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

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)

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

Konjunktivitli Van kedilerinde felid herpesvirus - 1 enfeksiyonu

Felid herpes virus -1 ( FeHV-1) kedilerde genellikle nasal akıntı, hapşırma, iştahsızlık, yüksek ateş ve konjunktivit ile seyreden üst solunum sistemi infeksiyonuna neden olur. Bu çalışmada Türkiye’de, FeHV-1 ile uyumlu klinik infeksiyon belirtileri gösteren ve yaşları 9 gün ile 1 yıl arasında değişen 20 adet Van kedisinden alınan göz sıvap örnekleri FeHV-1 varlığı yönünden incelendi. Virus tespiti için Polimerize Zincir Reaksiyonu (PZR) yönteminden yararlanıldı. İncelenen kedilerin 9 (45%)’ undan alınan örneklerde FeHV-1 spesifik amplikon (737 bç) tespit edildi. Elde edilen amplikonların sekans analizinde örnekler arasında fark bulunamadı. Sonuç olarak; Van kedisi neslinin korunması için daha etkili tedbirler alınması ile beraber rutin aşılamaların yapılması kanaatine varıldı.Felid herpesvirus 1 (FeHV-1) infecting in felidae can cause severe upper respiratory tract disease with clinical symptoms including nasal discharge, sneezing, inappetence, pyrexia and conjunctivitis. In this study, 20 ocular swab samples were obtained from Van cats, in age from 9 days to 1 year, presented signs of FeHV-1 infection. Polymerase Chain Reaction (PCR) method was used for the detection of virus. FeHV-1 specific amplicon (737 bp) was detected in 9 (45%) cases. Sequence analysis revealed that there is no diversity between the amplicons detected in samples. In conclusion, more effective preventive precautions should also perform for continuity of Van cat lineage in addition to routinely vaccination

Tick survey and detection of Crimean-Congo hemorrhagic fever virus in tick species from a non-endemic area, South Marmara region, Turkey

Crimean-Congo hemorrhagic fever (CCHF) is an increasing health concern in Turkey since 2002. There were also some recent human cases from the South Marmara region of Turkey; thus, a tick survey was performed, and possible vector tick species for the CCHF virus were determined in the region. A total of 740 adult ticks were collected from infested livestock from five locations: Canakkale-Biga, Bursa-Orhaneli, Bursa-Keles, BalA +/- kesir and Bilecik. Total of 11 tick species from the genera Hyalomma, Rhipicephalus, Dermacentor, Ixodes and Haemaphysalis were identified. Rhipicephalus ticks were dominant in the region; the most frequently observed tick species was R. turanicus, (53.1 %), and only 15.4 % of the identified ticks were H. marginatum. The occurrence of H. rufipes infestation in the region fort he first time. A total of 73 pools of adult ticks were tested with both an antigen-detecting ELISA and RT real-time PCR (RT rt PCR). The presence of the CCHF virus was demonstrated in 9 (12.3 %) of the tested tick pools. Although seven of the tick pools were positive for the CCHF virus with both of the methods, one pool was positive only with RT rt PCR and the other pool was only positive with the ELISA. Positive results were obtained from ticks collected from cattle, sheep and goats from two locations, Bursa-Orhaneli and Bilecik. The CCHF virus was detected in R. turanicus (n = 3), R. bursa (n = 2), H. marginatum (n = 2) and D. marginatus (n = 2) ticks. The results of this study confirm the presence of the CCHF virus and present preliminary data on the vector tick species in the southern Marmara region of Turkey