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.

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.

Detection and characterization of a novel rhabdovirus in Aedes cantans mosquitoes and evidence for a mosquito-associated new genus in the family Rhabdoviridae.

Thanks to recent advances in random amplification technologies, metagenomic surveillance expanded the number of novel, often unclassified viruses within the family Rhabdoviridae. Using a vector-enabled metagenomic (VEM) tool, we identified a novel rhabdovirus in Aedes cantans mosquitoes collected from Germany provisionally named Ohlsdorf virus (OHSDV). The OHSDV genome encodes the canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORF in the P gene. Sequence analysis indicated that OHSDV exhibits a similar genome organization and characteristics compared to other mosquito-associated rhabdoviruses (Riverside virus, Tongilchon virus and North Creek virus). Complete L protein based phylogeny revealed that all four viruses share a common ancestor and form a deeply rooted and divergent monophyletic group within the dimarhabdovirus supergroup and define a new genus, tentatively named Ohlsdorfvirus. Although the Ohlsdorfvirus clade is basal within the dimarhabdovirus supergroup phylogeny that includes genera of arthropod-borne rhabdoviruses, it remains unknown if viruses in the proposed new genus are vector-borne pathogens. The observed spatiotemporal distribution in mosquitoes suggests that members of the proposed genus Ohlsdorfvirus are geographically restricted/separated. These findings increase the current knowledge of the genetic diversity, classification and evolution of this virus family. Further studies are needed to determine the host range, transmission route and the evolutionary relationships of these mosquito-associated viruses with those infecting vertebrates.

Host-feeding patterns of mosquito species in Germany.

BACKGROUND: Mosquito-borne pathogens are of growing importance in many countries of Europe including Germany. At the same time, the transmission cycles of most mosquito-borne pathogens (e.g. viruses or filarial parasites) are not completely understood. There is especially a lack of knowledge about the vector capacity of the different mosquito species, which is strongly influenced by their host-feeding patterns. While this kind of information is important to identify the relevant vector species, e.g. to direct efficient control measures, studies about the host-feeding patterns of mosquito species in Germany are scarce and outdated. METHODS: Between 2012 and 2015, 775 blood-fed mosquito specimens were collected. Sampling was conducted with Heavy Duty Encephalitis Vector Survey traps, Biogents Sentinel traps, gravid traps, hand-held aspirators, sweep nets, and human-bait collection. The host species for each mosquito specimen was identified with polymerase chain reactions and subsequent Sanger sequencing of the cytochrome b gene. RESULTS: A total of 32 host species were identified for 23 mosquito species, covering 21 mammalian species (including humans) and eleven bird species. Three mosquito species accounted for nearly three quarters of all collected blood-fed mosquitoes: Aedes vexans (363 specimens, 46.8 % of all mosquito specimens), Culex pipiens pipiens form pipiens (100, 12.9 %) and Ochlerotatus cantans (99, 12.8 %). Non-human mammals dominated the host species (572 specimens, 73.8 % of all mosquito specimens), followed by humans (152, 19.6 %) and birds (51, 6.6 %). The most common host species were roe deer (Capreolus capreolus; 258 mosquito specimens, 33.3 % of all mosquito specimens, 65 % of all mosquito species), humans (Homo sapiens; 152, 19.6 %, 90 %), cattle (Bos taurus; 101, 13.0 %, 60 %), and wild boar (Sus scrofa; 116, 15.0 %, 50 %). There were no statistically significant differences in the spatial-temporal host-feeding patterns of the three most common mosquito species. CONCLUSIONS: Although the collected blood-fed mosquito species had a strong overlap of host species, two different host-feeding groups were identified with mosquito species feeding on (i) non-human mammals and humans or (ii) birds, non-human mammals, and humans, which make them potential vectors of pathogens only between mammals or between mammals and birds, respectively. Due to the combination of their host-feeding patterns and wide distribution in Germany, Cx. pipiens pipiens form pipiens and Cx. torrentium are potentially most important vectors for pathogens transmitted from birds to humans and the species Ae. vexans for pathogens transmitted from non-human mammals to humans. Finally, the presented study indicated a much broader host range compared to the classifications found in the literature for some of the species, which highlights the need for studies on the host-feeding patterns of mosquitoes to further assess their vector capacity and the disease ecology in Europe.

Surveillance of maternal antibodies against West Nile virus in chicken eggs in South-West Germany.

OBJECTIVE: The emergence of West Nile virus (WNV) in several European countries increases the risk of its introduction to Germany. This study evaluated a new method for WNV surveillance by testing for maternal antibodies in chicken eggs. METHODS: A total of 1,990 eggs were collected in 35 sampling sites in the south-west of Germany and tested for WNV-specific antibodies. RESULTS: The results did not indicate evidence for WNV circulation in the study area. CONCLUSION: This work serves as a proof-of-concept that such a method is useful and a potential alternative to use of sentinel chicken for regular WNV surveillance.

Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa.

UNLABELLED: Usutu virus (USUV), one of the most neglected Old World encephalitic flaviviruses, causes epizootics among wild and captive birds and sporadic infection in humans. The dynamics of USUV spread and evolution in its natural hosts are unknown. Here, we present the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution. The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow. Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks. Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles. IMPORTANCE: Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection.

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25 °C as well as 18 °C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25 °C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18 °C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.

Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal.

BACKGROUND: In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal. FINDINGS: From May to October 2007-2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment. CONCLUSIONS: We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.

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.

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.

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.

Cyclovirus CyCV-VN species distribution is not limited to Vietnam and extends to Africa.

Cycloviruses, small ssDNA viruses of the Circoviridae family, have been identified in the cerebrospinal fluid from symptomatic human patients. One of these species, cyclovirus-Vietnam (CyCV-VN), was shown to be restricted to central and southern Vietnam. Here we report the detection of CyCV-VN species in stool samples from pigs and humans from Africa, far beyond their supposed limited geographic distribution.

Field evaluation of four widely used mosquito traps in Central Europe.

BACKGROUND: To monitor adult mosquitoes several trapping devices are available. These are differently constructed and use various mechanisms for mosquito attraction, thus resulting in different trapping sensitivities and efficacies for the various species. Mosquito monitoring and surveillance programs in Europe use various types of mosquito traps, but only a few comparisons have been conducted so far. This study compared the performance of four commercial trapping devices, which are commonly used in Europe. METHODS: Four different traps, Biogents Sentinel trap (BG trap), Heavy Duty Encephalitis Vector Survey trap (EVS trap), Centres for Disease Control miniature light trap (CDC trap) and Mosquito Magnet Patriot Mosquito trap (MM trap) were compared in a 4 × 4 latin square study. In the years 2012 and 2013, more than seventy 24-hour trap comparisons were conducted at ten different locations in northern and southern Germany, representing urban, forest and floodplain biotopes. RESULTS: Per 24-hour trapping period, the BG trap caught the widest range of mosquito species, the highest number of individuals of the genus Culex as well as the highest number of individuals of the species Ochlerotatus cantans, Aedes cinereus/geminus, Oc. communis and Culex pipiens/torrentium. The CDC trap revealed best performance for Aedes vexans, whereas the MM trap was most efficient for mosquitoes of the genus Anopheles and the species Oc. geniculatus. The EVS trap did not catch more individuals of any genus or species compared to the other three trapping devices. The BG trap caught the highest number of individuals per trapping period in urban environments as well as in wet forest, while the CDC trap caught the highest number of individuals in the floodplain biotopes. Additionally, the BG trap was most efficient for the number of mosquito species in urban locations. CONCLUSION: The BG trap showed a significantly better or similar performance compared to the CDC, EVS or MM trap with regard to trapping efficacy for most common mosquito species in Germany, including diversity of mosquito species and number of mosquitoes per trapping period. Thus, the BG trap is probably the best solution for general monitoring or surveillance programs of adult mosquitoes in Central Europe.

The use of morphometric wing characters to discriminate female Culex pipiens and Culex torrentium.

The reliability of the length of wing radial vein r(2/3) as a character for the morphological discrimination of the two potential arbovirus vectors Culex pipiens s.s. and Cx. torrentium from Germany was reassessed, after this character had been neglected for more than 40 years. Additionally, multivariate morphometric analyses were applied to evaluate wing shape variation between both species. Although high-throughput molecular tools are now available to differentiate the two species, a simple, low-cost routine alternative may be useful in the absence of a molecular laboratory, such as under semi-field conditions. A thin-plate splines transformation confirmed that primarily the shrinkage of vein r(2/3) is responsible for the wing differences between the two species. In the bivariate analysis, the r(2/3)/r3 indices of Cx. pipiens s.s. and Cx. torrentium were 0.185 and 0.289, respectively, resulting in a correct classification of more than 91% of all tested specimens. Using the absolute length of vein r(2/3) alone still allowed for more than 90% accurate discrimination. Furthermore, classification accuracy of linear discriminant analysis exceeded 97%.

First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids in Germany.

Mosquitoes and other arthropods may transmit medically important pathogens, in particular viruses such as West Nile virus. The presence of suitable hosts and competent vectors for those zoonotic viruses is essential for an enzootic transmission, which is a prerequisite for epidemics. To establish reliable risk projections, it is an urgent need for an exact identification of mosquito species, which is especially challenging in the case of sibling species, such as Culex. pipiens pipiens biotypes pipiens and molestus. To facilitate detection of different Culex pipiens forms and their hybrids we established a multiplex real-time PCR. Culex pipiens samples were obtained by egg raft collection and rearing until imago stage or adult sampling using CO2 baited traps and gravid traps. In total, we tested more than 16,500 samples collected all over Germany in the years 2011 and 2012. The predominant species in Germany are Culex pipiens pipiens biotype pipiens and Culex. torrentium, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes at sites where both species occur sympatrically. This report of a potentially important bridge vector for West Nile virus might have major impact in the risk projections for West Nile virus in Germany.