Water samples were collected monthly between May 2012 and April 2013 from four sites along East Lake in Wuhan, China. The environmental characteristics of the sampling sites are described in our previous report(Xia et al., 2013).
To quantify virus concentration, 100-L water samples were collected from the surface to a depth of 0.5 m at the four sites. Water samples were then mixed, transported to the laboratory in polyethylene bottles, and treated within 2 h, as previously described(Thurber et al., 2009). In brief, to remove zooplankton, phytoplankton, and large particles, the sample was serially prefiltered through 16 layers of gauze and a plankton net(200 μm mesh size). To remove bacteria, the preliminarily filtered water was then filtered with 1-and 0.45-μm nominal pore size polycarbonate filters using a peristaltic pump. The filtrate was concentrated approximately 50–100-fold to a final volume of 200–250 mL using a 10 kDa-cutoff tangential-flow ultrafiltration system(Millipore, USA). Particles of less than 0.45 μm were further concentrated by ultrafiltration at 111, 000 × g for 2 h at 4℃(Beckman Coulter Optima, SW-28, USA) and resuspended with 100–200 μL precooled phosphate buffer saline. The prepared viral concentrates( < 0.45 μm)were stored immediately in the dark at −80 ℃ until further processing.
Primer sets targeting the genes encoding minor capsid assembly proteins(g20)of cyanomyoviruses(Sullivan et al., 2008), DNA polymerases(polAL)of cyanopodoviruses(Chen et al., 2009), and DNA polymerases(polB)or major capsid proteins(mcp)of phycodnaviruses(Chen and Suttle, 1995; Larsen et al., 2008)were used in this study. No conserved gene has been found for the five known cyanosiphovirus genomes. Based on published data and sequences obtained from our previous metagenomic data(unpublished), three degenerate primer sets separately targeting ribonucleotide reductase genes(RNR), large terminase subunit genes(terL), and major capsid protein genes(mcp)of cyanosiphoviruses were designed(Table 1). These three primer sets covered the three subtypes of cyanosiphovirus, i.e., S-CBS4, S-CBS2/PSS2, and S-CBS1/S-CBS3, respectively(Huang et al., 2012).
Gene Virus Primer name Sequence (5'-3') TM1/TM2 (℃) References g20 Cyanomyovirus CPS1.1 F
35/46 (Sullivan et al., 2008) polA Cyanopodovirus CP-DNAP-349F
48/58 (Chen et al., 2009) RNR S-CBS4 subtype RNR F
43/55 This study terL S-CBS1/S-CBS3 subtype TerL F
37/50 This study mcp S-CBS2/PSS2 subtype MCP F
42/56 This study mcp Phycodnavirus mcp F
45/58 (Larsen et al., 2008) polB Phycodnavirus AVS1
40/52 (Chen and Suttle, 1995) Note: g20: minor capsid assembly protein genes of cyanomyoviruses; polA: DNA polymerase I genes of cyanopodoviruses; RNR, terL, and mcp: ribonucleotide reductase genes, large terminase subunit genes, and major capsid protein genes of cyanosiphoviruses, respectively; polB and mcp: DNA polymerase genes of family B and major capsid protein genes of phy-codnavirus, respectively
Table 1. Primer sets used for amplification of algal virus-like sequences from East Lake.
Viral concentrates were digested with 0.1 U DNase(Promega, China) and 0.1 μg RNase(Fermentas, LTU)to remove host nucleic acid, according to our previously reported protocol(Xia et al., 2013). Fifty microliters of viral DNA extracted from a 140-μL sample using the QIAamp MinElute Virus Spin Kit(Qiagen, Germany)was used for detection. PCR was performed in a 50-μL reaction mix containing 5–10 ng nucleic acid for each sample, 25 × Mix Buffer, and 10 pmol of each primer(Beijing CoWin Bioscience Co., Ltd., China). Negative controls were set up using 1 μL of nuclease-free water as a template. A touch-up PCR program with a lower annealing temperature was used to improve the amplification efficiency. The PCR amplifications were carried out with a thermocycler(Bio-Rad, Hercules, CA, USA)with PCR programs as follows: denaturing at 95 ℃ for 5 min, followed by 45 cycles of denaturing at 95 ℃ for 30 s, annealing for 45 s, and extension at 72 ℃ for 1 min, with a final extension at 72 ℃ for 10 min. Different annealing temperatures were used for different primer sets. The annealing temperature was ramped from TM1 by 0.5 ℃ every cycle for first 20 cycles, followed by 25 cycles at constant annealing temperature TM2(Table 1). Each reaction was optimized to produce positive clones from the East Lake samples. The PCR products were analyzed by electrophoresis onto 1.5% agarose gels and stained with ethidium bromide. For sequencing, PCR products were excised from the agarose gel, purified with Gel Extraction Kits(OMEGA EZNA, Omega Bio-tek, USA), and cloned to T-vectors using the pGEM-T Easy System(Promega, Madison, WI, USA)following the manufacturer's instructions. About 10–15 r and om positive clones from each PCR product with expected sizes were sequenced with a universal primer set(M13)at Sangon Biotech(Shanghai, China).
The sequences were analyzed using BLAST at the NCBI website(http://blast.ncbi.nlm.nih.gov/Blast.cgi). All sequences were deposited in GenBank with accession numbers: KP775010–KP775086, KP775191–KP775342, and KP775408–KP775536. Multiple sequence alignments of amino acid consensus sequences were carried out using ClustalW and the Gonnet protein weight matrix.
Amino acid sequences from the same clone library with identities over 99% were removed before further analysis. Phylogenetic reconstruction was conducted using molecular evolutionary genetic analysis software(MEGA 5.0)(Kumar et al., 2008)with the neighbor joining(NJ)algorithmic method with 1000-fold bootstrap support and maximum likelihood(ML)phylogeny with 100 bootstrap replicates. Both NJ and ML methods for the seven genes were determined by the Jones-Taylor-Thornton(JTT)model and gamma-distributed rate heterogeneity among amino acid substitution(Zhong and Jacquet, 2013).