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.

Mosquitoes from Europe Are Able to Transmit Snowshoe Hare Virus.

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.

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.

Vector Competence of Northern European Culex pipiens Biotype pipiens and Culex torrentium to West Nile Virus and Sindbis Virus.

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.

Vector Competence of Mosquitoes from Germany for Sindbis Virus.

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.

Artificial resting sites: An alternative sampling method for adult mosquitoes.

Mosquito collections are commonly conducted with baited traps predominantly attracting host-seeking females. In contrast, resting sites are generally colonized by a broader range of the mosquito population, including a higher proportion of males and blood-engorged females. This study evaluates the sampling success of different artificial resting sites, attached to a deciduous or coniferous tree at different heights. As standard sampling method, carbon dioxide-baited Biogents Sentinel traps (BG traps) were operated in parallel. BG traps caught a higher number of specimens compared to the resting sites. However, the proportion of blood-engorged females and males was higher in resting sites. More Culiseta spp. specimens were collected in resting sites compared to BG traps, but less Aedes spp. specimens. In general, fewer specimens and species were recorded in small resting sites and at top height level compared to medium or large resting sites at medium or ground level. The proportion of males was highest at the ground, while the proportion of engorged females was highest at medium and top level. Due to the higher proportion of blood-engorged females, artificial resting sites are especially useful for studies of host-feeding patterns or xenosurveillance. Low costs and efforts allow a cost-effective increase of the number of resting sites per sampling site to collect more mosquitoes.

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.

Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells.

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.

Discovery and genetic characterization of a novel orthonairovirus in Ixodes ricinus ticks from Danube Delta.

Different arthropod species are vectors of a wide array of arboviruses (arthropod-borne viruses) and have likely been central to viral evolution. To better understand the extent of arthropod-borne pathogens, as well as their origin and evolutionary history, it is crucial to uncover the full range of microbial agents, including viruses associated with arthropods. In this study, a collection of ticks obtained in 2016 directly from mammal and bird hosts from several rural and natural sites of Danube Delta was subjected to transcriptome sequencing and amplification assays. Vector surveillance revealed the presence of a novel orthonairovirus species, designated Sulina virus, in Ixodes ricinus ticks. Phylogenetic clustering of each viral protein consistently placed the new virus in the Orthonairovirus genus as a new genogroup closely related to Tamdy orthonairovirus, a genogroup comprising both pathogenic and tick-associated orthonairoviruses. The serological testing of engorged ticks and blood of infected hosts, along with the inoculation of vertebrate cells and mice found no specific antibodies or viral replication, suggesting that Sulina virus is an orthonairovirus associated with the virome of Ixodes ricinus. Finally, the characterization of a novel orthonairovirus identified using high throughput sequencing will advance our knowledge of interactions between viruses and tick vectors, expanding our perspective on fundamental questions regarding orthonairovirus evolution, diversity, ecology and potential of emergence as pathogens.

Safety and immunogenicity of a primary yellow fever vaccination under low-dose methotrexate therapy-a prospective multi-centre pilot study1.

BACKGROUND: More people on immunosuppression live in or wish to travel to yellow fever virus (YFV)-endemic areas. Data on the safety and immunogenicity of yellow fever vaccination (YFVV) during immunosuppression are scarce. The aim of this study was to compare the safety and immunogenicity of a primary YFVV between travellers on methotrexate and controls. METHODS: We conducted a prospective multi-centre controlled observational study from 2015 to 2017 in six Swiss travel clinics. 15 adults (nine with rheumatic diseases, five with dermatologic conditions and one with a gastroenterological disease) on low-dose methotrexate (≤20 mg/week) requiring a primary YFVV and 15 age and sex-matched controls received a YFVV. Solicited/unsolicited adverse reactions were recorded, YFV-RNA was measured in serum samples on Days 3, 7, 10, 14, 28 and neutralizing antibodies on Days 0, 7, 10, 14, 28. RESULTS: Patients´ and controls' median ages were 53 and 52 years; 9 patients and 10 controls were female. 43% of patients and 33% of controls showed local side effects (P = 0.71); 86% of patients and 66% of controls reported systemic reactions (P = 0.39). YFV-RNA was detected in patients and controls on Day 3-10 post-vaccination and was never of clinical significance. Slightly more patients developed YFV-RNAaemia (Day 3: n = 5 vs n = 2, Day 7: n = 9 vs n = 7, Day 10: n = 3 vs n = 2, all P > 0.39). No serious reactions occurred. On Day 10, a minority of vaccinees was seroprotected (patients: n = 2, controls: n = 6). On Day 28, all vaccinees were seroprotected. CONCLUSIONS: First-time YFVV was safe and immunogenic in travellers on low-dose methotrexate. Larger studies are needed to confirm these promising results.

Microsatellite typing of Aedes albopictus (Diptera: Culicidae) populations from Germany suggests regular introductions.

The global spread of the Asian tiger mosquito Aedes albopictus is of concern, as this mosquito species constitutes an important vector of a number of emerging pathogens including dengue virus, chikungunya virus and Zika virus. Since its first appearance in Albania (1979) and Italy (1990), the species has been reported from more than twenty-five European countries. However, the dispersion process in Europe is largely unknown, as information on population genetic structure is lacking, which is relevant to understand the observed spread. In order to determine whether the ten Ae. albopictus populations detected in Germany until 2017 originate from a single introduction event or from independent importations, genetic analyses with a set of sixteen microsatellite markers were performed. The samples included specimens from three locations with potentially overwintering populations, collected in three consecutive years. The results indicate a heterogeneous population structure consisting of two clusters with significant substructuring, suggesting regular, independent introductions instead of a continuous spread across Germany originating from one or few sites. Moreover, the analyses provide further evidence for Ae. albopictus overwintering in Germany as samples from identical locations collected in three consecutive years had a relatively high genetic similarity. However, the population structure is probably influenced by local mosquito control activities. The results presented provide further evidence for regular introductions of Ae. albopictus specimens into Germany, probably leading to local establishment north of the Alps. This highlights the need for constant surveillance and control of Ae. albopictus not only in southern, but also in Central Europe.

Ecology of West Nile Virus in the Danube Delta, Romania: Phylogeography, Xenosurveillance and Mosquito Host-Feeding Patterns.

The ecology of West Nile virus (WNV) in the Danube Delta Biosphere Reserve (Romania) was investigated by combining studies on the virus genetics, phylogeography, xenosurveillance and host-feeding patterns of mosquitoes. Between 2014 and 2016, 655,667 unfed and 3842 engorged mosquito females were collected from four sampling sites. Blood-fed mosquitoes were negative for WNV-RNA, but two pools of unfed Culex pipiens s.l./torrentium collected in 2014 were tested positive. Our results suggest that Romania experienced at least two separate WNV lineage 2 introductions: from Africa into Danube Delta and from Greece into south-eastern Romania in the 1990s and early 2000s, respectively. The genetic diversity of WNV in Romania is primarily shaped by in situ evolution. WNV-specific antibodies were detected for 19 blood-meals from dogs and horses, but not from birds or humans. The hosts of mosquitoes were dominated by non-human mammals (19 species), followed by human and birds (23 species). Thereby, the catholic host-feeding pattern of Culex pipiens s.l./torrentium with a relatively high proportion of birds indicates the species' importance as a potential bridge vector. The low virus prevalence in combination with WNV-specific antibodies indicate continuous, but low activity of WNV in the Danube Delta during the study period.

Comparative analysis of subsampling methods for large mosquito samples.

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 < 0.001), but the number of species per sample was underestimated by 28% on average. Selecting adjacent grid cells instead of sampling randomly chosen grid cells and using dry weight instead of wet weight did not increase the accuracy of estimates. CONCLUSIONS: Different subsampling methods have various advantages and disadvantages. However, the area-based analysis of 20% of the sample is probably the most suitable approach for most kinds of mosquito studies, giving sufficiently precise estimations of the number of specimens and species, which is slightly less laborious compared to the other methods tested.

Culex torrentium: A Potent Vector for the Transmission of West Nile Virus in Central Europe.

The continuous circulation of West Nile virus (WNV) in Central, South and East Europe and its recent detection in several dead birds and two horses in Germany highlights the need for information on WNV vector competence of mosquitoes from Central Europe. Therefore, three common Culex species (Culex pipiens biotype pipiens, Culex pipiens biotype molestus and Culex torrentium) from Germany were orally infected with WNV and kept at 18 °C, 21 °C, 24 °C or 27 °C for 14 or 21 days post infection (dpi). Thereafter viable WNV was present in the saliva in all tested taxa, but only at incubation temperatures of 24 °C or 27 °C and predominantly at the extended incubation period of 21 dpi. Highest transmission efficiency rates of 17 % (24 °C) and 24% (27 °C) were found for Cx. torrentium. Culex p. pipiens and Cx. p. molestus showed low transmission efficiencies with a maximum of only 3%. Consequently, temperatures above 21 °C support transmission of WNV, which matches the predominant distribution of human WNV cases around the Mediterranean Sea and in South-East Europe. Culex torrentium has been identified as a potent vector for WNV in Central and Northern Europe, which highlights the need for surveillance of mosquito-borne viruses north of the Alps.

Detection, infection dynamics and small RNA response against Culex Y virus in mosquito-derived cells.

Many insect cell lines are persistently infected with insect-specific viruses (ISV) often unrecognized by the scientific community. Considering recent findings showing the possibility of interference between arbovirus and ISV infections, it is important to pay attention to ISV-infected cell lines. One example is the Entomobirnavirus, Culex Y virus (CYV). Here we describe the detection of CYV using a combination of small RNA sequencing, electron microscopy and PCR in mosquito cell lines Aag2, U4.4 and C7-10. We found CYV-specific small RNAs in all three cell lines. Interestingly, the magnitude of the detected viral RNA genome is variable among cell passages and leads to irregular detection via electron microscopy. Gaining insights into the presence of persistent ISV infection in commonly used mosquito cells and their interactions with the host immune system is beneficial for evaluating the outcome of co-infections with arboviruses of public health concern.

Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany.

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.

Male offspring born to mildly ZIKV-infected mice are at risk of developing neurocognitive disorders in adulthood.

Congenital Zika virus (ZIKV) syndrome may cause fetal microcephaly in ~1% of affected newborns. Here, we investigate whether the majority of clinically inapparent newborns might suffer from long-term health impairments not readily visible at birth. Infection of immunocompetent pregnant mice with high-dose ZIKV caused severe offspring phenotypes, such as fetal death, as expected. By contrast, low-dose (LD) maternal ZIKV infection resulted in reduced fetal birth weight but no other obvious phenotypes. Male offspring born to LD ZIKV-infected mothers had increased testosterone (TST) levels and were less likely to survive in utero infection compared to their female littermates. Males also presented an increased number of immature neurons in apical and basal hippocampal dendrites, while female offspring had immature neurons in basal dendrites only. Moreover, male offspring with high but not very high (storm) TST levels were more likely to suffer from learning and memory impairments compared to females. Future studies are required to understand the impact of TST on neuropathological and neurocognitive impairments in later life. In summary, increased sex-specific vigilance is required in countries with high ZIKV prevalence, where impaired neurodevelopment may be camouflaged by a healthy appearance at birth.

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 °C or 27 °C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 °C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

Experimental risk assessment for chikungunya virus transmission based on vector competence, distribution and temperature suitability in Europe, 2018.

BackgroundOver the last decade, the abundant distribution of the Asian tiger mosquito Aedes albopictus in southern Europe and the import of chikungunya virus (CHIKV) by infected travellers has resulted in at least five local outbreaks of chikungunya fever in France and Italy. Considering the ongoing spread of Ae. albopictus to central Europe, we performed an analysis of the Europe-wide spatial risk of CHIKV transmission under different temperature conditions. Methods:Ae. albopictus specimens from Germany and Italy were orally infected with CHIKV from an outbreak in France and kept for two weeks at 18 °C, 21 °C or 24 °C. A salivation assay was conducted to detect infectious CHIKV. Results: Analyses of mosquito saliva for infectious virus particles demonstrated transmission rates (TRs) of > 35%. Highest TRs of 50% for the mosquito population from Germany were detected at 18 °C, while the Italian population had highest TRs of 63% at 18 °C and 21 °C, respectively. Temperature data indicated a potential risk of CHIKV transmission for extended durations, i.e. sufficiently long time periods allowing extrinsic incubation of the virus. This was shown for areas already colonised by Ae. albopictus, as well as for large parts of central Europe that are not colonised. Conclusion: The current risk of CHIKV transmission in Europe is not primarily restricted by temperature, which allows extrinsic incubation of the virus, but rather by the vector distribution. Accordingly, all European countries with established populations of Ae. albopictus should implement respective entomological surveillance and monitoring systems, as basis for suitable control measures.