Novel Tick Phlebovirus Genotypes Lacking Evidence for Vertebrate Infections in Anatolia and Thrace, Turkey

We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kirklareli, and Tekirdag (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Sanliurfa (Southeastern Anatolia) provinces from 2013-2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequences in three pools. A comprehensive analysis revealed three distinct, monophyletic virus genotypes, comprised of previously-described viruses from Anatolia and the Balkans, with unique fingerprints in conserved amino acid motifs in viral replicase. A novel tick phlebovirus group was discovered circulating in the Balkans and Turkey, with at least three genotypes or species. No evidence for replication in vertebrates or infections in clinical cases could be demonstrated.Armed Forces Health Surveillance Board, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), United States of America [P0034_18_WR]; US ArmyUnited States Department of Defense [W911QY-16-C-0160]The study was supported in part by the Armed Forces Health Surveillance Board, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), United States of America (FY18 award P0034_18_WR (PI: Yvonne-Marie Linton) under US Army subcontract W911QY-16-C-0160)

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

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

Intersection problem for Droms RAAGs

We solve the subgroup intersection problem (SIP) for any RAAG G of Droms type (i.e., with defining graph not containing induced squares or paths of length 3): there is an algorithm which, given finite sets of generators for two subgroups H,K of G, decides whether $H \cap K$ is finitely generated or not, and, in the affirmative case, it computes a set of generators for $H \cap K$. Taking advantage of the recursive characterization of Droms groups, the proof consists in separately showing that the solvability of SIP passes through free products, and through direct products with free-abelian groups. We note that most of RAAGs are not Howson, and many (e.g. F_2 x F_2) even have unsolvable SIP.Comment: 33 pages, 12 figures (revised following the referee's suggestions

Utility of a Sequence-Independent, Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens

Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.Peer Reviewe

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

History and classification of Aigai virus (formerly Crimean-Congo haemorrhagic fever virus genotype VI)

Crimean-Congo haemorrhagic fever virus (CCHFV) is the medically most important member of the rapidly expanding bunyaviral family Nairoviridae. Traditionally, CCHFV isolates have been assigned to six distinct genotypes. Here, the International Committee on Taxonomy of Viruses (ICTV) Nairoviridae Study Group outlines the reasons for the recent decision to re-classify genogroup VI (aka Europe-2 or AP-92-like) as a distinct virus, Aigai virus (AIGV)

ICTV Virus Taxonomy Profile: Nairoviridae.

Members of the family Nairoviridae produce enveloped virions with three single-stranded RNA segments comprising 17.1 to 22.8 kb in total. These viruses are maintained in arthropods and transmitted by ticks to mammals or birds. Crimean-Congo hemorrhagic fever virus is tick-borne and is endemic in most of Asia, Africa, Southern and Eastern Europe whereas Nairobi sheep disease virus, which is also tick-borne, causes lethal haemorrhagic gastroenteritis in small ruminants in Africa and India. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nairoviridae, which is available at ictv.global/report/nairoviridae

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

Correction to: 2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales. Archives of Virology (2021) 166:3567–3579. https://doi.org/10.1007/s00705-021-05266-wIn March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.This work was supported in part through Laulima Government Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC under Contract No. HHSN272201800013C. This work was also supported in part with federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Contract No. 75N91019D00024, Task Order No. 75N91019F00130 to I.C., who was supported by the Clinical Monitoring Research Program Directorate, Frederick National Lab for Cancer Research. This work was also funded in part by Contract No. HSHQDC-15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges partial support from the Special Research Initiative of Mississippi Agricultural and Forestry Experiment Station (MAFES), Mississippi State University, and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project 1021494. Part of this work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001030), the UK Medical Research Council (FC001030), and the Wellcome Trust (FC001030).S