DWV infection model.

<p>(A) Bee pupae at the day p.i.. A black arrow at one bee pupa marks the injury caused by injection. (B) Typical outcome of infections with rDWV-A 1414 and wtDWV-A 1414: (B1) Healthy bee, which emerged at day 21 of development after mock infection. (B2) Bee pupa, which died three days after infection with wtDWV-A 1414. (B3) Nonviable adult bee, which emerged at day 21 after infection with rDWV-A 1414 showing typical wing and limb deformities.</p

Recombinant DWV VP1 (rVP1) and VP1 specific antibodies.

<p>(A) rVP1 was expressed in <i>E</i>. <i>coli</i> via plasmid pL443. Total protein from non-induced (NI) and induced (I) cultures was separated by SDS-PAGE and either stained with Coomassie blue or probed with an anti His-tag antibody. (B) IMAC purified rVP1 revealed by Coomassie stain and western blot analysis. (C) Reactivity and specificity of two mouse monoclonal antibodies is shown using <i>E</i>. <i>coli</i> expressing rVP1. Arrows indicate the protein bands of rVP1 and rVP1 dimer.</p

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

<div><p>European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of <i>Deformed wing virus</i> (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete <i>in vitro</i> model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.</p></div

Pathohistological analyses of tumor tissues.

<p>(<b>2A</b>) Sections of pancreatic tumors derived of the indicated pancreatic cell lines were stained with haematoxylin and eosin. The Hs-766T specimen was dissected from a subcutaneous tumor; all other samples were taken from orthotopically grown tumors. (<b>2B</b>) Different sections of a BxPC-3-derived tumor were subjected to immunohistological analysis with antibodies against Mucin-1 (MUC-1), pan-cytokeratin (Pan-CK), and α-smooth muscle actin (SMA). The black bar represents a length of 53 µm, except for anti-MUC-1 staining (27 µm).</p

Cytochrome 2B1 protein expression in PCCWmCMV-transduced and non-transduced parental cell lines.

<p>Total cellular lysates from 8×10⁵ cells were separated on a 10% polyacrylamide gel under denaturing conditions. After blotting, CYP2B1 protein was detected with a CYP-specific antibody.</p

Virions of rDWV (A) and wtDWV (B) visualized by transmission electron microscopy.

<p>Proteinacious aggregates (white box) and single virions (black box) are enlarged for both preparations. The bar is 100 nm in the left and 25 nm in the right panels. (C) The protein content of purified rDWV virions is presented after SDS-PAGE in Coomassie stain and Western blot (DVWVP1A1). The apparent bands of DWV structural proteins are indicated.</p

Summary of orthotopical tumor growth.

<p>Number of tumors growing after i.p. injection into mice and percentage of tumors that infiltrated the pancreatic tissue.</p>a<p>infiltrative tumor growth rate into the tail of the pancreas; percentages indicate the proportion of mice with infiltrative tumor growth among those with orthotopic tumor growth.</p

Sequence analyses of DWV-A 1414.

<p>(A) DWV 5’-NTR sequences were obtained from tailing RACE-PCRs. Numbers of clones containing the respective 5’-terminal nucleotides are indicated below the sequence. (B) Sequence comparison of the 5’-terminus of DWV-A 1414 and related Iflaviruses. Genbank entries are provided behind the strain designations. (C) Neighbor joining analysis using genomic sequences of different Iflaviruses. The phylogenetic analysis documents a close relationship of DWV-A 1414 to other DWV-A isolates from Europe and America. The number of substitutions per site is given as a scale and bootstrap values for 1,000 replicates were indicated above all nodes.</p

Cytosine deaminase gene expression in PCCDWmCMVpuro-transduced and in parental cell lines.

<p>Total cellular RNA was isolated from indicated cell lines and mRNA was reverse transcribed using oligo(dT) primers. CD- as well as GAPH-specific gene fragments were amplified using specific primers and separated on an agarose gel showing the expected sizes of ∼480 bp for CD and ∼350 bp for GAPDH. The cDNA amount used for PCR amplification was adjusted to yield comparable amounts of the GAPDH gene fragment.</p

Sensitivity of human pancreatic cancer cell lines towards ifosfamide.

<p>Cells stably transduced with the CYP2B1 gene as well as non-transduced parental cells (mock) were cultured in increasing concentrations of ifosfamide for five days as described in Materials and Methods. LD₅₀ values were calculated using Excel Fit software. Each experiment was performed in quadruplicates. Data from three independent experiments are shown.</p