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

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

doi:10.3390/v10090496

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.

Highlights

Viruses represent a non-cellular biological component of aquatic microbial communities [1].By outnumbering cellular organisms in the same ecosystem [2,3], viruses exert top-down control on microbial community dynamics and drive carbon and nutrient cycling [4,5,6,7]
None of the primer pairs corresponded to the polB sequence from Catovirus (CTV), which is a close relative of KNV and was not included in the MEGAPRIMER design
In silico polymerase chain reaction (PCR) against all the 921 reference polB sequences and their phylogeny indicated that reference sequences that can be amplified by a specific primer pair are not necessarily similar to each other along their length (Figure S2)

Summary

Introduction

Viruses represent a non-cellular biological component of aquatic microbial communities [1]. By outnumbering cellular organisms in the same ecosystem [2,3], viruses exert top-down control on microbial community dynamics and drive carbon and nutrient cycling [4,5,6,7]. The recently proposed “Megaviridae” family is emerging as an important viral group in marine ecosystems [12,13]. Members of this viral family are often called giant viruses [14,15,16,17,18,19]

Methods

Results

Conclusion