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

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 southeastern 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.Peer Reviewe

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

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

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

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