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)

İstanbul İli ve Çevresinde Toplanan Kenelerde, Nairo, Flebo ve Flavivirusların Araştırılması

Emanet, N., Investigation of Nairo, Phlebo and Flavivirus in Ticks Collected around Istanbul Province Hacettepe University Graduate School Health Scıences, MSc, thesis in Microbiology, Ankara, 2020. In this study, we aimed to screen tick-borne viruses, to determine the epidemiology of tick-borne diseases and to identify tick species in samples collected from around Istanbul province. Among arboviruses under the order Bunyavirales, Nairoviridae, Peribunyaviridae families and Flaviviridae family there are Nairovirus, Phlebovirus and Flavivirus genus which include tick-borne viruses with significant global health impact, such as Crimean- Congo hemorrhagic fever virus (CCHFV), severe fever with thrombocytopenia syndrome virus and tick-borne encephalitis virus. Arthropod surveillance provides information on regional vector-borne or non-pathogenic virus circulation, for the understanding virus epidemiology and risk assessment. In this study, we carried out nairovirus, phlebovirus and flavivirus screening in ticks via generic PCR, around Istanbul province which has a significant part of the urban population of Turkey. The detected strains were characterized via sequencing. A total of 7154 ticks were collected by flagging and animal sampling from 37 localities in Istanbul, Kırklareli, Edirne and Tekirdağ provinces, during 2013, 2014, 2015 and 2018 years. Morphological identification revealed Ixodes sp. (30.92%) to be the most abundant species, followed by Hyalomma marginatum (14.29%), Hyalomma scupense (13.45%), Haemaphysalis parva (9.40%), Rhipicephalus turanicus (8.17%) and others. The individuals were pooled in a total of 572 specimens, according to species, sex and collection sites. Nairovirus screening provided positive results in 0.7% (4/572) and phlebovirus screening provided positive results in 3.3% (19/572) of the pools. However, flavivirus RNA could not be detected in any tick pools. CCHFV and CCHFV-AP92 isolates were detected in the region. In addition, we obtained data on new tick-borne phleboviruses previously reported from Turkey. This is the first report from the region of tick-borne phleboviruses. Key words: Tick, Nairovirus, Phlebovirus, Flavivirus, PCREmanet, N., İstanbul İli ve Çevresinde Toplanan Kenelerde Nairo, Flebo ve Flavivirusların Araştırılması, Hacettepe Üniversitesi Sağlık Bilimleri Enstitüsü Mikrobiyoloji Programı Yüksek Lisans Tezi, Ankara, 2020. Bu çalışmada, İstanbul ili ve çevresinden toplanan örneklerde kene kaynaklı virüslerin taranması, kene kaynaklı hastalıkların epidemiyolojisinin belirlenmesi ve kene türlerinin tespit edilmesi amaçlanmıştır. Arbovirüsler içerisinde Bunyavirales takımında Nairoviridae, Peribunyaviridae ailelerinde ve Flaviridae ailesinde yer alan Nairovirüs, Flebovirüs ve Flavivirüs cinsleri, kenelerle bulaşma gösteren, aralarında Kırım-Kongo Kanamalı Ateşi (KKKAV), trombositopeni sendromlu yüksek ateş ve kene kaynaklı ensefalit virüsü gibi, tüm dünyada önemli sağlık sorunları oluşturmuş virüsleri içermektedir. Belirli bir bölgede vektör kaynaklı ya da patojen olmayan virüs aktivitesinin bilinmesi, hastalık riskinin değerlendirilebilmesi ve epidemiyolojik özelliklerin incelenebilmesi için, eklembacaklıların sürveyansı önem taşımaktadır. Bu çalışmada, ülkemizde nüfus yoğunluğu en fazla İstanbul ili ve çevresinden toplanan kene örneklerinde jenerik PZR yöntemleri ile nairo-, flebo- ve flavivirüsler araştırılmış, saptanan virüsler DNA dizi analizi ile tanımlanmış ve bulgular tartışılmıştır. Çalışma kapsamında 2013, 2014, 2015 ve 2018 yıllarında İstanbul, Kırklareli, Edirne ve Tekirdağ illerinden 37 lokasyondan bayraklama ve hayvan örneklemesi ile 7154 kene toplanmıştır. Morfolojik incelemeler, örnekler arasında en sık saptanan türün Ixodes sp. (%30,92) olduğunu göstermiş, bunu Hyalomma marginatum (%14,29), Hyalomma scupense (%13,45), Haemaphysalis parva (%9,40), Rhipicephalus turanicus (%8,17) ve diğerleri takip etmiştir. Toplanan örnekler; lokasyon, cinsiyet ve türe göre toplam 572 havuz olarak virüs taramasına alınmış, %0,7’sinde (4/572) nairovirüs ve %3,3’ünde (19/572) flebovirüs pozitifliği saptanmıştır. Ancak havuzlarda flavivirüs RNA’sı tespit edilememiştir. Bölgede KKKAV ve KKKAV-AP92 izolatı saptanmıştır. Bunun yanı sıra ülkemizden daha önce bildirilmiş olan yeni kene kaynaklı flebovirüslere dair veriler elde edilmiştir. Bu veriler kene kaynaklı flebovirüsler ile ilgili bölgeden yapılmış olan ilk bildirimdir

Multiple orthonairoviruses including Crimean-Congo hemorrhagic fever virus, Tamdy virus and the novel Meram virus in Anatolia

We conducted orthonairovirus RNA screening of 7043 tick specimens-representing 16 species-collected from various regions of Anatolia. In 602 pools, Crimean-Congo hemorrhagic fever virus (CCHFV) Europe 1 and 2 lineages were detected in seven pools (1.1 %) comprising Hyalomma marginatum, Hyalomma scupense, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks. In pools of Hyalomma aegyptium, we detected Tamdy virus (TAMV) and an unclassified nairovirus sequence. Next-generation se-quencing revealed complete coding regions of three CCHFV Europe 2 (AP92-like) viruses, TAMV and the novel orthonairovirus, tentatively named herein as Meram virus. We further performed in silico functional analysis of all available CCHFV Europe 2, TAMV, Meram and related virus genomes. The CCHFV Europe 2 viruses possessed several conserved motifs, including those with OTU-like cysteine protease activity. Probable recombinations were identified in L genome segments of CCHFV and TAMV. Through phylogeny reconstruction using individual genome segments, Meram virus emerged as a distinct virus among species within the Orthonairovirus genus. It further exhibited conserved motifs associated with RNA binding, encapsidation, signal peptidase cleavage, post -translational modification, RNA-dependent RNA polymerase and OTU-like activities. Bole tick virus 3 was also detected in two pools with CCHFV reactivity. Hereby, we describe a novel tick-associated orthonairovirus, in a CCHFV-endemic region with confirmed TAMV activity. Human and animal health impact of these viruses need to be addressed.Armed Forces Health Surveillance Board, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), United States of America (FY18 award US Army) [P0034_18_WR, W911QY-16-C-0160]; Food and Agriculture Organization of the United Nations (FAO) [GCP/TUR/055/GFF]The study was funded 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). Some of the field collections were performed within the project The inventory and management plan of biological diversity in Konya and Karaman provinces, conducted by Nature Conservation Center Foundation one of the project partners supported by the Food and Agriculture Organization of the United Nations (FAO) (GCP/TUR/055/GFF). Neither funder/supporter had any role in study design, data collection, analysis, decision to publish, or manuscript preparation. The presented material reflects the views of the authors and should not be construed to represent those of the U.S. Department of the Army or the U.S. Department of Defense

Humoral Immune Response to CoronaVac in Turkish Adults

While most approved vaccines are based on the viral spike protein or its immunogenic regions, inactivated whole-virion vaccines (e.g., CoronaVac) contain additional antigens that may enhance protection. This study analyzes short-term humoral responses against the SARS-CoV-2 spike (S1) and nucleocapsid (NCP) protein in 50 Turkish adults without previous SARS-CoV-2 infection after CoronaVac immunization. Samples were collected before vaccination (t0), 28–29 days after the first vaccine dose and prior to the second dose (t1), as well as 14–15 days after the second dose (t2). Anti-S1 IgG and IgA as well as anti-NCP IgG were quantified using ELISA. At t1, seroconversion rates for anti-S1 IgG, anti-S1 IgA and anti-NCP IgG were 30.0%, 28.0% and 4.0%, respectively, increasing significantly to 98.0%, 78.0% and 40.0% at t2. The anti-NCP IgG median (t2) was below the positivity cut-off, while anti-S1 IgG and IgA medians were positive. Anti-S1 IgG levels strongly correlated with anti-S1 IgA (rs = 0.767, p s = 0.683, p < 0.001). In conclusion, two CoronaVac doses induced significant increases in antibodies against S1 and NCP. Despite strong correlations between the antibody concentrations, the median levels and seroconversion rates of S1-specific responses exceed those of NCP-specific responses as early as two weeks after the second vaccine dose

Humoral Immune Response to CoronaVac in Turkish Adults

While most approved vaccines are based on the viral spike protein or its immunogenic regions, inactivated whole-virion vaccines (e.g., CoronaVac) contain additional antigens that may enhance protection. This study analyzes short-term humoral responses against the SARS-CoV-2 spike (S1) and nucleocapsid (NCP) protein in 50 Turkish adults without previous SARS-CoV-2 infection after CoronaVac immunization. Samples were collected before vaccination (t0), 28&ndash;29 days after the first vaccine dose and prior to the second dose (t1), as well as 14&ndash;15 days after the second dose (t2). Anti-S1 IgG and IgA as well as anti-NCP IgG were quantified using ELISA. At t1, seroconversion rates for anti-S1 IgG, anti-S1 IgA and anti-NCP IgG were 30.0%, 28.0% and 4.0%, respectively, increasing significantly to 98.0%, 78.0% and 40.0% at t2. The anti-NCP IgG median (t2) was below the positivity cut-off, while anti-S1 IgG and IgA medians were positive. Anti-S1 IgG levels strongly correlated with anti-S1 IgA (rs = 0.767, p &lt; 0.001) and anti-NCP IgG (rs = 0.683, p &lt; 0.001). In conclusion, two CoronaVac doses induced significant increases in antibodies against S1 and NCP. Despite strong correlations between the antibody concentrations, the median levels and seroconversion rates of S1-specific responses exceed those of NCP-specific responses as early as two weeks after the second vaccine dose

Survey And Characterization Of Jingmen Tick Virus Variants

We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. We subsequently screened 7223 ticks, representing 15 species in five genera, collected from various regions in Anatolia and eastern Thrace, Turkey. Moreover, we tested specimens from various patient cohorts (n = 103), and canine (n = 60), bovine (n = 20) and avian specimens (n = 65). JMTV nucleic acids were detected in 3.9% of the tick pools, including those from several tick species from the genera Rhipicephalus and Haemaphysalis, and Hyalomma marginatum, the main vector of CCHFV in Turkey. Phylogenetic analysis supported two separate clades, independent of host or location, suggesting ubiquitous distribution in ticks. JMTV was not recovered from any human, animal or bird specimens tested. Near-complete viral genomes were sequenced from the prototype isolate and from three infected tick pools. Genome topology and functional organization were identical to the members of Jingmen group viruses. Phylogenetic reconstruction of individual viral genome segments and functional elements further supported the close relationship of the strains from Kosovo. We further identified probable recombination events in the JMTV genome, involving closely-related strains from Anatolia or China.PubMedWoSScopu

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, Kırklareli, and Tekirdağ (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Şanlıurfa (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.Peer Reviewe

Multiple orthonairoviruses including Crimean-Congo hemorrhagic fever virus, Tamdy virus and the novel Meram virus in Anatolia

We conducted orthonairovirus RNA screening of 7043 tick specimens-representing 16 species-collected from various regions of Anatolia. In 602 pools, Crimean-Congo hemorrhagic fever virus (CCHFV) Europe 1 and 2 lineages were detected in seven pools (1.1 %) comprising Hyalomma marginatum, Hyalomma scupense, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks. In pools of Hyalomma aegyptium, we detected Tamdy virus (TAMV) and an unclassified nairovirus sequence. Next-generation se-quencing revealed complete coding regions of three CCHFV Europe 2 (AP92-like) viruses, TAMV and the novel orthonairovirus, tentatively named herein as Meram virus. We further performed in silico functional analysis of all available CCHFV Europe 2, TAMV, Meram and related virus genomes. The CCHFV Europe 2 viruses possessed several conserved motifs, including those with OTU-like cysteine protease activity. Probable recombinations were identified in L genome segments of CCHFV and TAMV. Through phylogeny reconstruction using individual genome segments, Meram virus emerged as a distinct virus among species within the Orthonairovirus genus. It further exhibited conserved motifs associated with RNA binding, encapsidation, signal peptidase cleavage, post -translational modification, RNA-dependent RNA polymerase and OTU-like activities. Bole tick virus 3 was also detected in two pools with CCHFV reactivity. Hereby, we describe a novel tick-associated orthonairovirus, in a CCHFV-endemic region with confirmed TAMV activity. Human and animal health impact of these viruses need to be addressed