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Targeting of an expressed neurotoxin by its recombinant baculovirus

Menashe Elazar, Rafi Levi and Eliahu Zlotkin^*

Department of Animal and Cell Biology, The Life Science Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

View larger version (30K): [in a new window]   Fig.1. Similarity of symptoms induced by the native toxin and by recombinant virus infection to silkworm larvae. The recombinant virus mimics the native toxin by inducing spasmodic contractions, progressive reduction of mobility and, finally, a complete paralysis in a contracted, shortened body shape (A,C); (A) paralyzed larva 5h after injection of 3µg100mg-1bodymass of AaIT; (B) larva injected with 5µl of distilled water as a control; (C) paralyzed larva 50h.p.i. with 105p.f.u. of the recombinant baculovirus, BmAaIT; (D) larva 50h.p.i. with 105p.f.u. of the wild-type BmM14 baculovirus. Scale bar, 1cm.

View larger version (147K): [in a new window]   Fig.2. Healthy and baculovirus-infected tracheal epithelium and fat body of silkworm larvae. Fourth instar silkworm larvae were infected with 105p.f.u. of RV or WV, or were mock-infected (H2O injection). At 50h.p.i., when RV-infected insects were fully paralyzed, insect tissues were processed for immunocytochemistry as described in Material and methods. (A) Typical trachea (T) from mock-infected larvae stained with Hematoxylin and Eosin. (B) Typical trachea from mock-infected larvae stained with the AaIT-specific immunoperoxidase (see Materials and methods). (C) Trachea from WV (BmM14)-infected larvae 50h.p.i. Notice the hypertrophied tracheal epithelium (TE) (compared with A and B). (D) Trachea from RV (BmAaIT)-infected and paralyzed larvae expressing AaIT at 50h.p.i. The expressed toxin, indicated by the brown pigmentation, is shown in the cytoplasm of tracheal epithelial cells. The swollen nuclei (TN) are devoid of the toxin. (E) Fat body of WV (BmM14)-infected larvae. Notice the swollen nuclei of the fat body cells (Fbc). (F) Fat body from non-infected larva paralyzed by injection (1.6µg100mg-1) of AaIT. (G) Fat body from larva 50h.p.i. by the RV, which was incubated with a serum that was quenched with 50µg AaIT for 2h and is devoid of the AaIT-specific antibodies. (H, I) Fat body from RV (BmAaIT)-infected and paralyzed larvae expressing AaIT (arrowheads) at 50h.p.i. Notice the swollen fat body nuclei (FN) that are devoid of toxin staining. Scale bar, 20µm (A–D) and 50µm (E–I).

View larger version (135K): [in a new window]   Fig.3. Recombinant AaIT in close vicinity to the CNS. Fourth instar silkworm larvae infected with 105p.f.u. of the RV (fully paralyzed; A,B) or WV (F) were processed as described in Materials and methods at 50h.p.i. (A) Section through an abdominal ganglion of RV-infected and paralyzed larvae showing the different regions of the ganglion, including the protective sheath composed of the neural lamella (NL) and the perineurium (PR) and the inner part, the neuropile (NP). Note the large trachea (T), which is an integral part of the neural lamella, and its epithelial cells (TE), filled with the recombinant toxin. (B) Small trachea (T) embedded within the neural lamella of the ganglion. The trachea is located at the site where the connective (C) attaches to the ganglion. Arrowheads indicate tracheal epithelial cells embedded in the neural lamella and show the presence of a recombinant AaIT. (C) An abdominal ganglion from a healthy larva fixed after being paralyzed by an injection of 3µg100mg-1 bodymass of AaIT. The dissected CNS was processed for immunohistoctemistry (see E). The ganglion is devoid of any immunochemically detectable AaIT. (D) A ganglion derived from a larva that was orally infected by the wild-type virus BmNPV (1000PBIs) and paralyzed by injection of 2µg100mg-1bodymass of AaIT 48h.p.i. The larva was fixed 6h after injection, at a stage of advanced paralysis, and its CNS dissected and processed for LM immunohistochemistry (see Materials and methods). 10µm thick sections were incubated with the AaIT antibody. (E) An inset from the peripheral segment of the ganglion boxed in D, close to the connective (C) branching site. The ganglion is devoid of any immunochemically detectable traces of AaIT. Peripheral tracheae (T) are surrounded by viral inclusion bodies (polh), indicating an advanced stage of infection. The injected toxin was not detected. (F) Transverse section through an abdominal ganglion of a WV-infected insect at 50h.p.i. Similar to A and B, the infection by WV reveals the location of small trachea (T) surrounded by the typically swollen epithelial cells (TE). G, inner part of the ganglion. Scale bar, 40µm (A,C,D,F); 20µm (B) and 25µm (C).

View larger version (167K): [in a new window]   Fig.4. Recombinant toxin inside an abdominal ganglion of the ventral nerve cord of silkworm larvae. The recombinant toxin is visualized by the immunogold method. (A) Tracheal epithelial cell (TE) in the ganglion beyond the neural lamella. (B) Magnification of the boxed area in A showing the massive expression of the toxin (arrowheads) in the epithelial cell in close vicinity to the nucleus (N). Note the viral particles (V) within the nucleus. (C) The toxin in the neuropile region of an abdominal ganglion (arrowheads). The presence of the toxin on the axonal membrane (am) in the neuropile is obvious. (D) Section from a ganglion in close vicinity to a tracheal cell at the perineural region from a healthy silkworm larva paralyzed by injection of an overdose (3µg100mg-1bodymass) of AaIT and visualised with immunogold labeling (see Materials and methods). (E) High magnification of the boxed area in D, where the injected AaIT was undetectable (compare to B). A, axon; am, axonal membrane; m, mitochondria; N, nucleus; s, synapse; TE, tracheal epithelium; V, viral particle; NL, neural lamella; LS, gleal lacunar system. Scale bar, 2µm (A), 0.35µm (B,E), 0.25µm (C) and 1.5µm (D).

View larger version (54K): [in a new window]   Fig.5. Spontaneous electrical activity in the VNCs of virus- infected silkworm larvae. The insects were infected with 105p.f.u. and, at the indicated times post-infection, were dissected to yield intact VNCs and bathed in saline. The RV-infected larvae revealed either a partial paralysis (slow mobility, spasmodic local contractions of the integument at 40–45h.p.i.) or full paralysis (in contracted body shape, as in Fig.1, at 45–52 h.p.i.). Extracellular recording was performed using a suction electrode between the 6th and 7th abdominal ganglion and data were collected during 5min of continuous recording. (A) Frequency analyses in the form of inter-spike interval (ISI) distribution histograms for individual insects were averaged and median ISI values were calculated (arrows). The total number of spikes recorded from the RV-infected VNC when compared to the WV were 9888±1646 (N=5) and 5141± 855 (N=6), respectively, at 40–45h.p.i. and 11485±1075 (N=7) and 5112±633 (N=3), respectively, at 45–52h.p.i. (B) A representative spontaneous electrical activity recorded from an RV- (upper trace) or WV-infected (lower trace) VNC. Notice the occurrence of bursts (underlined) in the recording from RV-infected (upper trace) insects at 50h.p.i. when compared to the homogeneous-scattered pattern of activity recorded from WV-infected insects at 50h.p.i. (lower trace). For technical reasons the amplitudes in such extracellular recordings are of no functional significance.