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17 pages, 3190 KB

Open AccessArticle

An Optimized Metabarcoding Method for Mimiviridae

by Florian Prodinger, Hisashi Endo, Yasuhiro Gotoh, Yanze Li, Daichi Morimoto, Kimiho Omae, Kento Tominaga, Romain Blanc-Mathieu, Yoshihito Takano, Tetsuya Hayashi, Keizo Nagasaki, Takashi Yoshida and Hiroyuki Ogata

Microorganisms 2020, 8(4), 506; https://doi.org/10.3390/microorganisms8040506 - 2 Apr 2020

Cited by 8 | Viewed by 5807

Abstract

Mimiviridae is a group of viruses with large genomes and virions. Ecological relevance of Mimiviridae in marine environments has been increasingly recognized through the discoveries of novel isolates and metagenomic studies. To facilitate ecological profiling of Mimiviridae, we previously proposed a meta-barcoding approach based on 82 degenerate primer pairs (i.e., MEGAPRIMER) targeting the DNA polymerase gene of Mimiviridae. The method detected a larger number of operational taxonomic units (OTUs) in environmental samples than previous methods. However, it required large quantities of DNA and was laborious due to the use of individual primer pairs. Here, we examined coastal seawater samples using varying PCR conditions and purification protocols to streamline the MEGAPRIMER method. Mixing primer pairs in “cocktails” reduced the required amount of environmental DNA by 90%, while reproducing the results obtained by the original protocol. We compared the results obtained by the meta-barcoding approach with quantifications using qPCR for selected OTUs. This revealed possible amplification biases among different OTUs, but the frequency profiles for individual OTUs across multiple samples were similar to those obtained by qPCR. We anticipate that the newly developed MEGAPRIMER protocols will be useful for ecological investigation of Mimiviridae in a larger set of environmental samples. Full article

(This article belongs to the Special Issue Molecular Ecology and Genetic Diversity of Marine Microorganisms)

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16 pages, 7694 KB

Open AccessArticle

Degenerate PCR Primers to Reveal the Diversity of Giant Viruses in Coastal Waters

by Yanze Li, Pascal Hingamp, Hiroyasu Watai, Hisashi Endo, Takashi Yoshida and Hiroyuki Ogata

Viruses 2018, 10(9), 496; https://doi.org/10.3390/v10090496 - 13 Sep 2018

Cited by 26 | Viewed by 10788

Abstract

“Megaviridae” is a proposed family of giant viruses infecting unicellular eukaryotes. These viruses are ubiquitous in the sea and have impact on marine microbial community structure and dynamics through their lytic infection cycle. However, their diversity and biogeography have been poorly characterized due to the scarce detection of Megaviridae sequences in metagenomes, as well as the limitation of reference sequences used to design specific primers for this viral group. Here, we propose a set of 82 degenerated primers (referred to as MEGAPRIMER), targeting DNA polymerase genes (polBs) of Megaviridae. MEGAPRIMER was designed based on 921 Megaviridae polBs from sequenced genomes and metagenomes. By applying this primer set to environmental DNA meta-barcoding of a coastal seawater sample, we report 5595 non-singleton operational taxonomic units (OTUs) of Megaviridae at 97% nucleotide sequence identity. The majority of the OTUs were found to form diverse clades, which were phylogenetically distantly related to known viruses such as Mimivirus. The Megaviridae OTUs detected in this study outnumber the giant virus OTUs identified in previous individual studies by more than an order of magnitude. Hence, MEGAPRIMER represents a useful tool to study the diversity of Megaviridae at the population level in natural environments. Full article

(This article belongs to the Special Issue Viruses of Microbes V: Biodiversity and Future Applications)

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18 pages, 1942 KB

Open AccessArticle

Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship

by Torill Vik Johannessen, Aud Larsen, Gunnar Bratbak, António Pagarete, Bente Edvardsen, Elianne D. Egge and Ruth-Anne Sandaa

Viruses 2017, 9(4), 84; https://doi.org/10.3390/v9040084 - 20 Apr 2017

Cited by 30 | Viewed by 7833

Abstract

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host–virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species. Full article

(This article belongs to the Special Issue Marine Viruses 2016)

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11 pages, 1704 KB

Open AccessArticle

Isolation and Characterization of a Double Stranded DNA Megavirus Infecting the Toxin-Producing Haptophyte Prymnesium parvum

by Ben A. Wagstaff, Iulia C. Vladu, J. Elaine Barclay, Declan C. Schroeder, Gill Malin and Robert A. Field

Viruses 2017, 9(3), 40; https://doi.org/10.3390/v9030040 - 9 Mar 2017

Cited by 22 | Viewed by 10916

Abstract

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms globally, leading to large-scale fish kills that have severe ecological and economic implications. For the model haptophyte, Emiliania huxleyi, it has been shown that large dsDNA viruses play an important role in regulating blooms and therefore biogeochemical cycling, but much less work has been done looking at viruses that infect P. parvum, or the role that these viruses may play in regulating harmful algal blooms. In this study, we report the isolation and characterization of a lytic nucleo-cytoplasmic large DNA virus (NCLDV) collected from the site of a harmful P. parvum bloom. In subsequent experiments, this virus was shown to infect cultures of Prymnesium sp. and showed phylogenetic similarity to the extended Megaviridae family of algal viruses. Full article

(This article belongs to the Special Issue Marine Viruses 2016)

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11 pages, 817 KB

Open AccessArticle

First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

by Mondher Boughalmi, Isabelle Pagnier, Sarah Aherfi, Philippe Colson, Didier Raoult and Bernard La Scola

Viruses 2013, 5(12), 2920-2930; https://doi.org/10.3390/v5122920 - 27 Nov 2013

Cited by 29 | Viewed by 11056

Abstract

Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection. Full article

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