Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey

Background: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. Results: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s. l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s. l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as Ochlerotatus caspius flavivirus Turkey, was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. Conclusions: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.U.S. Armed Forces Health Surveillance Board Global Emerging Infections Surveillance and Response System (AFHSB-GEIS) research; Walter Reed Army Institute of Research; Smithsonian InstitutionSmithsonian Institution; Georg Forster Research Fellowship (HERMES); Alexander von Humboldt Foundation, GermanyAlexander von Humboldt FoundationA U.S. Armed Forces Health Surveillance Board Global Emerging Infections Surveillance and Response System (AFHSB-GEIS) research award (to YML) supported this study. This research was performed in part under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organisations. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The material to be published reflects the views of the authors and should not be construed to represent those of the United States Department of the Army or the United States Department of Defense. KE was a 2015 recipient of the Georg Forster Research Fellowship (HERMES) for Experienced Researchers, of the Alexander von Humboldt Foundation, Germany

Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses frommosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed amonophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored. (C) 2016 Elsevier B.V. All rights reserved.Armed Forces Health Surveillance Center, Global Emerging Infections Surveillance and Response System (AFHSC-GEIS), United States [W81XWH-11-2-0174]; Georg Forster Research Fellowship (HERMES) for Experienced Researchers by Alexander von Humboldt Foundation; National Research Council (NRC) Research Associateship Award at the Walter Reed Army Institute of ResearchThis study was partially supported by The Armed Forces Health Surveillance Center, Global Emerging Infections Surveillance and Response System (AFHSC-GEIS), United States (W81XWH-11-2-0174) (with Yvonne-Marie Linton as the principal investigator). KE is a recipient of the Georg Forster Research Fellowship (HERMES) for Experienced Researchers by the Alexander von Humboldt Foundation, 2015. This manuscript was prepared whilst YML held a National Research Council (NRC) Research Associateship Award at the Walter Reed Army Institute of Research. This research was performed in part under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The material to be published reflects the views of the authors and should not be construed to represent those of the US Department of the Army or the US Department of Defense

Variante morfológica de adultos hembras de Anopheles benarrochi (Diptera: Culicidae) en Putumayo, Colombia

A survey of Anopheles mosquitoes was undertaken in Putumayo, southern Colombia, to clarify the identity of specimens classified as Anopheles (N) evansae in the region. Female anophelines were captured on human bait; then blood fed on small mammals to obtain eggs for progeny broods. Morphological characteristics of the eggs. larvae, pupae and adults of both sexes, including male genitalia were examined. Eggs were obtained from 247 females provisionally identified as A. (N.) evansae Morphology of associated life stages of the progeny broods was compared among 27 families. The morphology of al1 stages corresponded to the published descriptions of Anopheles (N.) benarrochi. However, morphologically variable adult females were noted. In hind tarsomere 2, the dark portion ranged from 0.17 to 0.33 of the tarsomere length; this is less than previously reported for A. (N) benarrochi and overlaps the ranges described for A. (N,) oswaldoi and A. (N) evansae This variable character probably has led to incorrect identifications of adult A. (N.) benarrochifemales as A. (N.) evansae or A. (N.) oswaldoi in specimens from southern Colombia. As a consequence, A. (N) benarrochi has not been previously recognized in Putumayo, and probably has been misidentified in other areas of northwestern South America.Con el propósito de ampliar el conocimiento sobre las especies de Anopheles presentes en el Putumayo, sur de Colombia, y para esclarecer la identidad de los ejemplares clasificados como Anopheles (Nyssorhynchus) evansae en esta región, se recolectaron mosquitos hembra en cebo humano, se alimentaron en pequeños mamíferos y se mantuvieron vivos para la cría de isofamilias. Se realizaron observaciones de las características morfológicas de los huevos, larvas, pupas y adultos de ambos sexos, incluidas las genitalias masculinas. Se obtuvieron 247 posturas de madres identificadas preliminarmente como A. (N) evansae A 27 de estas familias se les estudió la morfología de los estadios asociados. Todos los especimenes fueron subsecuentemente identificados como Anopheles (N) benarrochi por la morfología de los huevos, larvas, pupas y genitalias masculinas, lo cual coincidió con las descripciones publicadas para esta especie. Sin embargo, los adultos hembra de Putumayo presentaron la proporción oscura en el tarsómero posterior 2, entre 0.17 y 0.33 de su longitud, inferior a lo informado para esta especie, lo cual se superpone con los rangos de Anopheles (N.) oswaldoi y A. (N.) evansae. Como resultado de la superposición en este carácter, es probable que hembras adultas de A. (N.) benarrochi hayan sido incorrectamente identificadas como A. (N) evansae y A. (N.) oswaldoi en el sur de Colombia. La presencia de esta variante morfológica ha dificultado la identificación de A. (N.) benarrochi en Putumayo y, probablemente, en otras regiones de Colombia y paises vecinos

Utilidad de la morfología de los huevos como un método indirecto para identificar Anopheles benarrochi Gabaldón,Cova García & López, Anopheles oswaldoi (Peryassu) y Anopheles rangeli Gabaldón, Cova García & López, (Diptera:Culicidae) en Putumayo, Colombia.

In the Department of Putumayo in southern Colombia, malaria transmission has continued in the absence of the 4 traditional Latin American vector species--Anopheles darlingi, Anopheles nuneztovari, Anopheles albimanus or Anopheles trinkae. Human bait collections yielded Anopheles mosquitoes and a morphological variant of Anopheles benarrochi, the adult females of which can easily be misidentified as Anopheles oswaldoi. Species identification of females of Anopheles in the subgenus Nyssorhynchus is generally difficult due to overlapping morphological characters; therefore, progeny of field collected females were link-reared to assess species identity. Herein a robust method is presented to identify the species Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli from southern Colombia, using the morphology of the eggs induced from wild-caught females. Eggs of A. rangeli and A. benarrochi were differentiated on the basis of the anterior crown. In A. rangeli, this feature is positioned apically with high walls. In A. benarrochi, anterior crown is positioned more ventrally with comparatively shorter walls. No crown is present in A. oswaldoi. These differences are clear with the aid of a dissecting microscope and make accurate species determination possible even in field conditions. Egg morphology is shown to be an accurate, albeit indirect, method for the taxonomic determination for the three southern Colombian species and may also be useful in other regions of Latin America where the morphological variant of A. benarrochi is sympatric with A. oswaldoi.La identificación correcta de las hembras es esencial para el éxito de cualquier estudio de epidemiología, resistencia a insecticidas o de control de vectores. En el departamento del Putumayo, en el sur de Colombia, la transmisión de malaria continúa siendo un problema, a pesar de la ausencia de los vectores principales de Latinoamérica (Anopheles darlingi Root, Anopheles nuneztovari Gabaldón, Anopheles albimanus Wideman, Anopheles trinkae Faran) en esta región. Se recolectaron. con cebo humano, hembras de Anopheles y se encontró una variante morfológica de Anopheles benarrochi, que en su estadio adulto fácilmente se confunde con Anopheles oswaldoi. La identificación de hembras de Anopheles, particularmente del subgénero Nyssorhynchus, es en general notoriamente difícil debido a la superposición de caracteres morfológicos en el estadio adulto; por tanto, las colecciones deben estar ligadas a la cría de material asociado para identificar correctamente las especies. Esto requiere tiempo y es difícil de obtener en muchas ocasiones. Se presenta un método indirecto de identificación de las especies A. benarrochi, A. oswaldoi y Anopheles rangeli del sur de Colombia usando la morfología de los huevos de hembras silvestres. Los huevos de A. rangeli y A. benarrochi se diferencian por la corona anterior, la cual es apical en A. rangeli y con paredes altas, mientras que en A. benarrochi es ventral y con paredes más cortas. Esta corona está ausente en A. oswaldoi. Estas diferencias fueron obvias incluso bajo un microscopio de luz, lo que hace posible una identificación correcta de estas especies en condiciones de campo. Se muestra cómo la observación de la morfología de los huevos puede permitir la determinación taxonómica correcta, aunque indirecta, de estas tres especies de Nyssorhynchus encontradas en el sur de Colombia, el cual puede ser útil también en otras regiones de Latinoamérica, en donde se encuentre la variante morfológica de A. benarrochi en simpatría con A. oswaldoi

Diversity, composition, altitude, and seasonality of high-altitude windborne migrating mosquitoes in the Sahel: Implications for disease transmission

Recent studies have reported Anopheles mosquitoes captured at high-altitude (40–290 m above ground) in the Sahel. Here, we describe this migration modality across genera and species of African Culicidae and examine its implications for disease transmission and control. As well as Anopheles, six other genera—Culex, Aedes, Mansonia, Mimomyia, Lutzia, and Eretmapodites comprised 90% of the 2,340 mosquitoes captured at altitude. Of the 50 molecularly confirmed species (N = 2,107), 33 species represented by multiple specimens were conservatively considered high-altitude windborne migrants, suggesting it is a common migration modality in mosquitoes (31–47% of the known species in Mali), and especially in Culex (45−59%). Overall species abundance varied between 2 and 710 specimens/species (in Ae. vittatus and Cx. perexiguus, respectively). At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one blood meal on a vertebrate host prior to their departure. Most taxa were more common at higher sampling altitudes, indicating that total abundance and diversity are underestimated. High-altitude flight activity was concentrated between June and November coinciding with availability of surface waters and peak disease transmission by mosquitoes. These hallmarks of windborne mosquito migration bolster their role as carriers of mosquito-borne pathogens (MBPs). Screening 921 mosquitoes using pan-Plasmodium assays revealed that thoracic infection rate in these high-altitude migrants was 2.4%, providing a proof of concept that vertebrate pathogens are transported by windborne mosquitoes at altitude. Fourteen of the 33 windborne mosquito species had been reported as vectors to 25 MBPs in West Africa, which represent 32% of the MBPs known in that region and include those that inflict the heaviest burden on human and animal health, such as malaria, yellow fever, dengue, and Rift Valley fever. We highlight five arboviruses that are most likely affected by windborne mosquitoes in West Africa: Rift Valley fever, O'nyong'nyong, Ngari, Pangola, and Ndumu. We conclude that the study of windborne spread of diseases by migrating insects and the development of surveillance to map the sources, routes, and destinations of vectors and pathogens is key to understand, predict, and mitigate existing and new threats of public health

Phylogenomics reveals the history of host use in mosquitoes

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188–250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes. © 2023, Springer Nature Limited

Nowa Konstytucja Państwa Litewskiego

Digitalizacja i deponowanie archiwalnych zeszytów RPEiS sfinansowane przez MNiSW w ramach realizacji umowy nr 541/P-DUN/201

Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex.\ud \ud \ud \ud Methods\ud DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results\ud Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.This study formed part of the PhD study of FRL conducted at the Natural History Museum, London, and awarded from Canterbury Christ Church University, Canterbury, Kent, U.K. This investigation received financial support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) (grant A50252 to YML), Canterbury Christ Church University (studentship to FRL). Additional funding was obtained through the Friends of the Natural History Museum, London to further the activities of the Mosquito Barcoding Initiative (to YML); the Consortium for the Barcode of Life (CBOL) (to YML and RCW) and the Sloane Foundation (to YML and RCW); the National Institute of Health (NIH), USA (grant 2R01AI054139 to Jan E. Conn) and COLCIENCIAS (grant 110134319196 to MLQ). We thank Dr. A. Papadopoulou for help with the MYC analysis and Dr. S. Mahamdallie for helpful suggestions and discussions during preparation of the manuscript.This manuscript was prepared in part whilst YML held a National Research Council Senior Research Associateship Award at the Walter Reed Army Institute of Research. This research was performed in part under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations. The material to be published reflects the views of the authors and should not be construed to represent those of the Department of the Army or the Department of Defense

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey

Background: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. Results: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as “Ochlerotatus caspius flavivirus Turkey”, was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. Conclusions: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected