Distribution of Usutu Virus in Germany and Its Effect on Breeding Bird Populations.

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.

Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016.

Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.

Putative Lineage of Novel African Usutu Virus, Central Europe.

We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe.

Characterization of a novel circo-like virus in Aedes vexans mosquitoes from Germany: evidence for a new genus within the family Circoviridae.

Over recent decades, metagenomic studies have expanded the number of newly described, often unclassified, viruses within the family Circoviridae. Using broad-spectrum circovirus and cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative ORFs shared very low amino acid identity with those of previously characterized circoviruses and cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defined, together with previously described mosquito- and bat faeces-derived circo-like viruses, a different genus, tentatively called Krikovirus, within the family Circoviridae. We further demonstrated that viruses of the putative genus Krikovirus all shared a genomic organization that was unique among the family Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the family Circoviridae.

Mammalophilic feeding behaviour of Culex quinquefasciatus mosquitoes collected in the cities of Chetumal and Cancun, Yucatán Peninsula, Mexico.

OBJECTIVE: The studie describes the blood-feeding behaviour of mosquitoes in Mexico, to understand host-vector relationships and dynamics of disease transmission. METHODS: From September 2012 to November 2012 and in November 2013, 911 blood-fed Cx. quinquefasciatus mosquitoes were collected with aspirators inside houses in Chetumal and Cancun. Blood meals were analysed by PCR and subsequent Sanger sequencing of the cytochrome b gene. RESULTS: 93.3% of mosquitoes fed on mammals, 6.5% on birds and 0.2% on reptiles. The most frequent vertebrate hosts were humans (65.4%), dogs (23.2%), chicken (5.4%), cattle (2.2%) and cats (1.8%). CONCLUSIONS: Cx. quinquefasciatus most frequently fed on humans and dogs in both studied cities, which is in contrast to a previous study that demonstrated lower prevalence of mammalian blood in engorged Cx. quinquefasciatus.

Evaluation of the vector competence for Batai virus of native Culex and exotic Aedes species in Central Europe.

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.

Large-scale performance assessment of the BG-Counter 2 used with two different mosquito traps.

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 (R2 = 0.16), but per individual trap the value could reach up to 0.62 (mean R2 = 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.

Host attraction and host feeding patterns indicate generalist feeding of Culex pipiens s.s. and Cx. torrentium.

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.

Genetic diversity and wing geometric morphometrics among four populations of Aedes aegypti (Diptera: Culicidae) from Benin.

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.

Using geometric wing morphometrics to distinguish Aedes japonicus japonicus and Aedes koreicus.

BACKGROUND: Aedes japonicus japonicus (Theobald, 1901) and Aedes koreicus (Edwards, 1917) have rapidly spread in Europe over the last decades. Both species are very closely related and occur in sympatry. Females and males are difficult to distinguish. However, the accurate species discrimination is important as both species may differ in their vectorial capacity and spreading behaviour. In this study, we assessed the potential of geometric wing morphometrics as alternative to distinguish the two species. METHODS: A total of 147 Ae. j. japonicus specimens (77 females and 70 males) and 124 Ae. koreicus specimens (67 females and 57 males) were collected in southwest Germany. The left wing of each specimen was removed, mounted and photographed. The coordinates of 18 landmarks on the vein crosses were digitalised by a single observer. The resulting two-dimensional dataset was used to analyse the differences in the wing size (i.e. centroid size) and wing shape between Ae. j. japonicus and Ae. koreicus using geometric morphometrics. To analyse the reproducibility of the analysis, the landmark collection was repeated for 20 specimens per sex and species by two additional observers. RESULTS: The wing size in female Ae. koreicus was significantly greater than in Ae. j. japonicus but did not differ significantly for males. However, the strong overlap in wing size also for the females would not allow to discriminate the two species. In contrast, the wing shape clustering was species specific and a leave-one-out validation resulted in a reclassification accuracy of 96.5% for the females and 91.3% for the males. The data collected by different observers resulted in a similar accuracy, indicating a low observer bias for the landmark collection. CONCLUSIONS: Geometric wing morphometrics provide a reliable and robust tool to distinguish female and male specimens of Ae. j. japonicus and Ae. koreicus.

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells.

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.

Special Issue "Mosquito-Borne Virus Ecology".

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

First Detection of the Invasive Mosquito Vector Aedes albopictus (Diptera: Culicidae) in Benin, West Africa, 2021.

Aedes albopictus (Skuse) is native to Southeast Asia and has colonized tropical and temperate regions worldwide in the last three to four decades. In Africa, data on its distribution is incomplete. Most studies having focused on the abundance, competition to other species, and phylogenetics of this vector are from the central African region. Here, we report the first detection of Ae. albopictus in Benin, West Africa. A total of 13 specimens were collected during the study period in 2021. The phylogenetic analysis of a cytochrome oxidase c subunit I gene fragment revealed a close relationship to populations from tropical India. Because of its close geographical proximity to areas where it has been found, it is assumed that the species was introduced several years before and is currently widely distributed in Benin. Additional studies are needed to explore its distribution, expansion range, and competitive effects on native species.

Arbovirus Epidemiology: The Mystery of Unnoticed Epidemics in Ghana, West Africa.

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.

Mosquito-Associated Viruses and Their Related Mosquitoes in West Africa.

Mosquito-associated viruses (MAVs), including mosquito-specific viruses (MSVs) and mosquito-borne (arbo)viruses (MBVs), are an increasing public, veterinary, and global health concern, and West Africa is projected to be the next front for arboviral diseases. As in-depth knowledge of the ecologies of both western African MAVs and related mosquitoes is still limited, we review available and comprehensive data on their diversity, abundance, and distribution. Data on MAVs' occurrence and related mosquitoes were extracted from peer-reviewed publications. Data on MSVs, and mosquito and vertebrate host ranges are sparse. However, more data are available on MBVs (i.e., dengue, yellow fever, chikungunya, Zika, and Rift Valley fever viruses), detected in wild and domestic animals, and humans, with infections more concentrated in urban areas and areas affected by strong anthropogenic changes. Aedes aegypti, Culex quinquefasciatus, and Aedes albopictus are incriminated as key arbovirus vectors. These findings outline MAV, related mosquitoes, key knowledge gaps, and future research areas. Additionally, these data highlight the need to increase our understanding of MAVs and their impact on host mosquito ecology, to improve our knowledge of arbovirus transmission, and to develop specific strategies and capacities for arboviral disease surveillance, diagnostic, prevention, control, and outbreak responses in West Africa.

Vector Competence of the Invasive Mosquito Species Aedes koreicus for Arboviruses and Interference with a Novel Insect Specific Virus.

The global spread of invasive mosquito species increases arbovirus infections. In addition to the invasive species Aedes albopictus and Aedes japonicus, Aedes koreicus has spread within Central Europe. Extensive information on its vector competence is missing. Ae. koreicus from Germany were investigated for their vector competence for chikungunya virus (CHIKV), Zika virus (ZIKV) and West Nile virus (WNV). Experiments were performed under different climate conditions (27 ± 5 °C; 24 ± 5 °C) for fourteen days. Ae. koreicus had the potential to transmit CHIKV and ZIKV but not WNV. Transmission was exclusively observed at the higher temperature, and transmission efficiency was rather low, at 4.6% (CHIKV) or 4.7% (ZIKV). Using a whole virome analysis, a novel mosquito-associated virus, designated Wiesbaden virus (WBDV), was identified in Ae. koreicus. Linking the WBDV infection status of single specimens to their transmission capability for the arboviruses revealed no influence on ZIKV transmission. In contrast, a coinfection of WBDV and CHIKV likely has a boost effect on CHIKV transmission. Due to its current distribution, the risk of arbovirus transmission by Ae. koreicus in Europe is rather low but might gain importance, especially in regions with higher temperatures. The impact of WBDV on arbovirus transmission should be analyzed in more detail.

Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany.

A molecular survey of 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 demonstrated the presence of Sindbis virus (SINV) in Culex spp. and Anopheles maculipennis sensu lato. Phylogenetic analysis of the German SINV strains linked them with Swedish SINV strains, the causative agent of Ockelbo disease in humans.

Blood-meal analysis of Culicoides (Diptera: Ceratopogonidae) reveals a broad host range and new species records for Romania.

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.

Phlebotomine sand flies in Southwest Germany: an update with records in new locations.

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.