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.
Faculty of Medicine, Department of Medical Microbiology, Virology Unit, Hacettepe University, Ankara, Turkey. ekoray@hacettepe.edu.tr.|Robert Koch Institute, Center for Biological Threats and Special Pathogens 1 (ZBS-1), Berlin, Germany. ekoray@hacettepe.edu.tr.
Koray
K
Ergünay
Robert Koch Institute, Center for Biological Threats and Special Pathogens 1 (ZBS-1), Berlin, Germany.
Nadine
N
Litzba
Robert Koch Institute, Center for Biological Threats and Special Pathogens 1 (ZBS-1), Berlin, Germany.
Annika
A
Brinkmann
Faculty of Sciences, Department of Biology, Division of Ecology, Hacettepe University, Ankara, Turkey.
Filiz
F
Günay
Faculty of Sciences, Department of Biology, Division of Ecology, Hacettepe University, Ankara, Turkey.
Yasemen
Y
Sarıkaya
Faculty of Arts and Sciences, Department of Biology, Namık Kemal University, Tekirdağ, Turkey.
Sırrı
S
Kar
Faculty of Medicine, Department of Medical Microbiology, Virology Unit, Hacettepe University, Ankara, Turkey.
Serra
S
Örsten
Faculty of Veterinary Medicine, Department of Parasitology, Istanbul University, Istanbul, Turkey.
Kerem
K
Öter
Robert Koch Institute, Center for Biological Threats and Special Pathogens 1 (ZBS-1), Berlin, Germany.
Cristina
C
Domingo
Faculty of Sciences, Department of Biology, Division of Ecology, Hacettepe University, Ankara, Turkey.
Özge
Ö
Erisoz Kasap
Department of Virology, Faculty of Veterinary Medicine, Ankara, Turkey.
Aykut
A
Özkul
Walter Reed Biosystematics Unit, Museum Support Center MRC-534, Smithsonian Institution, Maryland, USA.
Luke
L
Mitchell
Robert Koch Institute, Center for Biological Threats and Special Pathogens 1 (ZBS-1), Berlin, Germany.
Andreas
A
Nitsche
Faculty of Sciences, Department of Biology, Division of Ecology, Hacettepe University, Ankara, Turkey.
Bülent
B
Alten
Walter Reed Biosystematics Unit, Museum Support Center MRC-534, Smithsonian Institution, Maryland, USA.|Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.|Department of Entomology, National Museum of Natural History, Smithsonian Institution, Washington DC, USA.
Yvonne-Marie
YM
Linton
D000330:Aedes / Q000145:classification / Q000821:virology*; D000818:Animals; D003465:Culex / Q000145:classification / Q000821:virology*; D005416:Flavivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018177:Flavivirus Infections / Q000453:epidemiology / Q000635:transmission / Q000821:virology*; D014644:Genetic Variation; D016679:Genome, Viral; D006801:Humans; D007303:Insect Vectors / Q000145:classification / Q000821:virology*; D010802:Phylogeny; D013045:Species Specificity; D014421:Turkey / Q000453:epidemiology; D014901:West Nile Fever / Q000453:epidemiology / Q000635:transmission / Q000821:virology*; D014902:West Nile virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology
Biosurveillance; Flavivirus; Insect-specific; Mosquito; Turkey; West Nile virus