurred in the patient with the highest viral load, shortest duration of disease, and most medical preconditions.

We developed an IgM-based ELISA that identifies the dengue virus serotype of recent infections. Dominant serotypes were detectable in 91.1% of samples from travelers and 86.5% of samples from residents of endemic regions; 97.1% corresponded to the serotype identified by PCR. This ELISA enables more accurate reporting of epidemiologic findings.

Abstract Background Different mosquito-borne pathogens are circulating in Iran including Sindbis virus, West Nile virus, filarioid worms and malaria parasites. However, the local transmission cycles of these pathogenic agents are poorly understood, especially because ecological data on vector species are scarce and there is limited knowledge about the host range; this understanding could help to direct species-specific vector control measurements or to prioritize research. Methods In the summers of 2015 and 2016, blood-fed mosquitoes were collected at 13 trapping sites on the coast of the Caspian Sea in northern Iran and at an additional trapping site in western Iran. Mosquitoes were generally collected with either a Biogents Sentinel trap or a Heavy Duty Encephalitis Vector Survey trap installed outside. A handheld aspirator was used at the trapping site in western Iran, in addition to a few samplings around the other trapping sites. On average, eight trapping periods were conducted per trapping site. The sources of blood meals were identified using a DNA barcoding approach targeting the cytochrome b or 16S rRNA gene fragment. Results The source of blood meals for 580 blood-fed mosquito specimens of 20 different taxa were determined, resulting in the identification of 13 different host species (9 mammals including humans, 3 birds and 1 reptile), whereby no mixed blood meals were detected. Five mosquito species represented more than 85.8% of all collected blood-fed specimens: Culex pipiens pipiens form pipiens (305 specimens, 55.7% of all mosquito specimens), Cx. theileri (60, 10.9%), Cx. sitiens (51, 9.3%), Cx. perexiguus (29, 5.3%) and Anopheles superpictus (25, 4.6%). The most commonly detected hosts of the four most abundant mosquito species were humans (Homo sapiens; 224 mosquito specimens, 40.9% of all mosquito specimens), cattle (Bos taurus; 171, 31.2%) and ducks (Anas spp.; 75, 13.7%). These four mosquito species had similar host-feeding patterns. The only exceptions were a relatively high proportion of birds for Cx. pipiens pipiens f. pipiens (23.2% of detected blood meal sources) and a high proportion of non-human mammals for Cx. theileri (73.4%). Trapping month, surrounding area, or trapping method had no statistically significant impact on the observed host-feeding patterns of Cx. pipiens pipiens f. pipiens. Conclusions Due to the diverse and overlapping host-feeding patterns, several mosquito species must be considered as potential enzootic and bridge vectors for diverse mosquito-borne pathogens in Iran. Most species can potentially transmit pathogens between mammals as well as between mammals and birds, which might be the result of a similar host selection or a high dependence on the host availability.

[This corrects the article DOI: 10.1371/journal.pntd.0002157.].

BACKGROUND:Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS:92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV μ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE:The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.

Mosquito-borne viruses (MBVs), also known as moboviruses, are associated mainly with mosquitoes and are able to infect humans and other vertebrates [...]

Abstract The invasive mosquito species Aedes japonicus japonicus (Ae. japonicus) is widely distributed in Central Europe and is a known vector of various arboviruses in the laboratory, including flaviviruses such as Japanese Encephalitis virus or West Nile virus. However, the vector competence of Ae. japonicus for the recently emerging Zika virus (ZIKV) has not been determined. Therefore, field-caught Ae. japonicus from Germany were orally infected with ZIKV and incubated at 21, 24, or 27 °C to evaluate the vector competence under climate conditions representative of the temperate regions (21 °C) in the species’ main distribution area in Europe and of Mediterranean regions (27 °C). Aedes japonicus was susceptible to ZIKV at all temperatures, showing infection rates between 10.0% (21 °C) and 66.7% (27 °C). However, virus transmission was detected exclusively at 27 °C with a transmission rate of 14.3% and a transmission efficiency of 9.5%. Taking into account the present distribution of Ae. japonicus in the temperate regions of Central Europe, the risk of ZIKV transmission by the studied Ae. japonicus population in Central Europe has to be considered as low. Nevertheless, due to the species’ vector competence for ZIKV and other mosquito-borne viruses, in combination with the possibility of further spread to Mediterranean regions, Ae. japonicus must be kept in mind as a potential vector of pathogens inside and outside of Europe.

Abstract Background Dirofilariosis is an emerging vector-borne parasitic disease in Europe. Monitoring of wild and domestic carnivores demonstrated circulation of Dirofilaria spp. in Romania in the past. For the implementation of control measures, knowledge on the native mosquito community responsible for Dirofilaria spp. transmission is required. Methods Mosquito samples originated from a longitudinal study previously performed in the Danube Delta Biosphere Reserve. Mosquito pools were screened for Dirofilaria immitis and Dirofilaria repens. The samples comprised 240,572 female mosquito specimens collected every ten days between April and September in 2014 at four different trapping sites. In addition, blood samples of 36 randomly selected dogs were collected in 2016 in each of the four mosquito sampling sites. A duplex real-time assay was used to detect the presence of one or both Dirofilaria species for each sample. This assay targets the cytochrome c oxidase subunit 1 and the 16S rRNA gene fragments to differentiate both parasites. Results Dirofilaria immitis and D. repens were detected in mosquito pools at all four trapping sites. In the 2118 mosquito pools tested, D. immitis was identified for eight and D. repens for six of the 14 screened mosquito taxa, with a higher prevalence of D. immitis (4.53% of analysed pools) compared to D. repens (1.09%). Dirofilaria spp. were also identified in dogs from the same sampling sites with a prevalence of 30.56%. For both Dirofilaria species, the highest estimated infection rates (EIRs) were found in Anopheles maculipennis (s.l.) (D. immitis: EIR = 0.206 per 100 specimens, D. repens: EIR = 0.066 per 100 specimens). In contrast, Coquillettidia richiardii and Anopheles hyrcanus as the most frequent taxa had infection rates which were significantly lower: Cq. richiardii (D. immitis: EIR = 0.021; D. repens: EIR = 0.004); An. hyrcanus (D. immitis: EIR = 0.028; D. repens: EIR = 0.006). The number of positive pools per calendar week was positively correlated with the number of screened pools per calendar week, suggesting constant Dirofilaria spp. transmission during the observation period. Conclusions This study further confirms significant circulation of Dirofilaria spp. in eastern Europe, with high parasite prevalence in domestic canids and mosquitoes. Therefore, systematic monitoring studies are required to better understand the environmental risk factors for Dirofilaria transmission, allowing the implementation of effective surveillance and control measures.

Semen from Zika virus infected individuals can contain high viral loads and can result in sexual transmission. Here, Müller et al. show that semen, and particularly seminal preparations containing extracellular vesicles, inhibit infection of Zika and other flaviviruses.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost’s squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost’s squirrel. Antibodies against bornaviruses were detected in the patient’s cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.

We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.

Usutu virus (USUV) is an emerging mosquitoborne flavivirus with an increasing number of reports from several countries in Europe, where USUV infection has caused high avian mortality rates. However, 20 years after the first observed outbreak of USUV in Europe, there is still no reliable assessment of the large-scale impact of USUV outbreaks on bird populations. In this study, we identified the areas suitable for USUV circulation in Germany and analyzed the effects of USUV on breeding bird populations. We calculated the USUV-associated additional decline of common blackbird (Turdus merula) populations as 15.7% inside USUV-suitable areas but found no significant effect for the other 14 common bird species investigated. Our results show that the emergence of USUV is a further threat for birds in Europe and that the large-scale impact on population levels, at least for common blackbirds, must be considered.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

We screened squirrels in Germany and the Netherlands for the novel zoonotic variegated squirrel bornavirus 1 (VSBV-1). The detection of VSBV-1 in 11 squirrels indicates a considerable risk for transmission to humans handling those animals. Therefore, squirrels in contact with humans should routinely be tested for VSBV-1.

During 2006–2015, we analyzed 70 dengue virus (DENV) strains isolated from febrile travelers returning to Germany. High genetic diversity, including multiple co-circulating DENV lineages and emerging new lineages of DENV-3 and DENV-4, was demonstrated. Our passive surveillance system based on returning travelers yielded substantial information on DENV diversity.