AEM Accepts, published online ahead of print on 14 March 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.00123-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved. 1 Diversity of prasinoviruses (Phycodnaviridae) in marine samples unveiled using next 2 generation sequencing analyses of PCR-amplified DNA polymerase and Major Capsid 3 Protein genes. 4 Running title: Diversity markers for DNA viruses 5 Camille Clerissi1,2, Nigel Grimsley1,2,*, Hiroyuki Ogata3,4, Pascal Hingamp5, Julie Poulain6, 6 Yves Desdevises1,2 7 1, Sorbonne Universités, UPMC Univ Paris 06, UMR 7232, Integrative Biology of Marine 8 Organisms, Observatoire Océanologique, Avenue du Fontaulé, F-66650, Banyuls-sur-Mer, 9 France. 10 2, Sorbonne Universités, CNRS, UMR 7232, Observatoire Océanologique, Integrative 11 Biology of Marine Organisms, Avenue du Fontaulé, F-66650, Banyuls-sur-Mer, France. 12 3, Education Academy of Computational Life Sciences, Tokyo, Institute of Technology, 2-12- 13 1, Ookayama, Meguro-ku, Tokyo, 152-8552, Japan. 14 4, Centre National de la Recherche Scientifique, France 15 5, CNRS, Université Aix-Marseille, Laboratoire Information Génomique et Structurale (UMR 16 7256), Mediterranean Institute of Microbiology (FR 3479), 13288, Marseille, France. 17 6, CEA, Institut de Génomique, Génoscope, 2 rue Gaston Crémieux, BP5706, Evry, FR- 18 91057, France. 19 * Corresponding author: Nigel Grimsley. Mailing address: UPMC, UMR 7232, Observatoire 20 Océanologique, Avenue du Fontaulé, 66650 Banyuls-sur-Mer, France. Phone: 21 +33468887396. Fax: +33468887398. E-mail: [email protected] 22 192 words for the abstract and 5075 words for the text 23 Keywords: PCR, 454-pyrosequencing, amplicon, virus, paralogs, community 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Abstract 43 Viruses strongly influence the ecology and evolution of their eukaryotic hosts in the marine 44 environment, but little is known about their diversity and distribution. Prasinoviruses infect an 45 abundant and widespread class of phytoplankton, the Mamiellophyceae, and thereby exert a 46 specific and important role in microbial ecosystems. However, molecular tools to specifically 47 identify this viral genus in environmental samples are still lacking. We developed two primer 48 sets, designed for use with polymerase-chain reactions and 454-pyrosequencing technologies, 49 to target two conserved genes, the DNA polymerase (PolB) and the Major Capsid Protein 50 (MCP). While only one copy of the PolB is present in Prasinovirus genomes, there are at least 51 seven paralogs for the MCP, the copy we named #6 being shared with other eukaryotic algae- 52 infecting viruses. Primer sets for PolB and MCP6 were thus designed and tested on 6 samples 53 from the Tara Oceans project. The results suggest that the MCP6 amplicons show greater 54 richness, but that PolB gave a wider coverage of the Prasinovirus diversity. As a 55 consequence, we recommend use of the PolB primer set, which will certainly reveal exciting 56 new insights about the diversity and distribution of prasinoviruses at the community scale. 57 58 59 60 61 62 63 64 Introduction 65 66 Members of the Phycodnaviridae family are classified in five genera according to the species 67 of eukaryotic algae that they are known to infect. Indeed, Chlorovirus, Raphidovirus, 68 Phaeovirus, Coccolithovirus and Prasinovirus respectively infect Chlorella, Raphidophytes, 69 Phaeophytes, Coccolithophores and Prasinophytes (known hosts of prasinoviruses now 70 belong to the new class Mamiellophyceae; reference 42, that remains in the subphylum 71 Prasinophytina, 57), (see 19; 59; 65; 69 for further information about these viral groups). 72 They are part of the nucleocytoplasmic large DNA viruses (NCLDV) and share nine 73 conserved core genes with other NCLDV (32). Among them, the DNA polymerase gene and 74 the Major Capsid Protein gene (hereafter named PolB and MCP, respectively) have been to 75 date the most commonly used molecular markers to investigate the diversity of 76 phycodnaviruses (e.g. 10; 11; 12; 36; 41; 64). 77 Prasinovirus was the first described phycodnaviral genus (43), and a metagenomic survey 78 recently proposed that they are the most abundant Phycodnaviridae in marine ecosystems 79 (31). Ostreococcus virus (OV), Micromonas virus (MpV) and Bathycoccus virus (BpV) 80 represent three major groups of prasinoviruses, and are known to infect the three most 81 widespread and abundant genera of Mamiellophyceae (42; 50; 66; 72). The PolB is the only 82 gene used so far to examine Prasinovirus diversity and previous analyses suggested that 83 Ostreococcus lucimarinus viruses show no biogeographic pattern, i.e. genetic and geographic 84 distances are not correlated (4), that there is a link between genetic distances and host range 85 (11) and that trophic conditions influence Prasinovirus abundances (5). 86 However, all PolB analyses performed so far were culture-dependant, and no studies took 87 advantage of deep sequencing amplicons using the next-generation sequencing such as 454 88 technology. Current primers to amplify PolB and MCP of phycodnaviruses (9, 36) are not 89 well suited to study the prasinovirus diversity using 454-pyrosequencing. Indeed, even though 90 the present technology allows up to 700-bp to be sequenced, in practice the sequencing of 91 multiplexed amplicons is limited to 350 to 400-bp . This limitation is particularly important in 92 the case of the PolB sequence, since ~900-bp selfish genetic elements called inteins were 93 found within this gene for Phycodnaviridae, as in other eukaryotic viruses (12; 14; 19; 36; 37; 94 47; 51). These elements are inserted at the conserved amino acid motif YGDTDS between the 95 AVS primers commonly used to study the Phycodnaviridae family (3; 9; 10; 11; 41; 52). 96 Moreover, current MCP primers do not seem optimal to investigate prasinoviruses diversity, 97 as observed by Larsen et al. (36) who failed to amplify MpV, while complete genome 98 sequencing revealed the existence of MCP paralogs in Chlorovirus and Prasinovirus (17; 20; 99 21; 22; 45). 100 As a consequence, we decided to design specific primers for prasinoviruses to solve these 101 issues and by targeting suitably sized parts of their PolB or MCP genes. This study aimed (i) 102 to analyze the phylogenetic diversity of MCP paralogs towards identifying orthologous copies 103 shared between Prasinovirus and other Phycodnaviridae, in order to design a specific primer 104 set for prasinoviruses, and (ii) to compare the diversity revealed by the Prasinovirus specific 105 PolB and MCP primers. Our results suggest that the capsid-like gene copy #6 (hereafter 106 named MCP6 because it encodes the 6th gene copy along the linear OtV5 genome showing 107 similarity to the MCP (17)) is shared with other eukaryotic algae-infecting viruses, including 108 PBCV where it is has been shown experimentally to function as the major capsid protein (29). 109 While the MCP6 primers revealed a higher richness, the use of PolB gave a wider coverage of 110 the actual Prasinovirus diversity. 111 112 Materials and methods 113 114 Sampling and DNA extraction. Seawater samples were collected at 2 depths from 3 stations 115 in the Indian Ocean: at the surface (about 5 metres deep), and at the DCM (deep chlorophyll 116 maximum, from 30-72 metres deep, depending on the station). Samples were collected by 117 pumping seawater up a tube immersed to the appropriate depth at the sampling location, using 118 a large peristaltic pump (A40, TECH-POMPES, Sens, France). Twenty litres of seawater 119 were first passed through 200 µm and 20 µm mesh filters to remove larger plankton, then in 120 series through 1.6, 0.22 and 0.1 µm filters (142 mm, GF/A glass microfibre pre-filter, 121 Whatman, Ref. 1825-142; 142 mm, 0.22 µm Express PLUS Membrane, Millipore, Ref. 122 GPWP14250; 142 mm, 0.1 µm MF-Membrane, Millipore, Ref. VCWP14250, respectively) 123 using a peristaltic pump (Masterflex, EW-77410-10). The filters were kept for one month at - 124 20°C onboard and at -80°C in the laboratory until the DNA extraction. DNA extractions were 125 done only on the 0.1 µm filters (0.1-0.2 µm fraction), following a modified CTAB protocol 126 (70): (i) the filters were crushed in liquid Nitrogen, (ii) incubated at 60 °C for one hour in a 127 CTAB buffer (2 % CTAB (hexadecyltrimethylammonium bromide); 100 mM TrisHCl 128 (pH=8); 20 mM EDTA; 1.4 M NaCl; 0.2 % β-mercaptoethanol; 0.1 mg/mL proteinase K; 129 10 mM DTT (dithiothreitol), (iii) DNA were purified using an equal volume of 130 chloroform/isoamylalcohol (24 :1) and a one-hour-long-RNase digestion step, (iv) DNA were 131 precipitated with a 2/3 volume of Isopropanol and washed with 1 mL of a solution containing 132 76 % v/v EtOH and 10 mM ammonium acetate solution. Finally, the extracted DNA samples 133 were dissolved in 100 µL of laboratory grade deionized water and stored at -20 °C until the 134 sequencing steps. An approximate yield of 65 ng/µL was obtained on average for each 135 sample. 136 Primer design, PCR, sequencing and data processing. Based on the amino acid sequences 137 of the PolB and the MCP6 of OtV1 (67), OtV5 (17), OlV1, MpV1, BpV1, BpV2 (45), 138 PBCV1, PBCV_NY2A, PBCV_AR158 (20; 28; 29), ATCV1 (21), PBCV_MT325, and 139 PBCV_FR483 (22), Prasinovirus-specific primer sets consisting of VpolAS4 (5’-GAR GGI 140 GCI ACI GTI YTN GA-3’) - VpolAAS1 (5’-CCI GTR AAI CCR TAI ACI SWR TTC AT- 141 3’), and VmcpAS3 (5’-GGI GGI CAR MGI RTI GAY AA-3’) - VmcpAAS1 (5’-TGI ACY 142 TGY TCD ATI ARR TAY TCR TG-3’) were inferred and designed to amplify and sequence 143 ~320 bp and ~350-bp long fragments of the genes, respectively.