Complete Genome Sequence of Alteromonas Virus vB_AspP-H4/4

Alteromonas virus vB_AspP-H4/4 is a member of the Podoviridae family and was isolated from North Sea water in the 1970s. The complete double-stranded DNA genome has 47,631 bp with 49 predicted genes

OrtSuite: from genomes to prediction of microbial interactions within targeted ecosystem processes

Published Online: 27 September, 2021The high complexity found in microbial communities makes the identification of microbial interactions challenging. To address this challenge, we present OrtSuite, a flexible workflow to predict putative microbial interactions based on genomic content of microbial communities and targeted to specific ecosystem processes. The pipeline is composed of three user-friendly bash commands. OrtSuite combines ortholog clustering with genome annotation strategies limited to user-defined sets of functions allowing for hypothesis-driven data analysis such as assessing microbial interactions in specific ecosystems. OrtSuite matched, on average, 96% of experimentally verified KEGG orthologs involved in benzoate degradation in a known group of benzoate degraders. We evaluated the identification of putative synergistic species interactions using the sequenced genomes of an independent study that had previously proposed potential species interactions in benzoate degradation. OrtSuite is an easy-to-use workflow that allows for rapid functional annotation based on a user-curated database and can easily be extended to ecosystem processes where connections between genes and reactions are known.This work was funded by the Helmholtz Young Investigator grant VH-NG-1248 Micro “Big Data.”info:eu-repo/semantics/publishedVersio

Evolutionary Relationships of Ljungan Virus Variants Circulating in Multi-Host Systems across Europe

The picornavirus named ‘Ljungan virus’ (LV, species Parechovirus B) has been detected in a dozen small mammal species from across Europe, but detailed information on its genetic diversity and host specificity is lacking. Here, we analyze the evolutionary relationships of LV variants circulating in free-living mammal populations by comparing the phylogenetics of the VP1 region (encoding the capsid protein and associated with LV serotype) and the 3Dpol region (encoding the RNA polymerase) from 24 LV RNA-positive animals and a fragment of the 5′ untranslated region (UTR) sequence (used for defining strains) in sympatric small mammals. We define three new VP1 genotypes: two in bank voles (Myodes glareolus) (genotype 8 from Finland, Sweden, France, and Italy, and genotype 9 from France and Italy) and one in field voles (Microtus arvalis) (genotype 7 from Finland). There are several other indications that LV variants are host-specific, at least in parts of their range. Our results suggest that LV evolution is rapid, ongoing and affected by genetic drift, purifying selection, spillover and host evolutionary history. Although recent studies suggest that LV does not have zoonotic potential, its widespread geographical and host distribution in natural populations of well-characterized small mammals could make it useful as a model for studying RNA virus evolution and transmission

Evolutionary Relationships of Ljungan Virus Variants Circulating in Multi-Host Systems across Europe

The picornavirus named ‘Ljungan virus’ (LV, species Parechovirus B) has been detected in a dozen small mammal species from across Europe, but detailed information on its genetic diversity and host specificity is lacking. Here, we analyze the evolutionary relationships of LV variants circulating in free-living mammal populations by comparing the phylogenetics of the VP1 region (encoding the capsid protein and associated with LV serotype) and the 3Dpol region (encoding the RNA polymerase) from 24 LV RNA-positive animals and a fragment of the 5′ untranslated region (UTR) sequence (used for defining strains) in sympatric small mammals. We define three new VP1 genotypes: two in bank voles (Myodes glareolus) (genotype 8 from Finland, Sweden, France, and Italy, and genotype 9 from France and Italy) and one in field voles (Microtus arvalis) (genotype 7 from Finland). There are several other indications that LV variants are host-specific, at least in parts of their range. Our results suggest that LV evolution is rapid, ongoing and affected by genetic drift, purifying selection, spillover and host evolutionary history. Although recent studies suggest that LV does not have zoonotic potential, its widespread geographical and host distribution in natural populations of well-characterized small mammals could make it useful as a model for studying RNA virus evolution and transmission

Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters

Viral ecology of terrestrial habitats is yet-to be extensively explored, in particular the terrestrial subsurface. One problem in obtaining viral sequences from groundwater aquifer samples is the relatively low amount of virus particles. As a result, the amount of extracted DNA may not be sufficient for direct sequencing of such samples. Here we compared three DNA amplification methods to enrich viral DNA from three pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory to evaluate potential bias created by the different amplification methods as determined by viral metagenomics. Linker amplification shotgun libraries resulted in lowest redundancy among the sequencing reads and showed the highest diversity, while multiple displacement amplification produced the highest number of contigs with the longest average contig size, suggesting a combination of these two methods is suitable for the successful enrichment of viral DNA from pristine groundwater samples. In total, we identified 27,173, 5,886 and 32,613 viral contigs from the three samples from which 11.92 to 18.65% could be assigned to taxonomy using blast. Among these, members of the Caudovirales order were the most abundant group (52.20 to 69.12%) dominated by Myoviridae and Siphoviridae. Those, and the high number of unknown viral sequences, substantially expand the known virosphere

Ljunganvirus - Prävalenz in Nagetieren und Viruspathogenese

Ljungan virus (LV) is a member of the Picornaviridae that was isolated from various species of voles and mice in Scandinavia, North America, and Italy, suggesting a wide host range and a world wide distribution of this virus. LV causes severe disease in its rodent reservoirs, such as diabetes and myocarditis. In addition, laboratory mice infected with LV suffer from encephalitis and fetal deaths indicating that LV might also induce this diseases in the wild. In addition, LV has been associated with human disease during pregnancy and of neonates, respectively. A real-time RT-PCR assay was established to identify and quantify LV in different types of sample. The method was evaluated using in vitro transcribed RNA and RNA extracted from cell culture supernatant and LV infected laboratory mice, respectively. The assay was specific to all known LV strains, but not to closely related picornaviruses. A linear detection range with high sensitivity (106 - 101 in vitro transcribed RNA molecules) was demonstrated. Furthermore, a melting curve analysis, pyrosequencing and a RT PCR assay targeting the LV VP1 region were established for characterisation and genotyping of LV positive samples. A panel of 22 monoclonal antibodies (mAbs) against LV genotypes 1 and 2 were produced by immunization of BALB/c mice with whole virus. Thirteen mAbs were class IgG antibodies and nine were class IgM antibodies, all contained kappa light chains. All mAbs were reactive by capture enzyme-linked immunosorbent assay and indirect immunofluorescent assay. In addition, 5 mAbs showed a positive staining in immunohistochemistry. No mAb bound to denatured capsid proteins as detected by immunoblotting. In contrast, the target capsid protein(s) of 20 mAbs were identified by immunoprecipitation, revealing the conformational nature of epitopes required for mAb binding. Furthermore, 7 mAbs were identified that inhibited LV infection to cell culture. Assays developed in this thesis should provide usefool tools for the development of diagnostic assays and the investigation of LV properties and its pathogenesis. Three different strains of laboratory rats were investigated for the presence of LV. LV specific RNA was found in brain and heart by RT-PCR. In addition, LV proteins were visualised in the pancreas by immunohistochemistry and specific antibodies were detected by indirect immunofluorescence test, suggesting that the laboratory rat is one of several rodent reservoirs of this new picornavirus. Wild rodents from Germany and Thailand were investigated for LV presence by RT PCR. In total, 454 (Germany) and 87 samples (Thailand), respectively, were analysed. A total of 44 (9.7 %) (Germany) and 14 (16.1 %) (Thailand) samples were positive for LV, belonging to the following species: Germany: Microtus agrestis, Microtus arvalis, Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Micromys minutus, Mus musculus, Rattus norvegicus; Thailand: Bandicota indica, Bandicota savilei, Mus caroli, Rattus rattus, Tupaia glis. LV positive animals were found in the German areas of Baden-Württemberg, Saxony-Anhalt, Brandenburg, Mecklenburg- Western Pomerania, the city of Cologne, and Thai provinces Bangkok, Buri Nam, and Prachuap Khiri Khan. A putative new LV genotype was found in a bank vole sample from Germany. The data present a so far unknown picture of LV prevalence in wild rodents also in Central Europe and Asia. In this thesis, organs of laboratory mice infected with LV were investigated for the presence of LV genome by real-time RT-PCR through time. The animals had clinical signs of encephalitis a few days post infection and developed diabetes later in life. All organs were found positive for LV over the whole period of 174 days. This work shows that LV causes a systemic persistent infection in laboratory mice that might support the onset of diabetes in adult individuals. Bank voles trapped in the wild were investigated for the presence of LV by real-time RT- PCR. Several organs were found positive with copy numbers close to those found in laboratory mice during persistent infection, indicating a systemic persistent LV infection also in wild bank voles. LV production was analysed in two cell lines. Both Vero B4 and BHK 21 cells were susceptible for LV infection. An increase of LV RNA and LV protein was detected in both cell lines. In contrast, only Vero B4 cell supernatant had an increasing titer of LV particles, indicating LV infection of BHK 21 cells is restricted. Furthermore, only Vero B4 cells showed a clear cytopathic effect post LV infection that was associated with induction of apoptosis. Apoptosis might play a role in LV induced onset of encephalitis, myocarditis, and diabetes, respectively.Ljunganvirus (LV) gehört zur Familie der Picornaviridae und wurde in verschiedenen Wühlmaus- und Mausspezies in Skandinavien, Nordamerika und Italien nach-gewiesen, was ein breites Wirtsspektrum sowie eine weltweite Verbreitung dieses Virus vermuten lässt. LV verursacht ernsthafte Erkrankungen, wie Diabetes und Myokarditis, in seinen Nagerreservoiren. LV infizierte Labormäuse entwickeln zudem neurologische Symptome und reproduktive Störungen. Diese Erkrankungen könnten ebenfalls bei Wildtieren durch LV induziert werden. Der Nachweis von LV im Menschen wurde mit Erkrankungen während der Schwangerschaft und von Neugeborenen assoziiert. In dieser Arbeit wurde eine real time RT PCR Methode etabliert, um LV in verschiedenem Probenmaterial nachzuweisen und zu quantifizieren. Die Methode wurde mittels in vitro transkribierter RNA, RNA aus Zellkulturüberständen und RNA aus LV infizierten Labormäusen evaluiert. Die RT PCR war spezifisch für alle bekannten LV Stämme, nicht jedoch für eng verwandte Picornaviren. Ein linearer Nachweisbereich mit hoher Sensitivität (106 - 101 in vitro transkribierte RNA Moleküle) konnte gezeigt werden. Für eine weitere Charakterisierung LV positiver Proben wurden eine Schmelzpunktanalyse, Pyrosequencing sowie eine RT PCR Methode zum Nachweis der LV VP1 Region etabliert. Ein Panel von 22 monoklonalen Antikörpern (mAk) wurde durch Immunisierung von BALB/c Mäusen mit Vollvirus produziert. Dreizehn mAk gehörten zur IgG Subklasse und neun mAk zur IgM Subklasse. Alle mAk waren positiv in einem capture enzyme linked immunosorbent assay und im Immunfluoreszenztest. Fünf der mAk detektierten ebenfalls LV Antigen in der Immunhistochemie. Keiner der mAk konnte denaturierte LV Kapsidproteine nachweisen, wohingegen die Interaktionspartner von 20 mAk durch Immunpräzipitation identifiziert werden konnten, was darauf schließen lässt, dass die mAk an Konformationsepitope der LV Kapsidproteine binden. Zusätzlich wurden sieben neutralisierende mAk identifiziert. Die in dieser Arbeit etablierten Methoden bieten die Grundlage für die Entwicklung diagnostischer Methoden zum LV Nachweis. Die Erforschung der biochemischen und physikalischen Eigenschaften des LV und der LV Pathogenese sollte mit Hilfe dieser Methoden gezielt durchgeführt werden können. Drei verschiedene Laborratten-Stämme wurden auf eine LV Infektion untersucht. LV spezifische RNA wurde mittels RT PCR in Hirnen und Herzen der Tiere nachgewiesen. LV Proteine wurden zudem im Pankreas infizierter Tiere mit Hilfe der Immunhistochemie gezeigt und spezifische anti LV Antikörper wurden durch einen Immunfluoreszenztest nachgewiesen. Diese Daten legen nahe, dass Laborratten eines von mehreren Nagerreservoiren für LV sind. In einem weiteren Projekt wurde die LV Prävalenz in Wildnagern aus Deutschland und Thailand untersucht. Insgesamt wurden 454 Tiere aus Deutschland und 87 Tiere aus Thailand mittels RT PCR getestet. Aus Deutschland waren 44 (9,7 %) Proben und aus Thailand 14 (16,1 %) Proben LV positiv. Folgende Spezies wurden LV positiv getestet: Deutschland: Microtus agrestis, Microtus arvalis, Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Micromys minutus, Mus musculus, Rattus norvegicus; Thailand: Bandicota indica, Bandicota savilei, Mus caroli, Rattus rattus, Tupaia glis. LV positive Tiere wurden in folgenden Regionen gefunden: Baden-Württemberg, Sachsen-Anhalt, Brandenburg, Mecklenburg-Vorpommern, Köln (Deutschland), Bangkok, Buri Nam und Prachuap Khiri Khan (Thailand). Ein vermutlich neuer LV Genotyp wurde in einer Rötelmausprobe aus Deutschland detektiert. Diese Daten repräsentieren eine bisher unbekannte Verbreitung von LV auch in Zentraleuropa und Asien. Organe von LV infizierten Labormäusen wurden im Zeitverlauf mittels real-time RT PCR auf LV-Präsenz untersucht. Die Tiere erkrankten wenige Tage nach der Infektion an einer Enzephalitis und entwickelten im späteren Verlauf klinische Symptome eines Diabetes. Alle Organe waren über den gesamten Zeitverlauf von 174 Tagen LV positiv. Es konnte somit gezeigt werden, dass LV eine systemische persistierende Infektion verursacht, die zum Ausbruch von Diabetes in erwachsenen Tieren beitragen dürfte. Organe von in der Wildnis gefangenen Rötelmäusen wurden ebenfalls getestet. Verschiedene Organe waren LV positiv. Die Anzahl der LV Kopien war dabei vergleichbar mit denen aus Labormäusen während der persistierenden Infektion, was darauf hinweist, dass LV auch in Wildnagern persistiert. Die LV-Produktion wurde in zwei verschiedenen Zellinien untersucht. Sowohl Vero B4 als auch BHK 21 Zellen waren für eine LV Infektion empfänglich. In beiden Zellinien wurde eine Zunahme von LV RNA und LV Protein im Zeitverlauf beobachtet. Ein ansteigender Virustiter konnte allerdings nur im Überstand von Vero B4 Zellen detektiert werden. Weiterhin wurde lediglich in Vero B4 Zellen ein virusinduzierter cytopathischer Effekt beobachtet, welcher durch Apoptose induzierende Effekte ausgelöst scheint. LV induzierte Apoptose könnte eine Rolle in der Entstehung von Enzephalitis, Myokarditis und Diabetes spielen

Mycelia-Assisted Isolation of Non-Host Bacteria Able to Co-Transport Phages

Recent studies have demonstrated that phages can be co-transported with motile non-host bacteria, thereby enabling their invasion of biofilms and control of biofilm composition. Here, we developed a novel approach to isolate non-host bacteria able to co-transport phages from soil. It is based on the capability of phage-carrying non-host bacteria to move along mycelia out of soil and form colonies in plaques of their co-transported phages. The approach was tested using two model phages of differing surface hydrophobicity, i.e., hydrophobic Escherichia virus T4 (T4) and hydrophilic Pseudoalteromonas phage HS2 (HS2). The phages were mixed into soil and allowed to be transported by soil bacteria along the mycelia of Pythium ultimum. Five phage-carrying bacterial species were isolated (Viridibacillus sp., Enterobacter sp., Serratia sp., Bacillus sp., Janthinobacterium sp.). These bacteria exhibited phage adsorption efficiencies of ≈90–95% for hydrophobic T4 and 30–95% for hydrophilic HS2. The phage adsorption efficiency of Viridibacillus sp. was ≈95% for both phages and twofold higher than T4-or HS2-adsorption to their respective hosts, qualifying Viridibacillus sp. as a potential super carrier for phages. Our approach offers an effective and target-specific way to identify and isolate phage-carrying bacteria in natural and man-made environments

Secondary structure of DNA released from purified capsids of human parvovirus B19 under moderate denaturing conditions

Parvovirus B19 (B19V) possesses a linear single-stranded DNA genome of either positive or negative polarity. Due to intramolecular sequence homologies, either strand may theoretically be folded in several alternative ways. Viral DNA, when extracted from virions by several procedures, presents as linear single-stranded and/or linear double-stranded molecules, except when one particular commercial kit is used. This protocol yields DNA with an aberrant electrophoretic mobility in addition to linear double-stranded molecules, but never any single-stranded molecules. This peculiar kind of DNA was found in all plasma or serum samples tested and so we decided to analyse its secondary structure. In line with our results for one- and two-dimensional electrophoresis, mobility shift assays, DNA preparation by an in-house extraction method with moderate denaturing conditions, density gradient ultracentrifugation, DNA digestion experiments and competition hybridization assays, we conclude that (i) the unique internal portions of this distinctive single-stranded molecules are folded into tight tangles and (ii) the two terminal redundant regions are associated with each other, yielding non-covalently closed pseudo-circular molecules stabilized by a short (18 nucleotides) intramolecular stem, whereas the extreme 3'- and 5'-ends are folded back on themselves, forming a structure resembling a twin hairpin. The question arises as to whether this fairly unstable structure represents the encapsidated genome structure. The answer to this question remains quite relevant in terms of comprehending the initiation and end of B19V genome replication

Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters

Viral ecology of terrestrial habitats is yet-to be extensively explored, in particular the terrestrial subsurface. One problem in obtaining viral sequences from groundwater aquifer samples is the relatively low amount of virus particles. As a result, the amount of extracted DNA may not be sufficient for direct sequencing of such samples. Here we compared three DNA amplification methods to enrich viral DNA from three pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory to evaluate potential bias created by the different amplification methods as determined by viral metagenomics. Linker amplification shotgun libraries resulted in lowest redundancy among the sequencing reads and showed the highest diversity, while multiple displacement amplification produced the highest number of contigs with the longest average contig size, suggesting a combination of these two methods is suitable for the successful enrichment of viral DNA from pristine groundwater samples. In total, we identified 27,173, 5,886 and 32,613 viral contigs from the three samples from which 11.92 to 18.65% could be assigned to taxonomy using blast. Among these, members of the Caudovirales order were the most abundant group (52.20 to 69.12%) dominated by Myoviridae and Siphoviridae. Those, and the high number of unknown viral sequences, substantially expand the known virosphere