The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.

Mosquito biosurveillance on Kyushu Island, Japan, with emphasis on Anopheles Hyrcanus Group and related species (Diptera: culicidae).

This report includes the distribution records of the Anopheles (Anopheles) Hyrcanus Group and associated species in Kyushu Island, Japan, based on our field collections from various localities of 4 prefectures (Fukuoka, Kumamoto, Nagasaki, Saga), primarily from 2002-2013. The status of common and potential mosquito vectors, particularly Anopheles species, in Japan are noted.

Plasmodium-specific molecular assays produce uninterpretable results and non-Plasmodium spp. sequences in field-collected Anopheles vectors.

The Malaria Research and Reference Reagent Resource-recommended PLF/UNR/VIR polymerase chain reaction (PCR) was used to detect Plasmodium vivax in Anopheles spp. mosquitoes collected in South Korea. Samples that were amplified were sequenced and compared with known Plasmodium spp. by using the PlasmoDB.org Basic Local Alignment Search Tool/n and the National Center for Biotechnology Information Basic Local Alignment Search Tool/n tools. Results show that the primers PLF/UNR/VIR used in this PCR can produce uninterpretable results and non-specific sequences in field-collected mosquitoes. Three additional PCRs (PLU/VIV, specific for 18S small subunit ribosomal DNA; Pvr47, specific for a nuclear repeat; and GDCW/PLAS, specific for the mitochondrial marker, cytB) were then used to find a more accurate and interpretable assay. Samples that were amplified were again sequenced. The PLU/VIV and Pvr47 assays showed cross-reactivity with non-Plasmodium spp. and an arthropod fungus (Zoophthora lanceolata). The GDCW/PLAS assay amplified only Plasmodium spp. but also amplified the non-human specific parasite P. berghei from an Anopheles belenrae mosquito. Detection of P. berghei in South Korea is a new finding.

Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

SandflyMap: leveraging spatial data on sand fly vector distribution for disease risk assessments.

We feature SandflyMap (www.sandflymap.org), a new map service within VectorMap (www.vectormap.org) that allows free public online access to global sand fly, tick and mosquito collection records and habitat suitability models. Given the short home range of sand flies, combining remote sensing and collection point data give a powerful insight into the environmental determinants of sand fly distribution. SandflyMap is aimed at medical entomologists, vector disease control workers, public health officials and health planners. Data are checked for geographical and taxonomic errors, and are comprised of vouchered specimen information, and both published and unpublished observation data. SandflyMap uses Microsoft Silverlight and ESRI's ArcGIS Server 10 software platform to present disease vector data and relevant remote sensing layers in an online geographical information system format. Users can view the locations of past vector collections and the results of models that predict the geographic extent of individual species. Collection records are searchable and downloadable, and Excel collection forms with drop down lists, and Excel charts to country, are available for data contributors to map and quality control their data. SandflyMap makes accessible, and adds value to, the results of past sand fly collecting efforts. We detail the workflow for entering occurrence data from the literature to SandflyMap, using an example for sand flies from South America. We discuss the utility of SandflyMap as a focal point to increase collaboration and to explore the nexus between geography and vector-borne disease transmission.