Assessing viral metagenomics for the diagnosis of acute undifferentiated fever in returned travellers: a multicenter cohort study.

BACKGROUND: Up to 45% of febrile returning travellers remain undiagnosed after a thorough diagnostic work-up, even at referral centres. Although metagenomic next-generation sequencing (mNGS) has emerged as a promising tool, evidence of its usefulness in imported fever is very limited. METHODS: Travellers returning with fever were prospectively recruited in three referral clinics from November 2017 to November 2019. Unbiased mNGS optimised for virus detection was performed on serum samples of participants with acute undifferentiated febrile illness (AUFI), and results were compared to those obtained by reference diagnostic methods (RDM). RESULTS: Among 507 returned febrile travellers, 433(85.4%) presented with AUFI. Dengue virus (n = 86) and Plasmodium spp. (n = 83) were the most common causes of fever. 103/433(23.8%) AUFI remained undiagnosed at the end of the follow-up.Metagenomic next-generation sequencing unveiled potentially pathogenic microorganisms in 196/433(38.7%) AUFI. mNGS identifications were more common in patients with a shorter duration of fever (42.3% in ≤5 days vs 28.7% in >5 days, P = 0.005). Potential causes of fever were revealed in 25/103(24.2%) undiagnosed AUFI and 5/23(21.7%) travellers with severe undiagnosed AUFI. Missed severe aetiologies included eight bacterial identifications and one co-infection of B19 parvovirus and Aspergillus spp.Additional identifications indicating possible co-infections occurred in 29/316(9.2%) travellers with AUFI, and in 11/128(8.6%) travellers with severe AUFI, who had received a diagnosis through RDM. The most common co-infections detected in severe AUFI were caused by Gram-negative bacteria. Serum mNGS was unable to detect >50% of infectious diagnoses achieved by RDM and also yielded 607 non-pathogenic identifications. DISCUSSION: mNGS of serum can be a valuable diagnostic tool for selected travellers with undiagnosed AUFI or severe disease in addition to reference diagnostic techniques, especially during the first days of symptoms. Nevertheless, mNGS results interpretation presents a great challenge. Further studies evaluating the performance of mNGS using different sample types and protocols tailored to non-viral agents are needed.

RT-PCR-based assessment of the SD Bioline Rota/Adeno Antigen-based test in infants with and without diarrhea.

BACKGROUND: Rotavirus A (RVA) infections remain a major cause of severe acute diarrhea affecting children worldwide. To date, rapid diagnostic tests (RDT) are widely used to detect RVA. However, paediatricians question whether the RDT can still detect the virus accurately. Therefore, this study aimed to evaluate the performance of the rapid rotavirus test in comparison to the one-step RT-qPCR method. METHODS: A cross-sectional study was conducted in Lambaréné, Gabon, from April 2018 to November 2019. Stool samples were collected from children under 5 years of age with diarrhoea or a history of diarrhoea within the last 24 h, and from asymptomatic children from the same communities. All stool samples were processed and analysed using the SD BIOLINE Rota/Adeno Ag RDT against a quantitative reverse transcription PCR (RT-qPCR), which is considered the gold standard. RESULTS: For a total of 218 collected stool samples, the overall sensitivity of the RDT was 46.46% (confidence interval (CI) 36.38-56.77), with a specificity of 96.64% (CI 91.62-99.08) compared to one-step RT-qPCR. After confirming the presence or absence of RVA gastroenteritis, the RDT showed suitable results in detecting rotavirus A-associated disease, with a 91% concordance with the RT-qPCR. Furthermore, the performance of this test varied when correlated with seasonality, symptoms, and rotavirus genotype. CONCLUSION: This RDT showed high sensitivity and was suitable for the detection of RVA in patients with RVA gastroenteritis, although some asymptomatic RVA shedding was missed by RT-qPCR. It could be a useful diagnostic tool, especially in low-income countries.

The Virome of Bats Inhabiting Brazilian Biomes: Knowledge Gaps and Biases towards Zoonotic Viruses.

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.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Serologic and Genomic Investigation of West Nile Virus in Kosovo.

The prevalence of West Nile virus (WNV) is increasing across Europe, with cases emerging in previously unaffected countries. Kosovo is situated in a WNV-endemic region where the seroepidemiological data on WNV in humans remains absent. To address this issue, we have conducted a seroepidemiological investigation of 453 randomly selected sera from a hospital in Kosovo, revealing a 1.55% anti-WNV IgG seroprevalence. Comparative and phylogeographic analyses of the WNV genomes obtained by sequencing archived samples from patients with West Nile fever indicate at least two recent and distinct introductions of WNV lineage 2 into Kosovo from neighboring countries. These findings confirm the eco-epidemiological status of WNV in southeast Europe, where long- and short-range dispersion of lineage 2 strains contributes to a wider circulation via central Europe. Our results suggest an increasing risk for WNV spreading in Kosovo, underscoring the need for an integrated national surveillance program targeting vectors and avian populations for early epidemic detection, as well as the screening of blood donors to gauge the impact of virus circulation on the human population.

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.

Molecular Characterization of Anopheles algeriensis Theobald, 1903 (Diptera: Culicidae) Populations from Europe.

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

Molecular surveillance and genetic divergence of rotavirus A antigenic epitopes in Gabonese children with acute gastroenteritis.

BACKGROUND: Rotavirus A (RVA) causes acute gastroenteritis in children <5 years of age in sub-Saharan Africa. In this study, we described the epidemiology and genetic diversity of RVA infecting Gabonese children and examined the antigenic variability of circulating strains in relation to available vaccine strains to maximize the public health benefits of introducing rotavirus vaccine through the Expanded Programme on Immunization (EPI) in Gabon. METHODS: Stool samples were collected consecutively between April 2018 and November 2019 from all hospitalized children <5 years with gastroenteritis and community controls without gastroenteritis. Children were tested for rotavirus A by quantitative RT-PCR and subsequently sequenced to identify circulating rotavirus A genotypes in the most vulnerable population. The VP7 and VP4 (VP8*) antigenic epitopes were mapped to homologs of vaccine strains to assess structural variability and potential impact on antigenicity. FINDINGS: Infections were mostly acquired during the dry season. Rotavirus A was detected in 98/177 (55%) hospitalized children with gastroenteritis and 14/67 (21%) of the control children. The most common RVA genotypes were G1 (18%), G3 (12%), G8 (18%), G9 (2%), G12 (25%), with G8 and G9 reported for the first time in Gabon. All were associated either with P[6] (31%) or P[8] (38%) genotypes. Several non-synonymous substitutions were observed in the antigenic epitopes of VP7 (positions 94 and 147) and VP8* (positions 89, 116, 146 and 150), which may modulate the elicited immune responses. INTERPRETATION: This study contributes to the epidemiological surveillance of rotavirus A required before the introduction of rotavirus vaccination in the EPI for Gabonese children.

Predominance of HBV Genotype B and HDV Genotype 1 in Vietnamese Patients with Chronic Hepatitis.

Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n = 115), liver cirrhosis (LC; n = 21), and hepatocellular carcinoma (HCC; n = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the "a" determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (n = 204) and 7% (n = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; n = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; n = 134), followed by HBV-C (31%; n = 64), HBV-D, and HBV-G (3%; n = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; n = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (n = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections.

Genomic surveillance of SARS-CoV-2 in the Republic of Congo.

OBJECTIVE: The aim of this study was to carry out whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using samples collected from Congolese individuals between April and July 2020. METHODS: Ninety-six samples were screened for SARS-CoV-2 using RT-PCR, and 19 samples with Ct values <30 were sequenced using Illumina Next-Generation Sequencing (NGS). The genomes were annotated and screened for mutations using the web tool 'coronapp'. Subsequently, different SARS-CoV-2 lineages were assigned using PANGOLIN and Nextclade. RESULTS: Eleven SARS-CoV-2 genomes were successfully sequenced and submitted to the GSAID database. All genomes carried the spike mutation D614G and were classified as part of the GH clade. The Congolese SARS-CoV-2 sequences were shown to belong to lineage B1 and Nextclade 20A and 20C, which split them into distinct clusters, indicating two separate introductions of the virus into the Republic of Congo. CONCLUSION: This first study provides valuable information on SARS CoV-2 transmission in the central African region, contributing to SARS CoV-2 surveillance on a temporal and spatial scale.

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.

Letea Virus: Comparative Genomics and Phylogenetic Analysis of a Novel Reassortant Orbivirus Discovered in Grass Snakes (Natrix natrix).

The discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. Arbovirus surveillance is not conducted regularly in Romania, being particularly very scarce in the remote and diverse areas like the Danube Delta. Here we describe the detection and genetic characterization of a novel orbivirus (Reoviridae: Orbivirus) designated as Letea virus, which was found in grass snakes (Natrix natrix) during a metagenomic and metatranscriptomic survey conducted between 2014 and 2017. This virus is the first orbivirus discovered in reptiles. Phylogenetic analyses placed Letea virus as a highly divergent species in the Culicoides-/sand fly-borne orbivirus clade. Gene reassortment and intragenic recombination were detected in the majority of the nine Letea virus strains obtained, implying that these mechanisms play important roles in the evolution and diversification of the virus. However, the screening of arthropods, including Culicoides biting midges collected within the same surveillance program, tested negative for Letea virus infection and could not confirm the arthropod vector of the virus. The study provided complete genome sequences for nine Letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. The phylogenetic associations warrant further screening of arthropods, as well as sustained surveillance efforts for elucidation of Letea virus natural cycle and possible implications for animal and human health.

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.

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.

Circulation of Dirofilaria immitis and Dirofilaria repens in the Danube Delta Biosphere Reserve, Romania.

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.

Detection of Dirofilaria repens and Dirofilaria immitis DNA in mosquitoes from Belarus.

During the last two decades, Belarus faces an increase of human cases of Dirofilaria (Nematoda, Spirurida, Onchocercidae) infections. However, comprehensive analyses explaining this development and the identification of mosquito vector species are missing. Here, we present results using temperature data from Belarus and show that the annual number of human Dirofilaria cases is significantly correlated with the yearly average temperatures (Spearman's rho = 0.49, p < 0.05) and the average sum of potential Dirofilaria transmission days (Spearman's rho = 0.46, p < 0.05), suggesting that autochthonous transmission is at least in part responsible for the increasing number of clinical Dirofilaria cases in the country. In addition, 467 female mosquitoes were collected from different sampling sites in the regions of Brest and Minsk, which were analyzed by molecular methods for the presence of Dirofilaria repens and Dirofilaria immitis DNA, respectively. Two pools (5.56 %) were tested positive for Dirofilaria (estimated infection rate per 100 specimens = 0.44, 95 % confidence interval = 0.08-1.43), comprising one Anopheles claviger s.l. pool that was positive for D. repens and one Culex pipiens s.l./Culex torrentium pool positive for D. immitis DNA. This, to our knowledge, is the first molecular evidence for the presence of Dirofilaria in mosquitoes from Belarus, suggesting a high probability of autochthonous Dirofilaria transmission in the country.

Pilot longitudinal mosquito surveillance study in the Danube Delta Biosphere Reserve and the first reports of Anopheles algeriensis Theobald, 1903 and Aedes hungaricus Mihályi, 1955 for Romania.

BACKGROUND: Mosquito-borne viruses (moboviruses) are of growing importance in many countries of Europe. In Romania and especially in the Danube Delta Biosphere Reserve (DDBR), mosquito and mobovirus surveillance are not performed on a regular basis. However, this type of study is crucially needed to evaluate the risk of pathogen transmission, to understand the ecology of emerging moboviruses, or to plan vector control programmes. METHODS: We initiated a longitudinal mosquito surveillance study with carbon dioxide-baited Heavy Duty Encephalitis Vector Survey traps at four sampling sites to analyse the spatio-temporal pattern of the (i) mosquito species composition and diversity, (ii) functional groups of mosquitoes (oviposition sites, overwintering stage, and number of generations), and (iii) the occurrence of potential West Nile virus (WNV) vectors. RESULTS: During 2014, a total of 240,546 female mosquitoes were collected. All species were identified using morphological characteristics and further confirmed by mitochondrial cytochrome c oxidase subunit I (COI) gene analysis of selected specimens. The two most common taxa were Coquilettidia richiardii (40.9 %) and Anopheles hyrcanus (34.1 %), followed by Culex pipiens (sensu lato) (s.l.)/Cx. torrentium (7.7 %), Aedes caspius (5.7 %), Cx. modestus (4.0 %), An. maculipennis (s.l.) (3.9 %), and Ae. vexans (3.0 %). A further seven species were less common in the area studied, including two new records for Romania: An. algeriensis and Ae. hungaricus. Phylogenetic analysis of COI gene demonstrated the evolutionary relatedness of most species with specimens of the same species collected in other European regions, except Ae. detritus and An. algeriensis, which exhibited high genetic diversity. Due to the dominance of Cq. richiardii and An. hyrcanus (75 % of all collected specimens), the overall phenology and temporal pattern of functional groups basically followed the phenology of both species. A huge proportion of the mosquito population in the course of the entire sampling period can be classified as potential WNV vectors. With 40 % of all collected specimens, the most frequent species Cq. richiardii is probably the most important vector of WNV in the DDBR. CONCLUSION: This is the first DNA-barcoding supported analysis of the mosquito fauna in the DDBR. The detection of two new species highlights the lack of knowledge about the mosquito fauna in Romania and in the DDBR in particular. The results provide detailed insights into the spatial-temporal mosquito species composition, which might lead to a better understanding of mobovirus activity in Romania and thus, can be used for the development of vector control programs.