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      Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia

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          Abstract

          This article describes Bacillus anthracis strains isolated during an outbreak of anthrax on the Yamal Peninsula in the summer of 2016 and independently in Yakutia in 2015. A common feature of these strains is their conservation in permafrost, from which they were extracted either due to the thawing of permafrost (Yamal strains) or as the result of paleontological excavations (Yakut strains). All strains isolated on the Yamal share an identical genotype belonging to lineage B.Br.001/002, pointing to a common source of infection in a territory over 250 km in length. In contrast, during the excavations in Yakutia, three genetically different strains were recovered from a single pit. One strain belongs to B.Br.001/002, and whole genome sequence analysis showed that it is most closely related to the Yamal strains in spite of the remoteness of Yamal from Yakutia. The two other strains contribute to two different branches of A.Br.008/011, one of the remarkable polytomies described so far in the B. anthracis species. The geographic distribution of the strains belonging to A.Br.008/011 is suggesting that the polytomy emerged in the thirteenth century, in combination with the constitution of a unified Mongol empire extending from China to Eastern Europe. We propose an evolutionary model for B. anthracis recent evolution in which the B lineage spread throughout Eurasia and was subsequently replaced by the A lineage except in some geographically isolated areas.

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          Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

          The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.
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            The microbial ecology of permafrost.

            Permafrost constitutes a major portion of the terrestrial cryosphere of the Earth and is a unique ecological niche for cold-adapted microorganisms. There is a relatively high microbial diversity in permafrost, although there is some variation in community composition across different permafrost features and between sites. Some microorganisms are even active at subzero temperatures in permafrost. An emerging concern is the impact of climate change and the possibility of subsequent permafrost thaw promoting microbial activity in permafrost, resulting in increased potential for greenhouse-gas emissions. This Review describes new data on the microbial ecology of permafrost and provides a platform for understanding microbial life strategies in frozen soil as well as the impact of climate change on permafrost microorganisms and their functional roles.
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              Global Genetic Population Structure of Bacillus anthracis

              Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064–6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: Writing – original draft
                Role: InvestigationRole: Writing – original draft
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Formal analysisRole: InvestigationRole: Visualization
                Role: InvestigationRole: Resources
                Role: InvestigationRole: Resources
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: SupervisionRole: VisualizationRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 May 2019
                2019
                : 14
                : 5
                : e0209140
                Affiliations
                [1 ] State Research Center for Applied Microbiology & Biotechnology (FBIS SRCAMB), Obolensk, Russia
                [2 ] Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
                Institut National de la Recherche Agronomique, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-9501-1380
                http://orcid.org/0000-0003-0913-194X
                Article
                PONE-D-18-34064
                10.1371/journal.pone.0209140
                6530834
                31116737
                5cc40a62-4de2-409a-8787-ac141dddd61d
                © 2019 Timofeev et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 November 2018
                : 7 May 2019
                Page count
                Figures: 2, Tables: 2, Pages: 21
                Funding
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: French Agence Nationale de la Recherche
                Award ID: ANR-14-ASMA-0002-02 “Microtype”
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: French Agence Nationale de la Recherche
                Award ID: ANR-14-ASMA-0002-02 “Microtype”
                Award Recipient :
                Funded by: French Agence Nationale de la Recherche
                Award ID: ANR-14-ASMA-0002-02 “Microtype”
                Award Recipient :
                Funded by: French Agence Nationale de la Recherche
                Award ID: ANR-14-ASMA-0002-02 “Microtype”
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                Funded by: Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor)
                Award ID: Sectoral Scientific Program «Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020)»
                Award Recipient :
                The work of TV, BI, MR, TG, LI, BogA, and BorA is supported by the Sectoral Scientific Program: Problem-oriented research in the epidemiological surveillance of infectious and parasitic diseases (2016-2020), of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor) ( http://www.rospotrebnadzor.ru/). The work of Vg and CD is supported by the French Agence Nationale de la Recherche grant ANR-14-ASMA-0002-02 “Microtype”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Bacillus
                Bacillus Anthracis
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