Additional file 1: Table S1. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

Predicted ORFs and repeat regions in the genome of DapuNPV. This file lists the ORFs predicted (with its direction and promoter sequences) in the genome of DapuNPV and their homologues in 15 other completely sequenced baculoviruses. (XLSX 31 kb

Additional file 2: Table S2. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

List of sequenced to date baculoviruses. This file lists all baculoviruses sequenced to date, with their accession number, genome length and source of the sequence. (DOCX 31 kb

Additional file 3: Figure S1. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

Alignment of OpMNPV p8.9 ORF and its homologue in DapuNPV genome. Large insertion after nucleotide 201 in DapuNPV gene does not change the reading frame, although it decreases total basicity of translated protein which is a specific feature of p8.9 protein from OpMNPV (MAFT multiple alignment with default settings, visualization in Geneious R7). (PNG 80 kb

Flow cytometry analysis of EBA-140 Region II binding to normal and variant human RBCs Gerbich phenotype containing a deletion variant of GPC (Δ36–63 a.a.).

<p>The black line corresponds to the control (RBCs incubated with the heat denatured Region II and then with anti-Region II rabbit serum); the grey peak corresponds to the binding of Region II to RBCs; the grey line corresponds to RBCs binding of MoAb anti-Ge3 recognizing a.a. residues 43–51 of GPC (clone 3C4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref038" target="_blank">38</a>], indicating normal or Gerbich-negative RBCs phenotype.</p

Expression and purification of the recombinant EBA-140 Region II.

<p>A)Western blotting of baculovirus-infected Sf9 cells at 50 h post-infection with MoAb anti-myc (clone 9E10); MW, protein molecular weight standards; L, cell lysate; S, soluble cell fraction; IS, insoluble cell fraction; CM, culture medium. B) Affinity purification of the recombinant EBA-140 Region II on Ni-NTA resin. SDS–PAGE of proteins in the gel stained by CBB; MW, protein molecular weight standards; L, cell lysate; CM, culture medium; FT, flow- through of resin. C) Western blotting of fractions eluted with 50mM (1,2) and 100mM (3, 4) imidazole, with MoAb anti-myc (clone 9E10); RII, Region II (~ 75 kDa).</p

The binding of EBA-140 Region II to GPC in Western blotting.

<p>Normal (N), Gerbich (Ge) RBCs membranes and purified RBCs glycophorins (GP) were fractionated in SDS-PAGE and transferred to NC. RBCs glycophorins were desialylated (GP’) by the incubation of the blots in sulfuric acid. The blots were overlaid with the solution of recombinant Region II detected with MoAb anti-myc (clone 9E10). The GPC, GPD and variant GPC Gerbich was detected with MoAbs specific to N-terminus (clone 1G4) or C-terminus (clone1F6) of GPC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref037" target="_blank">37</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref038" target="_blank">38</a>]. GPC is visible on the blots as a double band stained with MoAb 1G4 or a single band stained with MoAb 1F6 due to a partial degradation of GPC at the C-terminus; M, protein molecular weight standards.</p

Analytical size exclusion chromatography of the EBA-140 Region II on Superdex 200 column.

<p>Purified protein was analyzed by SDS-PAGE in the gel stained with CBB and Western blotting with anti-myc MoAb (clone 9E10); peak 1 corresponds to the Region II (13,5ml elution volume); MW, protein molecular weight standards; RII, Region II (~ 75 kDa); elution volumes of standards used in column calibration are marked by the dashed lines.</p

Flow cytometry analysis of EBA-140 Region II binding to the native and neuraminidase (RBCs/neu) or trypsin (RBCs/trypsin)-treated human erythrocytes (RBCs).

<p>The black line corresponds to control (RBCs incubated with the heat denatured Region II and then with anti-Region II rabbit serum); the grey line (peak) corresponds to Region II of EBA-140 antigen.</p

Circular dichroism spectrum (195–270 nm) of EBA-140 Region II.

<p>Solution of Region II (0.5 µM) in 50 mM Tris-HCl, 300 mM NaCl, 10% glycerin, pH 7.5 was used.</p

Surface plasmon resonance analysis of human RBCs binding to the recombinant EBA-140 Region II.

<p>The solid lines correspond to RBCs binding to NTA flow cell covered with the functional Region II; the dashed lines correspond to RBCs binding to NTA flow cell covered with the denatured RII; analysis was performed in PBS buffer with 0.5% BSA, pH 7.2 on Biacore T200.</p