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      Oyster RNA-seq Data Support the Development of Malacoherpesviridae Genomics

      research-article
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      Frontiers in Microbiology
      Frontiers Media S.A.
      Malacoherpesviridae, RNA-seq, viromes, bivalve, OsHV-1, Herpesvirales

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          Abstract

          The family of double-stranded DNA (dsDNA) Malacoherpesviridae includes viruses able to infect marine mollusks and detrimental for worldwide aquaculture production. Due to fast-occurring mortality and a lack of permissive cell lines, the available data on the few known Malacoherpesviridae provide only partial support for the study of molecular virus features, life cycle, and evolutionary history. Following thorough data mining of bivalve and gastropod RNA-seq experiments, we used more than five million Malacoherpesviridae reads to improve the annotation of viral genomes and to characterize viral InDels, nucleotide stretches, and SNPs. Both genome and protein domain analyses confirmed the evolutionary diversification and gene uniqueness of known Malacoherpesviridae. However, the presence of Malacoherpesviridae-like sequences integrated within genomes of phylogenetically distant invertebrates indicates broad diffusion of these viruses and indicates the need for confirmatory investigations. The manifest co-occurrence of OsHV-1 genotype variants in single RNA-seq samples of Crassostrea gigas provide further support for the Malacoherpesviridae diversification. In addition to simple sequence motifs inter-punctuating viral ORFs, recombination-inducing sequences were found to be enriched in the OsHV-1 and AbHV1-AUS genomes. Finally, the highly correlated expression of most viral ORFs in multiple oyster samples is consistent with the burst of viral proteins during the lytic phase.

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          Most cited references49

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          Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008.

          Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype. (c) 2010 Elsevier B.V. All rights reserved.
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            A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses.

            We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.
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              Transcribed dark matter: meaning or myth?

              Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic 'dark matter' transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                09 August 2017
                2017
                : 8
                : 1515
                Affiliations
                Department of Biology, University of Padua Padua, Italy
                Author notes

                Edited by: Akio Adachi, Tokushima University, Japan

                Reviewed by: Joaquin Martinez Martinez, Bigelow Laboratory for Ocean Sciences, United States; Timothy James Green, Macquarie University, Australia; Tristan Renault, French Research Institute for Exploitation of the Sea, France

                *Correspondence: Umberto Rosani umberto.rosani@ 123456unipd.it

                This article was submitted to Virology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.01515
                5552708
                28848525
                380dbbbe-ea03-4724-bbe7-098645b6aa20
                Copyright © 2017 Rosani and Venier.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 26 April 2017
                : 27 July 2017
                Page count
                Figures: 6, Tables: 8, Equations: 0, References: 65, Pages: 15, Words: 9647
                Funding
                Funded by: Horizon 2020 Framework Programme 10.13039/100010661
                Award ID: n°678589
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                malacoherpesviridae,rna-seq,viromes,bivalve,oshv-1,herpesvirales
                Microbiology & Virology
                malacoherpesviridae, rna-seq, viromes, bivalve, oshv-1, herpesvirales

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