ABSTRACT
Sharp-spined notothen (Trematomus pennellii) is an icefish endemic to the southern ocean. From the stomach of an individual, we identified the genomes of 51 microviruses (family Microviridae). The major capsid proteins of most of these share the closest similarities to those identified in other marine organisms.
ANNOUNCEMENT
The sharp-spined notothen (Trematomus pennellii) is a member of the Nototheniidae family and endemic to the southern ocean (1). There is limited information on viruses associated with Antarctic fish in general. Prior research on Antarctic fish identified a papillomavirus in emerald notothen (Trematomus bernacchii) (2) and two polyomaviruses in an emerald notothen and a sharp-spined notothen (3, 4).
A sharp-spined notothen was caught in the McMurdo Sound (Ross Sea, Antarctica) using traditional hook and line fishing during the 2012–2013 austral summer. The fish was caught under the 2011/08R animal ethics permit (University of Canterbury, New Zealand). Approximately 0.5 cm3 of stomach was dissected and homogenized in 20 mL of SM buffer (0.1 M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM MgSO4) with mortar and pestle. The homogenate was centrifuged at 6,000 × g for 10 min to pellet cell debris. The supernatant was sequentially filtered through 0.45- and 0.22-μm syringe filters, and viral particles in the filtrate were precipitated with 15% (wt/vol) polyethylene glycol 8000 (PEG 8000). The resulting solution was centrifuged at 6,000 × g for 20 min, and the pellet was resuspended in 1 mL of SM buffer. Viral DNA was extracted from 200 μL solution using the High Pure viral nucleic acid kit (Roche Diagnostics, Germany). Circular DNA was preferentially amplified using rolling circle amplification (RCA) with a TempliPhi 100 kit (GE Healthcare, USA). A library using the TruSeq Nano DNA kit (Illumina, USA) was prepared from the RCA products and sequenced on an Illumina HiSeq 4000 sequencer at Macrogen Inc. (Korea). The sequence reads (23,631,809 paired-end reads; average read length, 101 nucleotides [nt]) were quality trimmed using Trimmomatic v0.39 (5) and then de novo assembled using metaSPAdes v3.14 (6). Circular genomes were identified based on terminal redundancy. All bioinformatic tools were run with default parameters.
Circular contigs (>1,000 nt in length) were analyzed using BLASTx (7) for viral-like sequences using a RefSeq viral protein database (RefSeq release 207). We identified 51 circular contigs (4,012 to 5,250 nt) with similarities to the microvirus proteins (family Microviridae). Microviruses are small circular single-stranded DNA bacteriophages in the order Petitvirales and phylum Phixviricota (8). These 51 microvirus genomes have coverage depth of 8.9× to 280.4×, number of mapped reads of 375 to 8,809, and GC content of 36.4 to 55.3% (Fig. 1). VIBRANT (9) was used to annotate the genomes with additional verification with BLASTp (7) similarity searches. Of these genomes, 82% have a similar organization of the relatively conserved open reading frames, i.e., major capsid protein, DNA pilot protein, replication initiator protein, internal scaffolding protein, and nonstructural protein (Fig. 1). The major capsid protein is the most conserved protein in all microviruses, and BLASTp analysis revealed that majority shared similarities with sequences of microviruses of marine organisms, i.e., abalone tissue (n = 1), crucian tissue (n = 1), red snapper tissue (n = 1), haddock tissue (n = 4), minnow tissue (n = 5), and sea quirt (n = 30), while there was a small number shared with those from cold methane seep sediment (n = 1), robin feces (n = 1), human gut sample (n = 1), lake water sample (n = 1), and sewage oxidation pond, sludge, and wastewater (n = 5) (Table 1) (10–15). The BLASTp hits ranged from 41% to 83% (Table 1). It is highly likely that the 51 microviruses infect gut-associated bacteria of the sharp-spined notothen or their diet.
FIG 1.
Genome organization of the 51 microviruses identified in the stomach of a sharp-spined notothen caught in the Ross Sea in east Antarctica. A summary of the GC%, read depth, and number of reads is provided for each genome.
TABLE 1.
Summary of the top BLASTx hit for the MCP of the 51 microviruses described in this study
| Query sequence (GenBank accession no.) | Data for major capsid protein top BLAST hit |
|||
|---|---|---|---|---|
| Best hit MCP (GenBank accession no.) | Virus | Microvirus source | % identity | |
| OP376967 | MH572390 | Microviridae sp. strain SD_SF_42 | Ciona robusta intestinal tract | 83.3 |
| OP376968 | MH572372 | Microviridae sp. strain SD_SC_58 | Ciona robusta intestinal tract | 56.4 |
| OP376969 | MH572461 | Microviridae sp. strain SD_MC_53 | Ciona robusta intestinal tract | 56.4 |
| OP376970 | MH572325 | Microviridae sp. strain SD_SC_10 | Ciona robusta intestinal tract | 72.3 |
| OP376971 | MT310128 | Microviridae sp. strain 6424_116 | Wastewater | 57.3 |
| OP376972 | MH572453 | Microviridae sp. strain SD_MC_6 | Ciona robusta intestinal tract | 72.0 |
| OP376973 | MH572365 | Microviridae sp. strain SD_SC_34 | Ciona robusta intestinal tract | 68.5 |
| OP376974 | MH616998 | Microviridae sp. strain ctdc857 | Minnow tissue | 61.1 |
| OP376975 | MH617576 | Microviridae sp. strain ctcc822 | Minnow tissue | 58.4 |
| OP376976 | MH572289 | Microviridae sp. strain SD_SC_80 | Ciona robusta intestinal tract | 74.1 |
| OP376977 | MH572441 | Microviridae sp. strain SD_MC_80 | Ciona robusta intestinal tract | 66.4 |
| OP376978 | MH617039 | Microviridae sp. strain ctda820 | Minnow tissue | 66.2 |
| OP376979 | MH572493 | Microviridae sp. strain SD_HF_33 | Ciona robusta intestinal tract | 61.4 |
| OP376980 | MT310213 | Microvirus sp. strain 1712115_898 | Sludge | 63.7 |
| OP376981 | MH616888 | Microviridae sp. strain cthi64 | Haddock tissue | 63.2 |
| OP376982 | MH617651 | Microviridae sp. strain ctcc35 | Red snapper tissue | 63.0 |
| OP376983 | MH572299 | Microviridae sp. strain SD_SC_76 | Ciona robusta intestinal tract | 66.0 |
| OP376984 | MT310091 | Microviridae sp. strain 6434_67 | Wastewater | 64.2 |
| OP376985 | MT309961 | Microviridae sp. strain BS1_412 | Sewage oxidation pond | 60.2 |
| OP376986 | MH572474 | Microviridae sp. strain SD_MF_21 | Ciona robusta intestinal tract | 62.5 |
| OP376987 | MH572345 | Microviridae sp. strain SD_SC_53 | Ciona robusta intestinal tract | 65.0 |
| OP376988 | MZ364287 | Robinz microvirus RP_160 | Feces of robin | 67.4 |
| OP376989 | MH572299 | Microviridae sp. strain SD_SC_76 | Ciona robusta intestinal tract | 72.7 |
| OP376990 | MH572365 | Microviridae sp. strain SD_SC_34 | Ciona robusta intestinal tract | 67.9 |
| OP376991 | MT310030 | Microviridae sp. strain 6538_71 | Wastewater | 61.3 |
| OP376992 | MH572501 | Microviridae sp. strain SD_HF_34 | Ciona robusta intestinal tract | 66.0 |
| OP376993 | MH616888 | Microviridae sp. strain cthi64 | Haddock tissue | 69.6 |
| OP376994 | MH616888 | Microviridae sp. strain cthi64 | Haddock tissue | 70.9 |
| OP376995 | MH552548 | Microviridae sp. strain ctjb11 | Abalone tissue | 57.8 |
| OP376996 | MH572501 | Microviridae sp. strain SD_HF_34 | Ciona robusta intestinal tract | 63.8 |
| OP376997 | MH572501 | Microviridae sp. strain SD_HF_34 | Ciona robusta intestinal tract | 66.9 |
| OP376998 | MH572299 | Microviridae sp. strain SD_SC_76 | Ciona robusta intestinal tract | 72.9 |
| OP376999 | MH572493 | Microviridae sp. strain SD_HF_33 | Ciona robusta intestinal tract | 61.8 |
| OP377000 | MH572365 | Microviridae sp. strain SD_SC_34 | Ciona robusta intestinal tract | 68.8 |
| OP377001 | MH572289 | Microviridae sp. strain SD_SC_80 | Ciona robusta intestinal tract | 59.3 |
| OP377002 | MH622906 | Microviridae sp. strain ctbj815 | Minnow tissue | 65.0 |
| OP377003 | MH617086 | Microviridae sp. strain ctcc904 | Minnow tissue | 64.4 |
| OP377004 | MH572365 | Microviridae sp. strain SD_SC_34 | Ciona robusta intestinal tract | 67.9 |
| OP377005 | MH616888 | Microviridae sp. strain cthi64 | Haddock tissue | 65.2 |
| OP377006 | MK012456 | Microviridae sp. strain ctcb6 | Crucian tissue | 63.4 |
| OP377007 | MW697696 | Arizlama microvirus AZLM_250 | Lake water sample | 68.3 |
| OP377008 | MH572481 | Microviridae sp. strain SD_MF_9 | Ciona robusta intestinal tract | 73.4 |
| OP377009 | MH572451 | Microviridae sp. strain SD_MC_7 | Ciona robusta intestinal tract | 73.2 |
| OP377010 | MH572325 | Microviridae sp. strain SD_SC_10 | Ciona robusta intestinal tract | 67.9 |
| OP377011 | MH572451 | Microviridae sp. strain SD_MC_7 | Ciona robusta intestinal tract | 71.5 |
| OP377012 | MH572337 | Microviridae sp. strain SD_SC_2 | Ciona robusta intestinal tract | 66.9 |
| OP377013 | MH572451 | Microviridae sp. strain SD_MC_7 | Ciona robusta intestinal tract | 64.4 |
| OP377014 | MH572451 | Microviridae sp. strain SD_MC_7 | Ciona robusta intestinal tract | 67.4 |
| OP377015 | MH572451 | Microviridae sp. strain SD_MC_7 | Ciona robusta intestinal tract | 72.1 |
| OP377016 | BK051953 | Microviridae sp. strain cttI53 | Human metagenome | 53.7 |
| OP377017 | KP087943 | Eel River Basin pequenovirus | Cold methane seep sediment | 41.4 |
Data availability.
The microvirus sequences have been deposited in NCBI databases under BioProject accession number PRJNA874327, BioSample accession number SAMN30543052, SRA accession number SRR21284442, and GenBank accession numbers OP376967 to OP377017.
ACKNOWLEDGMENTS
The field work in the Ross Sea was supported by a grant (K057) awarded to W.D. from Antarctica New Zealand. The molecular work described in this study is supported by a Center of Evolution and Medicine Venture Fund (Center of Evolution and Medicine, Arizona State University, USA) grant awarded to A.V.
Contributor Information
Arvind Varsani, Email: arvind.varsani@asu.edu.
John J. Dennehy, Queens College Department of Biology
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The microvirus sequences have been deposited in NCBI databases under BioProject accession number PRJNA874327, BioSample accession number SAMN30543052, SRA accession number SRR21284442, and GenBank accession numbers OP376967 to OP377017.