(A) Results of real-time PCR with specific primers. (B) Real-time PCR of C005 (left) and C112 (right). (A) Specific primers for C005 (lanes 1 and 2) and C112 (lanes 3 and 4) produced 438- and 380-bp amplicons, respectively. Viral DNA extracted from concentrated virus preparations (lanes 1 and 3) and DNA amplified by MDA without a denaturing step (lanes 2 and 4) were used as templates. Lane L, 100-bp ladder. (B) Circle a, DNA after MDA without the denaturing step (used for the RX library); circle b, DNA after MDA with the denaturing step (RH library); circle c, DNA before MDA. Similar concentrations of DNA (1.4, 1.3, and 1.0 ng/μl for circles a, b, and c, respectively) were used as templates. Nonlinear points indicated by red filled circles in the left-hand standard curve were not included in the standard curve. R² values are 0.982 (left) and 0.988 (right).

Virus-like particles (A) and bacteria (B) from rice paddy soil stained with Sybr gold and observed by EFM. Scale bars, 5 μm. (A) No bacterial contamination is observed.

Genome organizations of the three putative circular genomic components reconstructed from soil viral metagenomic sequences. The components show a genomic organization similar to that of Porcine circovirus2 (PCV2), a representative circovirus.

(A) Preferential amplification of circular DNA during MDA without the denaturing step. (a) Plasmids left uncut (circular DNA) and cut with the Eam1105I restriction enzyme (linear DNA). Lane 1, S11 and uncut DNA; lane 2, S11 and cut DNA; lane 3, S36 and uncut DNA; lane 4, S36 and cut DNA; lane L1, 1,000-bp ladder. (b) PCR products amplified from plasmids cut with Eam1105I. Specific primers for S11 (lane 5) and S36 (lane 6) produced 391- and 378-bp amplicons, respectively. Lane L1, 100-bp ladder. (B) Quantification of circular and linear DNAs during MDA without the denaturing step. A 0.037-ng/μl concentration of each circular or linear DNA was mixed with 10 ng/μl lambda DNA, and MDA was performed without the heating step. Concentrations of circular and linear DNAs were estimated by real-time PCR. Filled circles, circular S11; filled squares, circular S36; empty circles, linear S11; empty squares, S16. Circular DNA was amplified much more efficiently than linear DNA. x axis, time in minutes; y axis, DNA concentration in picograms per microliter.

Phylogenetic trees of the amino acid sequences from the Rep_Viral (A) and Gemini_AL3 (B) domains obtained in this study and related members with those domains. The alignment lengths for tree construction were 75 and 108 amino acids for trees A and B, respectively. The scale bars indicate the distance score calculated with the scoring matrix BLOSUM62. The prefix C in the sequence name means that the sequence is from a contig, and the prefixes RH or RX indicates the library where the sequence was retrieved. (A) Reference sequences: CFDV, Coconut foliar decay virus (accession no. Q66005); SCSVa, -b, and -c, Subterranean clover stunt virus (accession no. Q87009, Q87013, and Q9ICP7); BBTVa, -b, and -c, Banana bunchy top virus (accession no. Q8QTK9, Q83026, and Q65378); PCV2, Porcine circovirus 2 (accession no. Q8BB16); GCV, Goose circovirus (accession no. Q8AYY2); CCV, Canary circovirus (accession no. Q912W1); CuCV, Columbid circovirus (accession no. Q91GA3); EHa, -b, and -c, Entamoeba histolytica HM-1 (accession no. XP_648754, XP_648748, XP_650115); CV, Canarypox virus (accession no. NP_955176); AYVV-aDNA1, Ageratum yellow vein virus-associated DNA 1 (accession no. Q8QME8); MVDVa and -b, Milk vetch dwarf virus; FBNYVa, -b, -c, Fava bean necrotic yellows virus (accession no. O91250, O91250, and Q66862); TCSV-aDNA1, Tobacco curly shoot virus-associated DNA 1 (accession no. Q7T7M5); GI, Giardia intestinalis (accession no. Q9NJY0); p4M, plasmid of Bifidobacterium pseudocatenulatum (accession no. NP_613078). (B) Reference sequences: CSMV, Chloris striate mosaic virus (accession no. P18921); PSV, Panicum streak virus (accession no. Q00338); MSV, Maize streak virus (accession no. P03568); WDV, Wheat dwarf virus (accession no. P06847); TYDV, Tobacco yellow dwarf virus (accession no. P31617); TYLCV, Tomato yellow leaf curl virus (accession no. P36279); PHYVV, Pepper huasteco yellow vein virus (accession no. Q06923); BGMV, Bean golden mosaic virus (accession no. P05175), ACMV, African cassava mosaic virus (accession no. P14982); SLCV, Squash leaf curl virus (accession no. P2904); BCTV, Beet curly top virus (accession no. P14991); pPASa and -b and pPAUa and -b, plasmids of Candidatus P. asterisasteris (accession no. Q2NIE5, Q2NIE4, Q0QLC1, and Q0QLC5). For the accession numbers of sequences from this study in the figures and alignments of the translated sequences, see the supplemental material.