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

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

IDENTIFICATION OF MOSQUITO SPECIES (DIPTERA: CULICIDAE) AROUND REFUGEE ZONES OF THE TURKISH BORDER WITH SYRIA AND ON MIGRATION ROUTES OF REFUGEES

The internal unrest in Syria forced 4,000,000 Syrian citizens to immigrate to neighbouring countries. This has increased the importance of monitoring the mosquitoes and mosquito-borne diseases in the region. In this context, mosquito samples were collected during the mosquito seasons on Southeast Anatolia where immigrants are intensively settled and western, northwestern borders, which are the pathway of immigration to Europe. During the study, samples were taken from 506 stations on 17 different cities. After all the collected mosquitoes were morphologically identified, some of them were kept for the molecular species identification through the sequence of the mitochondrial COI gene region, whereas all the remaining samples were sent to the virus analysis. As a result of the morphological identification, 30,940 collected mosquitoes were found to contain 6 genera and 22 specimens, and the identification of selected specimens and especially individuals belonging to species complexes was confirmed by molecular methods. The presence of the species Aedes albopictus, Ae. aegypti, Anopheles sacharovi and Culex pipiens s.l. in the study area, which are known to be vectors, has become even more important with the advent of the immigrants. This thesis is the prelude to a surveillance exercise for the region concerned.İÇİNDEKİLER Sayfa ÖZET i ABSTRACT iii TEŞEKKÜR v İÇİNDEKİLER vi ÇİZELGELER DİZİNİ viii ŞEKİLLER DİZİNİ ix SİMGELER VE KISALTMALAR x 1. GİRİŞ 1 2. GENEL BİLGİ 4 2.1. Suriyeli Mülteciler ve Türkiye’nin Vektöriyel Açıdan Etkileşimi 4 2.2. Türkiye’deki Vektör Türler, Taşıdıkları Patojen ve Virüsler 5 2.3. DNA Barkodlama ve Surveyans Çalışmalarındaki Yeri 8 3. YÖNTEM 10 3.1. Arazi Çalışmaları ve Örneklem Oluşturulması 10 3.2. Moleküler Analizler ve Filogenetik Verinin Elde Edilmesi 12 3.2.1 DNA İzolasyonu ve COI Gen Bölgesinin Amplifikasyonu 12 3.2.2. Sekansların Yorumlanması ve Filogenetik Verinin Elde Edilmesi 14 4. BULGULAR 16 4.1. Anophelinae Alt Ailesine İlişkin Bulgular 16 4.2. Culicinae Alt Ailesine İlişkin Bulgular 19 4.2.1. Aedes Cinsine İlişkin Bulgular 19 4.2.2. Culex Cinsine İlişkin Bulgular 22 4.2.3. Coquillettidia, Culiseta, Uranotaenia Cinslerine İlişkin Bulgular 24 5. TARTIŞMA 27 KAYNAKLAR 35 EKLER 45 ÖZGEÇMİŞ 47Suriye’deki iç huzursuzluklar 4.000.000 Suriye vatandaşını komşu ülkelere göçe zorlamıştır. Bu durum, bölgedeki sivrisinekler ve sivrisinek kaynaklı hastalıkların sürveyansının önemini arttırmıştır. Bu çerçevede, Türkiye’de göçmenlerin yoğun olarak yerleştikleri veya geçici olarak konakladıkları Güneydoğu Anadolu Bölgesi ve Avrupa’ya göç etmek için ilk durakları olan batı ve kuzeybatı sınırlarında, sivrisinek üreme sezonları boyunca arazi çalışmaları yapılmış ve örnekler toplanmıştır. Çalışmalar sırasında 17 ayrı ilde 506 istasyondan ergin ve larva örneklemesi yapılmıştır. Toplanan sivrisineklerin tamamı morfolojik olarak teşhis edilmiştir. Örneklerin bir kısmı, mitokondriyal COI gen bölgesinin sekansı yoluyla moleküler tür teşhisi için ayrılıp, kalan örneklerin tamamı tespit edilen sivrisinek türlerin taşıdıkları virüslerin belirlenmesi amacıyla analize gönderilmiştir. Morfolojik teşhisler sonucunda, toplanan 30.940 adet sivrisineğin 6 cins ve 22 türün örneklerini içerdiği ortaya konmuş, seçilen örneklerin ve özellikle tür komplekslerine ait bireylerin teşhisleri moleküler yöntemlerle doğrulanmıştır. Bu türlerden birincil vektör olduğu bilinen Aedes albopictus, Ae. aegypti, Anopheles sacharovi ve Culex pipiens s.l. türlerinin çalışma alanındaki varlığı, Suriyeli göçmenlerin de bölgeye gelişiyle daha da önem kazanmıştır. Bu tez, söz konusu bölgeler için yapılacak geniş kapsamlı bir sürveyans çalışmasının önayağıdır

West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n = 3), Cx. pipiens s. l. (n = 1) and Aedes caspius (n = 1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s. l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate.Armed Forces Health Surveillance Center, Global Emerging Infections Surveillance and Response System (AFHSC-GEIS), United States [W911QY-16-C-0160-P00001]; Georg Forster Research Fellowship (HERMES) for Experienced Researchers, of the 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) (W911QY-16-C-0160-P00001), United States (with Yvonne Marie-Linton as the principal investigator). KE is a recipient of the Georg Forster Research Fellowship (HERMES) for Experienced Researchers, of 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