Chen et al. BMC Genomics (2018) 19:49 DOI 10.1186/s12864-018-4432-4 RESEARCH ARTICLE Open Access The genome of a prasinoviruses-related freshwater virus reveals unusual diversity of phycodnaviruses Hao Chen1, Weijia Zhang1,5, Xiefei Li1, Yingjie Pan1,3, Shuling Yan1,4 and Yongjie Wang1,2,3* Abstract Background: Phycodnaviruses are widespread algae-infecting large dsDNA viruses and presently contain six genera: Chlorovirus, Prasinovirus, Prymnesiovirus, Phaeovirus, Coccolithovirus and Raphidovirus. The members in Prasinovirus are identified as marine viruses due to their marine algal hosts, while prasinovirus freshwater relatives remain rarely reported. Results: Here we present the complete genomic sequence of a novel phycodnavirus, Dishui Lake Phycodnavirus 1 (DSLPV1), which was assembled from Dishui Lake metagenomic datasets. DSLPV1 harbors a linear genome of 181,035 bp in length (G + C content: 52.7%), with 227 predicted genes and 2 tRNA encoding regions. Both comparative genomic and phylogenetic analyses indicate that the freshwater algal virus DSLPV1 is closely related to the members in Prasinovirus, a group of marine algae infecting viruses. In addition, a complete eukaryotic histone H3 variant was identified in the genome of DSLPV1, which is firstly detected in phycodnaviruses and contributes to understand the interaction between algal virus and its eukaryotic hosts. Conclusion: It is in a freshwater ecosystem that a novel Prasinovirus-related viral complete genomic sequence is discovered, which sheds new light on the evolution and diversity of the algae infecting Phycodnaviridae. Keywords: Phycodnaviridae, Prasinovirus, DSLPV1, Histone H3, Diversity Background algae-infecting large viruses are active players in the The phycodnaviruses comprise genetically diverse, mor- aquatic ecosystem [4–6]. phologically similar, large icosahedral (100 ~ 200 nm), The hosts of phycodnaviruses are abundant and wide- and double strand DNA (180 ~ 560 kbp) viruses that spread in the natural environments, while almost all phy- infect eukaryotic algae from both fresh and marine codnaviruses, including members of the Coccolithovirus, waters, and presently contain six genera: Chlorovirus, Phaeovirus, Prasinovirus, Prymnesiovirus and Raphidovirus Prasinovirus, Prymnesiovirus, Phaeovirus, Coccolithovirus genera, infect marine algae except for chloroviruses and Raphidovirus [1]. The family of Phycodnaviridae is (Chlorovirus)thattargetfreshwateralgae[7,8].Themem- placed within a major, monophyletic assemblage of large bers of Prasinovirus are the most studied among these eukaryotic DNA viruses, termed the nucleo-cytoplasmic marine algae-infecting viruses because the hosts of prasi- large DNA viruses (NCLDVs), which was recently noviruses are worldwide distributed and play a central role proposed to group into a new viral order Megavirales in the oceanic carbon cycle [9]. All known prasinoviruses [2, 3]. Accumulating evidence suggests that these infect marine photosynthetic picoeukaryotic algae in the class Mamiellophyceae, which mainly contains three gen- era: Ostreococcus, Micromonas and Bathycoccus [10, 11]. * Correspondence: [email protected] Thus far, several prasinoviruses have been isolated and se- 1College of Food Science and Technology, Shanghai Ocean University, quenced. For instance, the Ostreococcus virus OtV5 Shanghai, China (Ostreococcus tauri virus 5), possessing 186,234 bp long 2Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China linear genome, was isolated from the green alga O. tauri Full list of author information is available at the end of the article [12], and the Micromonas virus MpV1 (Micromonas sp. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. BMC Genomics (2018) 19:49 Page 2 of 12 RCC1109 virus 1) was isolated from M. sp. RCC1109 in program. One top hit to virus and/or non-virus was re- eutrophic northwestern Mediterranean coastal lagoons corded. Functional annotation of ORFs was performed with a genome size of 184,095 bp [13]. Interestingly, in using the InterProScan program (http://www.ebi.ac.uk/ our previous work of culture-independent metagenomic Tools/pfa/iprscan/) [17], and conserved domain was analyses, genomes of prasinoviruses-related viruses, determined with both the NCBI server and the HHpred Yellowstone Lake Phycodnaviruses (YSLPVs), were dis- server (http://toolkit.tuebingen.mpg.de/hhpred) [18]. Trans- covered in Yellowstone Lake [14], a freshwater lake in fer RNA (tRNA) sequences were identified by using the Yellowstone National Park, Wyoming. tRNAscan-SE tool [19]. Repetitive sequences were detected In this study, we assemble the complete genome of a with both the Geneious Pro software and the Reputer pro- Prasinovirus-related novel large phycodnavirus from gram (http://bibiserv.techfak.uni-bielefeld.de/reputer/) [20]. freshwater metagenomic datasets in Shanghai, China. Wholegenomealignmentatthenucleotidelevelwasper- Comparative genomic and phylogenetic analyses reveal formed with the MAUVE software [21]. unique features of this new member of Phycodnaviridae and shed light on the evolution, diversity and distribu- Phylogenetic analysis tion of phycodnaviruses. Maximum likelihood phylogenetic trees were recon- structed based on homologs of DNA polymerase B Methods family (PolB) gene, nicotinamide adenine dinucleotide Metagenomic data source (NAD)-dependent epimerase/dehydratase gene and his- The Dishui Lake (DSL) metagenomic datasets were gener- tone H3 gene, respectively. Homolog counterparts from ated with Illumina Miseq sequencer and subjected to a a range of representative viruses, bacteria and eukaryotes series of processes for quality controlling as described pre- were downloaded from the NCBI protein database viously [15]. In brief, water samples were taken from neritic (https://www.ncbi.nlm.nih.gov/protein). Amino acid se- area of Dishui Lake (121°55′27.00′′ N30°53′56.00′′ E) in quences were multiply aligned with the MUSCLE program, 2013. Microbial biomasses were collected onto 0.22-μm followed by tree reconstruction with the MEGA 7 software membrane filters, from which over 6 μg genomic DNA by using the JTT model and bootstrap value of 100 [22]. was extracted and used for metagenomic sequencing. Raw reads were firstly analyzed with FastQC and PCR for the histone gene NGS QC Toolkit prior to de novo sequence assembly. To avoid any possibilities of the misassembly of the Detail information of the datasets was shown in histone gene in the assembled viral genome, a pair of Additional file 1: Table S1. PCR primers (5’-CTTGAGTATGGCACCCTTGG-3′ and 5’-TCGCTTGGCGTCTTTCAAGG-3′) respectively Metagenomic sequence assembly of viral genome targeting the up- and downstream of the histone gene All high-throughput sequenced reads in the DSL metage- were designed based on the assembled vial genomic se- nomic paired-end libraries were assembled into contigs by quence. PCR was then performed with the same DNA using Newbler v2.6 (Roche) with default parameters. samples as applied to the metagenomic sequencing. The Among 1949 contigs with the length of more than 10 kb, reaction (25 μL) contained 0.4 mM of forward and re- one was initially confirmed to be related to algal large verse primers, 12.5 μLofTaq PCR master mix (2×) viruses with BLASTP searching of NCBI nr database and (Tiangen Biotech), and 20–25 ng of DNA. The amplifi- subsequently used as the template in reference assembly cation started with the program: initial denaturation at as previously described [16]. Briefly, this contig was used 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, as the reference sequence to which all reads in the DSL 58 °C for 30 s, and 72 °C for 45 s in an Eppendorf metagenomic paired-end datasets were assembled with a Mastercycler machine. PCR products were cloned and minimum overlap length of 25 bp and minimum overlap sequenced as previously described [15]. Sequences were identity of 95%. The reference assembly was repeated until aligned to the assembled genome by using Geneious R9 the assembled sequence stopped extending. All assemblies (Biomatters, https://www.geneious.com). were performed with the bioinformatics software Geneious R9 (Biomatters, https://www.geneious.com). Homology modeling of Histone H3 in DSLPV1 The three dimensional structure of histone fold H3 in Genome analysis DSLPV1_013 was modeled on the online protein homology Geneious R9 software (Biomatters) was used to predict modeling SWISS-MODEL server (www.swissmodel. open reading frames (ORFs) by defining a start codon of expasy.org) with the template of the crystal structure of ATG and a minimum 150 bp. Translated amino acid human histone H3 (PDB id 3lel.1.E, amino acid sequence − sequences were used to search (E-value < 10 3) for identity 83.82%). The structure was visualized and homologs in NCBI nr database by using the BLASTP analyzed with the PyMOL software (www.pymol.org) [23]. Chen et al. BMC Genomics (2018)