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. 2021 May 13;10(19):e00373-21. doi: 10.1128/MRA.00373-21

Genome Sequences of Microviruses Identified in a Sample from a Sewage Treatment Oxidation Pond

Simona Kraberger a,✉, Joshua Schreck a, Craig Galilee b, Arvind Varsani a,c,✉
Editor: John J Dennehyd
PMCID: PMC8142586  PMID: 33986100

Oxidation ponds are often used in the treatment of sewage as an aeration step prior to discharge. We identified 99 microvirus genomes from a sample from a sewage oxidation pond. This diverse group of microviruses expands our knowledge of bacteriophages associated with sewage oxidation pond ecosystems.

ABSTRACT

Oxidation ponds are often used in the treatment of sewage as an aeration step prior to discharge. We identified 99 microvirus genomes from a sample from a sewage oxidation pond. This diverse group of microviruses expands our knowledge of bacteriophages associated with sewage oxidation pond ecosystems.

ANNOUNCEMENT

Treated and untreated sewage or wastewater contains a multitude of microorganisms that are excreted from humans and the environment. Given the rich bacterial communities present in sewage, viruses that infect bacteria, called bacteriophages, are common (1, 2). Bacteriophages play an important role in the treatment process (3–6) by modulating bacterial populations. They have also been used as an indicator of fecal pollution (7, 8). Many studies have also identified bacteriophages from sewage as potential therapeutics against antibiotic-resistant bacteria (9, 10).

In September 2012, a 50-ml sample was collected from an oxidation pond at the Christchurch wastewater treatment plant in New Zealand (Aotearoa), transported on ice, and processed. Nucleic acids were isolated as described by Kraberger et al. (11). A total 50 ml of the sample was sequentially filtered through a 0.45-μm and 0.2-μm syringe filter, and the filtrate was precipitated with 15% (wt/vol) polyethylene glycol at 4°C overnight. The precipitate was pelleted by centrifugation at 10,000 × g for 10 min and then resuspended in 1 ml of SM buffer (0.1 M NaCl and 50 mM Tris-HCl [pH 7.4]), and 200 μl of this suspension was used to extract viral DNA using the High Pure viral nucleic acid kit (Roche Diagnostics, USA). The extracted circular viral DNA was preferentially amplified by rolling circle amplification (RCR) with the TempliPhi 100 kit (GE Healthcare, USA). The RCR products were sequenced at Beijing Genomics Institute (Hong Kong, China) on the Illumina HiSeq 2000 platform using the propriety library preparation workflow with an insert size of 170 bp with 91-bp paired-end reads. Raw reads (26,757,090) were quality trimmed with Trimmomatic v 0.39 (12) and de novo assembled using metaSPAdes v 3.12.0 (13). The eukaryotic viruses, mainly those in the phylum Cressdnaviricota (14), from this sample have been previously described by Kraberger et al. (11). To determine the prokaryotic viruses in this sample, contigs were analyzed using VirSorter 2.2.1. (15). Contigs with terminal redundancy were deemed complete circular genomes, and reads were mapped back to full genomes using BBMap (16) for verification and to determine the number of mapped reads and depth of coverage. Default parameters were used for all software unless otherwise specified. Ninety-nine full unique microvirus genomes were identified, with an average genome coverage ranging from 9- to 2,879-fold (Fig. 1).

FIG 1.

FIG 1

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Genome organization (linear depiction) of the 99 microviruses identified in this study.

Microviridae is a family of singled-stranded DNA viruses with circular genomes (17) that have been identified in a broad range of samples types and environments (18–31), including sewage (3, 32, 33). Two subfamilies have been established in the family, namely, Bullavirinae and Gokushovirinae. In this study, we aimed to expand our knowledge on microviruses in sewage by specifically looking at those in the secondary stages of sewage treatment where it is aerated in an oxidation pond prior to being dispersed into the environment.

The microvirus genomes range in length from 3,797 to 5,043 nucleotides (nt), with a GC content of ∼33% to 60% (Table 1). Open reading frames were determined using RASTtk (34), with annotation being determined using BLASTp (35) similarities with GenBank-curated microvirus RefSeq proteins. All genomes encode at least a major capsid protein (MCP) and a replication initiator protein (Fig. 1). Additional proteins were also identified in some of the genomes, which include DNA pilot proteins, nonstructural proteins, and internal scaffolding proteins. A data set of the MCP sequences from the microviruses in this study and those available in GenBank (17 February 2021) was assembled. Analysis of this data set revealed that major capsid proteins from this study share 36.9% to 98.4% pairwise amino acid identity, as determined using BLASTp (35), with all others in the data set (Table 1).

TABLE 1.

Organization summary of the 99 microvirus genomes identified from a sewage oxidation pond sample

Accession no. Isolate name Genome length (nt) GC content (%) No. of reads Coverage depth (×) Top BLASTp hit of MCPa
Accession no. % pairwise identity
MT309919 BS1_596 3,797 50.1 782 19 MK765641 42.50
MT309920 BS1_594 3,808 36 573 13 MT309929 50.50
MT309921 BS1_567 3,926 41.7 10,000 228 MH617746 59.10
MT309922 BS1_560 3,940 33.8 7,405 169 MT309982 53.40
MT309923 BS1_557 3,955 52.6 962 22 MH617134 60.60
MT309924 BS1_556 3,959 34.4 16,751 379 MT309925 67.10
MT309925 BS1_555 3,986 39.5 616 14 MT309934 67.50
MT309926 BS1_554 3,982 47 21,416 481 MH992209 55.60
MT309927 BS1_546 4,015 36.8 1,305 29 MH617584 76.70
MT309928 BS1_540 4,058 49.4 484 11 MT309925 64.30
MT309929 BS1_533 4,062 39.1 794 18 MH617567 58.00
MT309930 BS1_525 4,090 56.5 3,747 82 MH616782 59.60
MT309931 BS1_516 4,173 58.5 440 9 MH617074 69.80
MT309932 BS1_515 4,119 36.2 874 23 MH992221 57.90
MT309933 BS1_508 4,136 42.6 956 21 MT309925 60.70
MT309934 BS1_502 4,214 43.9 710 15 MT309925 67.70
MT309935 BS1_501 4,165 39.4 22,154 477 MK765646 52.70
MT309936 BS1_499 4,175 41.6 48,158 1,037 MK496825 84.30
MT309937 BS1_492 4,243 47.9 515 11 MT309925 63.70
MT309938 BS1_489 4,219 42 40,633 863 MT309937 54.70
MT309939 BS1_487 4,265 42.5 136,661 2,879 KP823396 83.40
MT309940 BS1_485 4,269 43.2 804 17 MK496822 76.30
MT309941 BS1_484 4,248 42.8 8,148 175 MT310016 88.40
MT309942 BS1_481 4,245 42 22,246 470 MH617700 63.40
MT309943 BS1_477 4,253 40.3 21,294 450 MH572427 85.70
MT309944 BS1_474 4,263 41.7 73,932 1,560 MH572292 87.20
MT309945 BS1_471 4,307 55.8 594 12 MH572285 63.30
MT309946 BS1_466 4,271 47.8 1,971 41 MH616977 65.70
MT309947 BS1_462 4,295 41.8 63,579 1,333 MT310014 90.80
MT309948 BS1_450 4,311 49.8 1,556 32 MH510271 84.20
MT309949 BS1_449 4,312 40.8 6,084 126 MH572283 81.80
MT309950 BS1_448 4,316 46.8 3,066 65 MH992170 79.10
MT309951 BS1_446 4,319 36.9 712 15 MH992221 51.80
MT309952 BS1_441 4,383 53 453 9 MH617103 62.40
MT309953 BS1_440 4,380 51.5 880 18 MH617138 66.50
MT309954 BS1_438 4,373 41.5 22,096 453 MT310014 85.50
MT309955 BS1_432 4,346 44.4 633 13 MN582062 70.20
MT309956 BS1_426 4,359 52.8 1,141 23 MT309988 79.90
MT309957 BS1_425 4,360 50.3 8,885 183 MH510271 78.20
MT309958 BS1_423 4,362 48.1 1,299 27 MT309957 74.70
MT309959 BS1_415 4,385 46.9 796 16 MT309983 59.90
MT309960 BS1_413 4,386 42.2 3,251 67 MK496825 74.80
MT309961 BS1_412 4,387 51.4 1,428 29 MH622930 89.80
MT309962 BS1_410 4,394 41.1 2,703 55 MH617735 72.80
MT309963 BS1_408 4,415 47 435 9 MT309928 44.40
MT309964 BS1_401 4,412 47.9 6,700 137 MT309999 75.90
MT309965 BS1_400 4,437 48.1 43,718 884 MT309968 87.40
MT309966 BS1_399 4,414 47.7 600 12 MT309984 67.50
MT309967 BS1_398 4,414 41.2 692 14 MK765582 58.30
MT309968 BS1_397 4,415 47.9 2,023 41 MT309965 87.40
MT309969 BS1_387 4,439 37.7 10,731 219 MH572285 48.48
MT309970 BS1_386 4,442 47.8 487 10 MH617685 77.60
MT309971 BS1_385 4,442 46.1 1,191 24 MT309978 62.80
MT309972 BS1_384 4,443 57.5 1,379 28 MK249220 59.60
MT309973 BS1_380 4,451 37.5 1,103 22 MT309992 72.70
MT309974 BS1_378 4,453 49 1,020 21 MK249212 72.10
MT309975 BS1_377 4,453 47.2 1,435 29 MH617260 67.20
MT309976 BS1_373 4,462 45.2 1,485 30 MK249225 68.30
MT309977 BS1_372 4,463 38.8 1,090 22 MT309935 47.50
MT309978 BS1_370 4,516 43 702 14 MT309984 73.50
MT309979 BS1_368 4,474 38.5 9,022 181 MH992203 66.10
MT309980 BS1_367 4,481 49 42,406 853 MT310017 89.50
MT309981 BS1_364 4,487 59.7 2,315 46 MH552548 62.40
MT309982 BS1_361 4,494 35.1 92,459 1852 MT309922 53.40
MT309983 BS1_360 4,497 44.3 3,457 69 MK249212 66.90
MT309984 BS1_352 4,516 43.6 731 15 MT309978 73.50
MT309985 BS1_351 4,516 47 2,031 41 MK249219 82.30
MT309986 BS1_350 4,519 48.9 4,940 98 MT310017 81.20
MT309987 BS1_348 4,524 58.1 26,775 532 MK249179 80.60
MT309988 BS1_347 4,555 53.1 854 17 MH616807 76.90
MT309989 BS1_340 4,539 47.7 872 17 MT310007 65.50
MT309990 BS1_339 4,591 58 1,020 20 MH617422 70.00
MT309991 BS1_336 4,546 46.9 1,163 23 MT309961 62.60
MT309992 BS1_330 4,552 36.9 16,467 325 MT309973 72.70
MT309993 BS1_328 4,561 49.5 1,440 28 KP087956 49.40
MT309994 BS1_322 4,632 52.6 9,817 190 MK249189 70.80
MT309995 BS1_320 4,578 53.2 1,410 28 MK249190 68.00
MT309996 BS1_313 4,587 46 2,754 54 MH617260 67.80
MT309997 BS1_312 4,589 47.3 906 18 MH992193 61.70
MT309998 BS1_311 4,591 48.6 5,310 104 MH617374 72.40
MT309999 BS1_308 4,596 47.5 1,450 28 MT309963 75.90
MT310000 BS1_305 4,605 48.9 1,519 30 MT309980 82.30
MT310001 BS1_300 4,620 50.9 1,239 24 MK249178 79.40
MT310002 BS1_295 4,635 49.1 702 14 MK249163 78.10
MT310003 BS1_294 4,636 46.9 2,299 45 MH572492 62.30
MT310004 BS1_286 4,666 49.1 6,498 125 MT309966 60.80
MT310005 BS1_285 4,683 33.3 633 14 MH617644 36.90
MT310006 BS1_280 4,701 48.8 3,097 59 MH616638 85.50
MT310007 BS1_270 4,727 45.8 811 15 MT309989 65.00
MT310008 BS1_264 4,765 51.7 1,699 32 MH616735 71.64
MT310009 BS1_260 4,789 50.7 1,003 19 MH572441 66.70
MT310010 BS1_250 4,918 43.1 2,415 44 MH992183 62.60
MT310011 BS1_235 5,009 33.8 855 17 MH617129 54.60
MT310012 BS1_228 5,043 46.1 777 14 KT264751 98.40
MT310013 BS1_206 4,600 46.7 67,641 1,324 MH616922 78.10
MT310014 BS1_34 4,361 43.1 17,779 366 MT309947 90.80
MT310015 BS1_33 4,363 42.1 38,905 803 MH572427 78.90
MT310016 BS1_31 4,230 42.6 8,662 185 MT309941 88.40
MT310017 BS1_11 4,520 48.7 41,024 819 MT309980 89.50
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a

MCP, major capsid protein.

This study adds to the growing data on microviruses from various ecosystems and certainly shows that this group of viruses is diverse with varied genome organization.

Data availability.

The sequences of microviruses in this study have been deposited in the NCBI SRA under project SRR11451582 and GenBank accession numbers MT309919 to MT310017.

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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 sequences of microviruses in this study have been deposited in the NCBI SRA under project SRR11451582 and GenBank accession numbers MT309919 to MT310017.

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