Expression of HA antigens in rVHSV-HA or rIHNV-HA infected cells.

<p>The expression of HA antigens was assessed by indirect immunofluorescence assays on EPC cells. The cells were infected with either rVHSV-HA or rIHNV-HA and incubated at 14°C. (A) At 24 h post-infection, cells were fixed and permeabilized with alcohol/acetone and HA expression was detected using a monoclonal antibody against HA1 (H36-26). (B) At 48 h post-infection, membrane expression of HA antigen was visualized on live cells in PBS using H36-26 monoclonal antibody (magnification x40).</p

Estimation of the amount of recombinant HA antigens expressed on recombinant viruses.

<p>Proteins of sucrose gradient-purified viral particles were separated on a 12% polyacrylamide gel. Serial amount of total viral proteins were loaded and migrated on SDS-PAGE. Proteins concentration was estimated using Micro BCA assay protein quantification Kit. The gel was electrotransfered onto a PVDF membrane and HA antigens were detected with a monoclonal antibody directed against HA.</p

Schematic representation of the engineered rIHNV-HA and rVHSV-HA genomes.

<p>GS: gene start; GE: gene end; SP: signal peptide; TM: transmembrane.</p

Detection of HA antigen at the surface rIHNV-HA particle through immunogold staining.

<p>Sucrose gradient-purified viral particles were adsorbed on electron microscopy nickel grids. After fixation, the glycoprotein G of IHNV and HA antigen were detected using specific mouse monoclonal antibodies and anti-mouse secondary antibody coupled with gold particles (black dots of 5 nm in diameter). After negative staining, recombinant viral particles were observed by transmission electron microscopy. Magnification x40 000.</p

Non-adjuvanted recombinant novirhabdoviruses induce a neutralizing immune response against HA A/PR/8/34 in BALB/c mice.

<p>A) Schedule of immunization and B) groups of BALB/c mice (n = 8) six-week old were immunized subcutaneously with 30 μg of purified recombinant novirhabdoviruses three times at two week interval. Mice sera were taken at day -3, day 21 and day 49. At day 59, mice were challenged with a lethal dose of 5x10⁴ PFU of influenza A/PR/8/34 virus. Course of the infection was monitor daily for two weeks. C) Seroneutralization antibody assay against influenza HA A/PR/8/34. Heat-inactivated sera of immunized mice at day 49 were used to evaluate the amount of serum neutralization antibodies against influenza virus. Titers are expressed as reciprocals of the highest antibody dilution at which cytopathic effects appeared. Bars represent the mean titer of 8 animals in each group.</p

Analysis of HA antigens incorporation in recombinant virus particles.

<p>Proteins of sucrose gradient-purified viral particles were separated on a 12% polyacrylamide gel. A) Six micrograms of total viral proteins were denaturated, loaded and migrated on SDS page gel. The gel was electrotransfered onto a PVDF membrane and HA antigens were detected with monoclonal antibody directed against HA. B) Three to six mg of total viral proteins were incubated at 37°C with or without 2 mg/mL of Trypsin-TPCK for 5 min prior denaturation, loading and migration on SDS-PAGE as in A.</p

Primers used in the study.

<p>Restriction enzyme sites generated by site-directed mutagenesis or by PCR are boldfaced and underlined.</p

Lethal challenge experiments.

<p>Mice previously vaccinated with non-adjuvanted recombinant viruses were challenged intranasally with a lethal dose of 5x10⁴ PFU of influenza A/PR/8/34 virus. A) Weight change and B) Clinical score over the course of the disease. Mice were weighted and followed daily for two weeks. In graphs A and B are presented the mean values of each group with standard error. C) Survival curves of mice immunized with rVHSV-HA and rIHNV-HA following challenge. D) Disease severity. Each box represents one mouse and the severity of its disease when clinical signs were detected for more than 24h. Black indicates severe (maximum clinical score from 5 to 7), dark gray: moderate (3–4), light gray: mild (1–2) and white indicates that the mouse did not show any clinical sign for more than 24h.</p