Differentialdiagnostik von pockentypischen Hautveränderungen mittels Metagenomanalyse und VAmpSeq

Jährlich erreichen das Konsiliarlabor für Pockenviren des Robert Koch-Instituts zahlreiche Proben von Patienten mit pockentypischen und pockenähnlichen Hautveränderungen zur Diagnostik von Ortho-, Para- und Molluscipockenviren. Nicht immer können die eingesandten Proben eindeutig diagnostiziert werden und der Ursprung der pockentypischen Hautläsionen bleibt unklar. Für die Diagnostik von Viren gibt es verschiedene molekularbiologische Methoden. Während mit der Polymerase-Kettenreaktion (PCR) spezifisch auf einzelne Erreger gescreent werden kann, wird mit der Hochdurchsatz-Sequenzierung (HTS) ein unspezifischer Blick in die gesamte DNA und RNA der untersuchten Probe und damit alle darin enthaltenen Erreger ermöglicht. Seit 2014 hat der MinION in vielen Laboren Einzug gehalten, ein handtellergroßer Hochdurchsatz-Sequenzierer, welcher die Analyse von DNA-Molekülen in Echtzeit ermöglicht. Diese Technologie hat völlig neue Möglichkeiten in der Erregerdiagnostik eröffnet, jedoch sind die Sensitivität der Methode und die Qualität der generierten Sequenzen noch immer gering. In dieser Studie wurden 49 Patientenproben mittels Illumina HTS sowie mit einer neu entwickelten Methode (VAmpSeq) auf die verursachenden Erreger der pockenähnlichen Hautläsionen untersucht. Dabei handelt es sich bei der VAmpSeq (Virus Amplification based Sequencing)-Methode um eine Multiplex-PCR mit anschließender MinION-Sequenzierung. Dies kombiniert die Vorteile der PCR (Sensitivität, Spezifität) und der HTS (ungerichtetes, simultanes Screening) und ermöglicht eine sensitive und simultane Diagnostik und Differentialdiagnostik von pockenähnlichen Hautläsionen. Ergänzend wurde die VAmpSeq-Methode weiterentwickelt (PAmpliSeq – Pox Amplification based Sequencing), um neben der Detektion von Viren auch eine Vollgenomsequenzierung von Orthopockenviren zu ermöglichen. Für die Analyse in Echtzeit während der MinION-Sequenzierung wurde eine Oberflächenanwendung entwickelt (VAmpSeeker und PAmpliSeeker), um die Interpretation der generierten Daten auch ohne bioinformatische Kenntnisse zu ermöglichen. Mittels Illumina HTS und der VAmpSeq-Methode konnten in den Patientenproben Herpes simplex-Virus Typ 1, Molluscum-contagiosum-Virus Typ 1 und Typ 2 sowie Orf-Virus diagnostiziert werden. Zudem konnte mit der Illumina HTS ein neuartiges (Para)pockenvirus, mit der VAmpSeq-Methode in weiteren Proben das Epstein-Barr-Virus und Parvovirus B19 identifiziert werden. Mit der VAmpSeq-Methode konnte damit in dieser Arbeit ein neues diagnostisches Verfahren entwickelt werden, welches es ermöglicht, auf mehrere Erreger in kurzer Zeit zu screenen und welches bereits für zahlreiche Anwendungen im Laboralltag weiterentwickelt wurde

Viral Metagenomics on Blood-Feeding Arthropods as a Tool for Human Disease Surveillance

Surveillance and monitoring of viral pathogens circulating in humans and wildlife, together with the identification of emerging infectious diseases (EIDs), are critical for the prediction of future disease outbreaks and epidemics at an early stage. It is advisable to sample a broad range of vertebrates and invertebrates at different temporospatial levels on a regular basis to detect possible candidate viruses at their natural source. However, virus surveillance systems can be expensive, costly in terms of finances and resources and inadequate for sampling sufficient numbers of different host species over space and time. Recent publications have presented the concept of a new virus surveillance system, coining the terms “flying biological syringes”, “xenosurveillance” and “vector-enabled metagenomics”. According to these novel and promising surveillance approaches, viral metagenomics on engorged mosquitoes might reflect the viral diversity of numerous mammals, birds and humans, combined in the mosquitoes’ blood meal during feeding on the host. In this review article, we summarize the literature on vector-enabled metagenomics (VEM) techniques and its application in disease surveillance in humans. Furthermore, we highlight the combination of VEM and “invertebrate-derived DNA” (iDNA) analysis to identify the host DNA within the mosquito midgut

Genomic Sequencing and Analysis of a Novel Human Cowpox Virus With Mosaic Sequences From North America and Old World Orthopoxvirus

Orthopoxviruses (OPXVs) not only infect their natural hosts, but some OPXVs can also cause disease in humans. Previously, we partially characterized an OPXV isolated from an 18-year-old male living in Northern Norway. Restriction enzyme analysis and partial genome sequencing characterized this virus as an atypical cowpox virus (CPXV), which we named CPXV-No-H2. In this study, we determined the complete genome sequence of CPXV-No-H2 using Illumina and Nanopore sequencing. Our results showed that the whole CPXV-No-H2 genome is 220,276 base pairs (bp) in length, with inverted terminal repeat regions of approximately 7 kbp, containing 217 predicted genes. Seventeen predicted CPXV-No-H2 proteins were most similar to OPXV proteins from the Old World, including Ectromelia virus (ECTV) and Vaccinia virus, and North America, Alaskapox virus (AKPV). CPXV-No-H2 has a mosaic genome with genes most similar to other OPXV genes, and seven potential recombination events were identified. The phylogenetic analysis showed that CPXV-No-H2 formed a separate clade with the German CPXV isolates CPXV_GerMygEK938_17 and CPXV_Ger2010_MKY, sharing 96.4 and 96.3% nucleotide identity, respectively, and this clade clustered closely with the ECTV-OPXV Abatino clade. CPXV-No-H2 is a mosaic virus that may have arisen out of several recombination events between OPXVs, and its phylogenetic clustering suggests that ECTV-Abatino-like cowpox viruses form a distinct, new clade of cowpox viruses

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

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

Genomic Sequencing and Phylogenomics of Cowpox Virus

Cowpox virus (CPXV; genus Orthopoxvirus; family Poxviridae) is the causative agent of cowpox, a self-limiting zoonotic infection. CPXV is endemic in Eurasia, and human CPXV infections are associated with exposure to infected animals. In the Fennoscandian region, five CPXVs isolated from cats and humans were collected and used in this study. We report the complete sequence of their genomes, which ranged in size from 220–222 kbp, containing between 215 and 219 open reading frames. The phylogenetic analysis of 87 orthopoxvirus strains, including the Fennoscandian CPXV isolates, confirmed the division of CPXV strains into at least five distinct major clusters (CPXV-like 1, CPXV-like 2, VACV-like, VARV-like and ECTV-Abatino-like) and can be further divided into eighteen sub-species based on the genetic and patristic distances. Bayesian time-scaled evolutionary history of CPXV was reconstructed employing concatenated 62 non-recombinant conserved genes of 55 CPXV. The CPXV evolution rate was calculated to be 1.65 × 10−5 substitution/site/year. Our findings confirmed that CPXV is not a single species but a polyphyletic assemblage of several species and thus, a reclassification is warranted

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

Auswirkungen von Kooperationsverhältnissen auf die wahrgenommene Nachhaltigkeit in Verbundprojekten

Kooperation ist seit vielen Jahrzehnten ein zentrales Thema in Forschung, Lehre und Weiterbildung, wobei insbesondere die Zusammenarbeit mit außerhochschulischen Partnern im Vordergrund steht. Ungeachtet der zahlreichen Herausforderungen sowie der bestehenden Konkurrenzverhältnisse, werden auch zunehmend Kooperationen zwischen wissenschaftlichen Weiterbildungseinrichtungen verschiedener Hochschulen eingegangen. Ein Grund hierfür könnte das Interesse von Fördermittelgebern an Verbünden bei der Beantragung und Umsetzung von Forschungs- und Entwicklungsprojekten sein, um zum einen Synergieeffekte zu nutzen und zum anderen die Vernetzung zu fördern und damit nachhaltige(re) Ergebnisse zu erreichen. Vor diesem Hintergrund widmet sich der vorliegende Beitrag der Fragestellung, welche Auswirkungen die Kooperation in geförderten Verbundprojekten auf die wahrgenommene Nachhaltigkeit hat. Zur Beantwortung der Frage werden im Rahmen des Bund-Länder-Wettbewerbs „Aufstieg durch Bildung: offene Hochschulen“ (2011-2020) erhobene quantitative Daten herangezogen

Sensitive on-site detection of SARS-CoV-2 by ID NOW COVID-19

Point of care detection of SARS-CoV-2 is one pillar in a containment strategy and important to break infection chains. Here we report the sensitive, specific and robust detection of SARS-CoV-2 and respective variants of concern by the ID NOW COVID-19 device.Peer Reviewe