Dataset: Circovirus DNA Sequence Information
Deployment: Tampa_Bay_Seawall_Breitbart

Zooplankton were collected using a 335-um plankton net, and copepods were individually picked from the bulk zooplankton samples, washed three times in 100-kDa filtered seawater, and incubated overnight in a fecatron to allow for gut clearing. After gut clearing, the copepods were rinsed three times in 100-kDa filtered seawater and then frozen at -80 deg C until further processing. For viral metagenomic analysis, L. aestiva and A. tonsa were collected from Bayboro Harbor in April 2009 and May 2010, respectively. For qPCR detection, L. aestiva were collected from multiple locations, including Bayboro Harbor, Eckerd Pier, the mouth of the Alafia River, and Fort Desoto Beach. To determine the temporal dynamics of viral infection in A. tonsa, samples were collected from Bayboro Harbor once a month throughout 2011.

Virus particles were purified from each copepod species based on size, density, and nuclease resistance, and viral metagenomes were sequenced according to standard protocols The copepods were homogenized in sterile SM buffer (50 mM TrisCl, 10 mM MgSO4, 0.1 M NaCl; pH 7.5), centrifuged at 10,000 x g for 10 min at 4 deg C to pellet animal tissues, and passed through a 0.22-um filter to remove bacteria and animal cells. The A. tonsa filtrate was also loaded onto a cesium chloride step gradient with 1 mL each of 1.2, 1.5, and 1.7 g mL-1 in SM buffer. After ultracentrifugation at 61,000 x g for 3 h at 4 deg C, the viral fraction (between the 1.2- and 1.5-g mL-1 density layers) was collected, then concentrated and washed twice on a Microcon YM-30 column (Millipore). Both the A. tonsa and L. aestiva viral fractions were treated with 0.2 volumes of chloroform for 10 min, and then incubated with 2.5 U DNase I per uL of sample for 3 h to eliminate free nucleic acids. After the reaction was stopped by incubation at 65 deg C for 10 min, viral DNA was extracted with the QIAmp MinElute Virus Spin Kit (Qiagen) and amplified with the strand-displacement method of the Genomiphi V2 DNA Amplification Kit (GE Healthcare). The GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich) was used to fragment and amplify the DNA, which was then cloned into the pCR4 vector using TOPO TA cloning (Invitrogen). A total of 38 transformants were sequenced for each copepod species using dideoxynucleotide sequencing, and the resulting metagenomic sequences were analyzed using tBLASTx against the GenBank nonredundant database. In this study, the main purpose of sequencing these small viral metagenomes was to identify putative viral targets for further quantitative ecological study.

Several sequences from the viral metagenomes of both A. tonsa and L. aestiva had tBLASTx similarities to viruses in the Circoviridae family. Given that known circoviruses have small circular genomes, back-to-back PCR primers (L. aestiva primers: 5'-CACCAGCAACTACAGCATCAA-3' and 5'-GTGACTATGATCCGCTTGGG-3'; A. tonsa primers: 5'-ACGAAGTAGCGCTCGAACTG-3' and 5'-CGTGAACTACGCTGGTCGTA-3') were designed from the metagenomic sequences using Primer 3 to amplify the complete circular genome of the copepod circo-like viruses directly from unamplified copepod DNA extracts through inverse PCR. The PCR reactions [containing 1 uM of each primer, 200 uM dNTPs, 1 U RedTaq DNA Polymerase (Sigma-Aldrich), 1 x Red Taq Reaction Buffer, and 5 uL of target DNA in a 50-uL reaction] were amplified as follows: 95 deg C for 5 min; 45 cycles of 94 deg C for 1 min, 58 deg C minus 0.2 deg C per cycle for 1 min, and 72 deg C for 3 min; and a final extension at 72 deg C for 10 min. The resulting whole genome PCR products were cloned into the pCR4 vector using TOPO TA cloning and sequenced to 3 x coverage. ORFs were predicted and annotated using SeqBuilder (DNASTAR), and stem- loop structures were manually annotated by locating complementary sections.