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15 pages, 1994 KiB

Open AccessArticle

Identification of Huge Phages from Wastewater Metagenomes

by René Kallies, Die Hu, Nafi’u Abdulkadir, Michael Schloter and Ulisses Rocha

Viruses 2023, 15(12), 2330; https://doi.org/10.3390/v15122330 - 28 Nov 2023

Cited by 4 | Viewed by 2526

Huge phages have genomes larger than 200 kilobases, which are particularly interesting for their genetic inventory and evolution. We screened 165 wastewater metagenomes for the presence of viral sequences. After identifying over 600 potential huge phage genomes, we reduced the dataset using manual curation by excluding viral contigs that did not contain viral protein-coding genes or consisted of concatemers of several small phage genomes. This dataset showed seven fully annotated huge phage genomes. The phages grouped into distinct phylogenetic clades, likely forming new genera and families. A phylogenomic analysis between our huge phages and phages with smaller genomes, i.e., less than 200 kb, supported the hypothesis that huge phages have undergone convergent evolution. The genomes contained typical phage protein-coding genes, sequential gene cassettes for metabolic pathways, and complete inventories of tRNA genes covering all standard and rare amino acids. Our study showed a pipeline for huge phage analyses that may lead to new enzymes for therapeutic or biotechnological applications. Full article

(This article belongs to the Special Issue Diversity and Evolution of Viruses in Ecosystem)

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13 pages, 1159 KiB

Open AccessArticle

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

by Xin You, Niclas Klose, René Kallies, Hauke Harms, Antonis Chatzinotas and Lukas Y. Wick

Viruses 2022, 14(2), 195; https://doi.org/10.3390/v14020195 - 20 Jan 2022

Cited by 6 | Viewed by 3799

Abstract

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. Full article

(This article belongs to the Special Issue Phage Ecology 2021)

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18 pages, 3503 KiB

Open AccessArticle

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

by Chiara Rossi, Nicola Zadra, Cristina Fevola, Frauke Ecke, Birger Hörnfeldt, René Kallies, Maria Kazimirova, Magnus Magnusson, Gert E. Olsson, Rainer G. Ulrich, Anne J. Jääskeläinen, Heikki Henttonen and Heidi C. Hauffe

Viruses 2021, 13(7), 1317; https://doi.org/10.3390/v13071317 - 7 Jul 2021

Cited by 3 | Viewed by 3713

Abstract

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 3D^pol 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. Full article

(This article belongs to the Special Issue Rodent-Borne Viruses)

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18 pages, 3976 KiB

Open AccessArticle

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

by René Kallies, Martin Hölzer, Rodolfo Brizola Toscan, Ulisses Nunes da Rocha, John Anders, Manja Marz and Antonis Chatzinotas

Viruses 2019, 11(6), 484; https://doi.org/10.3390/v11060484 - 28 May 2019

Cited by 27 | Viewed by 6785

Abstract

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. Full article

(This article belongs to the Special Issue Virus Bioinformatics)

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12 pages, 701 KiB

Open AccessShort Communication

Genetic Characterization of Goutanap Virus, a Novel Virus Related to Negeviruses, Cileviruses and Higreviruses

by René Kallies, Anne Kopp, Florian Zirkel, Alejandro Estrada, Thomas R. Gillespie, Christian Drosten and Sandra Junglen

Viruses 2014, 6(11), 4346-4357; https://doi.org/10.3390/v6114346 - 12 Nov 2014

Cited by 59 | Viewed by 9301

Abstract

Pools of mosquitoes collected in Côte d’Ivoire and Mexico were tested for cytopathic effects on the mosquito cell line C6/36. Seven pools induced strong cytopathic effects after one to five days post infection and were further investigated by deep sequencing. The genomes of six virus isolates from Côte d’Ivoire showed pairwise nucleotide identities of ~99% among each other and of 56%–60% to Dezidougou virus and Wallerfield virus, two insect-specific viruses belonging to the proposed new taxon Negevirus. The novel virus was tentatively named Goutanap virus. The isolate derived from the Mexican mosquitoes showed 95% pairwise identity to Piura virus and was suggested to be a strain of Piura virus, named C6.7-MX-2008. Phylogenetic inferences based on a concatenated alignment of the methyltransferase, helicase, and RNA-dependent RNA polymerase domains showed that the new taxon Negevirus formed two monophyletic clades, named Nelorpivirus and Sandewavirus after the viruses grouping in these clades. Branch lengths separating these clades were equivalent to those of the related genera Cilevirus, Higrevirus and Blunervirus, as well as to those within the family Virgaviridae. Genetic distances and phylogenetic analyses suggest that Nelorpivirus and Sandewavirus might form taxonomic groups on genus level that may define alone or together with Cilevirus, Higrevirus and Blunervirus a viral family. Full article

(This article belongs to the Special Issue Impact of the Insect Microbiome on Arbovirus Transmission)

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