Folding of the 5′ and 3′ NCRs of the DpCPV-MC genome segments.

<p>The RNAfold program was used to predict the secondary structures formed by the 16 DpCPV-MC genome segments. The panhandle structure was formed by base-pairing between the 5′ and 3′ ends, and the stem–loop structure was formed by either the 5′ or 3′ terminal sequence. The panhandle structure and the stem–loop structure are marked with long brackets.</p

Neighbor-joining tree constructed from polyhedrin amino acid sequences of representative CPVs.

<p>Sequences were aligned with the multiple sequence alignment program ClustalX2. The neighbor-joining method was used to construct the phylogenetic tree of the derived polyhedrin protein sequences with the MEGA software version 5.2. The black triangle shows the position of DpCPV-MC. Bootstrap percentage values are indicated on the left. All reference sequences used for the construction of this tree were retrieved from GenBank with their corresponding accession numbers: <i>Antheraea assamensis cypovirus 4</i>, AY212275; <i>Antheraea mylitta cypovirus 4</i>, AY212273; <i>Antheraea proylei cypovirus 4</i>, AY212276; <i>Dendrolimus punctatus cypovirus 1</i>, AY204879; <i>Bombyx mori cypovirus 1</i>, D37770; <i>Culex restuans cypovirus 17</i>, DQ212785; <i>Choristoneura occidentalis cypovirus 16</i>, EU201043; <i>Euxoa scandens cypovirus 5</i>, J04338; <i>Heliothis assulta cypovirus 14</i>, DQ077914; <i>Heliothis armigera cypovirus 5</i>, DQ077912; <i>Lymantria dispar cypovirus 1</i>, AF389471; <i>Operophtera brumata cypovirus 18</i>, DQ192250; <i>Operophtera brumata cypovirus 19</i>, DQ192254; <i>Simulium ubiquitum cypovirus 20</i>, DQ834386; <i>Trichoplusia ni cypovirus 15</i>, NC_002565; <i>Uranotaenia sapphirina cypovirus 17</i>, AY876384; <i>Lymantria dispar cypovirus 14</i>, AF389461; <i>Choristoneura fumiferana cypovirus 16</i>, U95954.</p

Primers used in this study.

<p>Primers used in this study.</p

Properties of the dsRNA segments of DpCPV-MC.

<p>Properties of the dsRNA segments of DpCPV-MC.</p

Electron micrographs of OBs of DpCPV-MC.

<p>(A) Transmission electron micrograph of ultrathin sections of OBs of DpCPV-MC. (B) Transmission electron micrograph of purified virions of DpCPV-MC.</p

Median lethal concentrations (LC₅₀) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.

a<p>Potency ratio was calculated by dividing the LC₅₀ of DpCPV-MC by that of DpCPV-1. Significant difference was based on whether the 95% confidence interval (CI) of the potency ratio included the value 1.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113201#pone.0113201-Robertson2" target="_blank">[47]</a>.</p><p>Median lethal concentrations (LC₅₀) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.</p

Southern blot analysis of genomic DNA of <i>S. exigua</i> larvae.

<p>A. Electrophoresis (left) and southern blot (right) of restriction endonuclease-digested genomic DNA of <i>S. exigua</i> larvae, detected by pheromone-binding protein 3 (PBP3) probe. Marker, phage λ DNA digested with <i>Eco</i>T14 I. XhoI, <i>Xho</i>I-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). BamHI, <i>Bam</i>HI-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). The arrows indicate hybridization bands in genomic DNA of <i>S. exigua</i> larvae. B. Electrophoresis (left) and southern blot (right) of restriction endonuclease-digested genomic DNA of <i>S. exigua</i> larvae, detected by probe mixture of six DpCPV-MC dsRNA segments (S6, S10, S11, S14, S15 and S16). The six plasmids containing full-length sequence of six segments was used as positive control. Marker, phage λ DNA digested with <i>Eco</i>T14 I. XhoI, <i>Xho</i>I-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). BamHI, <i>Bam</i>HI-digested genomic DNA of <i>S. exigua</i> larvae (2 µg). pMD18-T-S6, pMD18-T vector containing full-length sequence of S6 (2 ng). pMD18-T-S10, pMD18-T vector containing full-length sequence of S10 (2 ng). pMD18-T-S11, pMD18-T vector containing full-length sequence of S11 (2 ng). pMD18-T-S14, pMD18-T vector containing full-length sequence of S14 (2 ng). pMD18-T-S15, pMD18-T vector containing full-length sequence of S15 (2 ng). pMD18-T-S16, pMD18-T vector containing full-length sequence of S16 (2 ng).</p

Northern blot analysis of six dsRNA segments of DpCPV-MC.

<p>Total genomic dsRNA of DpCPV-MC was separated on 1% agarose gel, transferred to nylon membrane, and hybridized with the corresponding DIG-labeled probes derived from each of the six segment sequences. The left part is agar gel stained with ethidium bromide. M, phage λ DNA digested with <i>Eco</i>T14 I. The right six lanes are northern blot results.</p