Decreased lethality in mice after intracranial injection with VSV-ΔP.

<p>ICR mice (5 mice/group) were lightly anesthetized with ether and then intracranially injected with either PBS or 1×10⁴ pfu of VSVs. (A) Body weight was measured daily and (B) survival was plotted using the Kaplan-Meier survival curve. Values are averages of five mice with error bars showing SD and are representative of two independent experiments. NS, not significant; *, p<0.5; **, p<0.05.</p

Induction of immune responses following immunization with VSV-ΔP expressing H1 and H7 HA.

<p>BALB/c mice (5 mice/group) were intravenously injected with 1×10⁷ pfu of VSVs in 100 μl at days 0 and 21. At day 28, sera were harvested to determine for H1N1-specific and H7N9-specific IgG levels at a titer of 5,120. Values are averages of two independent experiments with error bars showing SD.</p

P gene deletion attenuated replication of recombinant virus.

<p>(A) BHK-21 and BHK-P cells were infected with VSV-ΔP at an MOI of 1 and observed for cytopathic effects (CPE). Infected cells were then subjected to flow cytometry to quantify the percentage of mCherry-expressing cells. The pictures are representative of triplicate samples. (B) BHK-P cells were infected with VSV or VSV-ΔP at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing standard deviation (SD). (C) Viruses were serially diluted for plaque titration, and plaques were stained with neutral red for visualization. Representative images of VSV and VSV-ΔP were selected for plaque size comparison.</p

Construction of recombinant VSVs with P gene deletion.

<p>(A) The top schematic shows the VSV genome layout and the naturally occurring restriction sites used for cloning. To construct VSV-ΔP, the VSV genome was digested with <i>Eco</i>RV, religated in the absence of the P gene fragment and the mCherry gene was then inserted between the G and L genes. The mCherry gene was replaced by HN, H1 or H7 HA to generate VSV-ΔP-HN, VSV-ΔP-HA1 and VSV-ΔP-HA7, respectively. (B) Supporting cells (BHK-P) were constructed by transducing BHK-21 cells with lentivirus bearing the P gene, and the selected clone expressing the emerald fluorescent protein was examined by (C) bright field and (D) fluorescence imaging. (E) BHK-P cells were infected with VSV or VSV-ΔP, and supernatants were harvested for viral genome extraction and RT-PCR. RBZ, hepatitis virus delta ribozyme; T7, T7 RNA polymerase leader; T7 ter, T7 terminator; LTR, long terminal repeat; ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; SFFV, spleen focus-forming virus (promoter); WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔU3, U3 deletion.</p

Expression of foreign antigens on the viral surface.

<p>(A) BHK-P cells were infected with VSVs at an MOI of 0.1. Supernatants were harvested at the indicated time points and were assessed using a MUNANA-based assay, (B) HA assay and (C) HAI assay. Supernatant and cell lysates were subjected to Western blot analysis using a β-actin monoclonal antibody as the primary antibody. Values are averages of triplicate wells with error bars showing SD. (D) 1×10⁷ purified VSVs were lysed and subjected to Western blot analysis using an HA (H1N1) polyclonal antibody and serum from VSV-immunized mice as the primary antibody. (E) To study the incorporation of the H7 HA gene in VSVs, RNA were extracted from purified VSVs and subjected to RT-PCR using primers specific for the N/M fragment or H7 HA genes. (F) BHK-P cells were infected with VSVs at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing SD. A.U., arbitrary units.</p

Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.

<p>Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.</p