Urtica dioica Agglutinin Prevents Rabies Virus Infection in a Muscle Explant Model

Abstract

Infection with the rabies virus (RABV) results in a 100% lethal neurological disease once symptoms develop. Post-exposure prophylaxis (PEP) consists of a combination of vaccination and anti-rabies immunoglobulins (RIGs); it is 100% effective if administered early after exposure. Because of its limited availability, alternatives for RIGs are needed. To that end, we evaluated a panel of 33 different lectins for their effect on RABV infection in cell culture. Several lectins, with either mannose or GlcNAc specificity, elicited anti-RABV activity, of which the GlcNAc-specific Urtica dioica agglutinin (UDA) was selected for further studies. UDA was found to prevent the entry of the virus into the host cell. To further assess the potential of UDA, a physiologically relevant RABV infection muscle explant model was developed. Strips of dissected swine skeletal muscle that were kept in a culture medium could be productively infected with the RABV. When the infection of the muscle strips was carried out in the presence of UDA, RABV replication was completely prevented. Thus, we developed a physiologically relevant RABV muscle infection model. UDA (i) may serve as a reference for further studies and (ii) holds promise as a cheap and simple-to-produce alternative for RIGs in PEP.

Keywords: antiviral; lectin; muscle explant; rabies virus.

Figure 1

Anti-RABV activity of UDA, BanLec, and PSA in BHK-21J cells. Serial dilutions of UDA (A), BanLec (B), and PSA (C) were added together with mCherry-RABV (MOI = 0.01) to BHK cells. On day 5 p.i., the antiviral activity was determined by the quantification of the virus-induced mCherry fluorescence. In a parallel experiment, the effects of the lectins on cell viability were determined using a viability staining (MTS). The averages and standard deviations of 3 independent experiments are presented. Fitting the dose–response curves indicates an EC₅₀ of UDA of 4.6 μg/mL and a CC₅₀ of 65 μg/mL; an EC₅₀ of BanLec of 6 μg/mL and a CC₅₀ of 36 μg/mL; an EC₅₀ of PSA of 18 μg/mL and a CC₅₀ of more than 100 μg/mL.

Figure 2

UDA blocks RABV entry. Time-of-drug-addition assay. BHK cells were incubated with RABV with or without UDA at 4 °C for 1 h (−1–0 h, indicated in red). After 1 h, unattached virus was washed away, and UDA was added to the cultures at different time points (0, 0.5, 1, 2, 4 h.p.i.). At 16 h.p.i., intracellular viral RNA was quantified by RT-qPCR. Infected and untreated samples collected at 1 h.p.i. were considered the virus background. Each condition was tested in 3 independent assays, and averages and STDEV are indicated. Tukey’s multiple comparisons test was used to calculate the statistical significance. (ns, not significant; * p < 0.05; *** p < 0.001, **** p < 0.0001).

Figure 3

UDA inhibits RABV infection by mainly interacting with the cell. UDA at a final concentration of 25 µg/mL was pre-incubated with RABV or cells at 37 °C for 2 h. Next, UDA was diluted to a non-inhibitory concentration (1 µg/mL), and cells were further incubated with virus (MOI 0.1) for 3 days at 37 °C. Virus infection (%) was determined relative to the untreated condition. Each condition was tested in at least 3 independent experiments, and averages and STDEV were calculated. Tukey’s multiple comparisons test was used to calculate the statistical significance. (** p < 0.01, **** p < 0.0001).

Figure 4

RABV replicates in swine skeletal muscle explants. (A) Picture of a swine muscle explant culture. The biceps femoris muscle was dissected from freshly euthanized pigs (3 months, female) and maintained under tension using pins. (B) Upon infection with RABV (SAD-B19-mCherry), culture supernatant was analyzed each day for levels of vRNA by RT-qPCR. Nine independent cultures were used; median and quartiles are indicated. Kruskal–Wallis test with Dunn’s multiple comparisons test was used to calculate the statistical significance. (ns, not significant; *, p-value < 0.05; ***, p-value < 0.001) (C) On day 6 p.i., the supernatants of the infected (RABV) and non-infected (Mock) muscle cultures were transferred to BHK cells, and after 7 days of incubation, the fluorescence of the cells was visualized by microscopy (the picture is a representative culture of 3 independent experiments). Scale bar: 100 µm.

Figure 5

UDA inhibited RABV replication in swine skeletal muscle explants. (A) Viability (using resazurin) of muscle explants that were incubated for 7 days with UDA (25 µg/mL) at 37 °C. The resazurin-based viability assay was conducted daily. Blank control: cultures without muscle strips. Three independent cultures were used; averages and STDEV are indicated. (B) Muscle explants were infected with RABV in the presence or absence of UDA (25 μg/mL). At 6 d.p.i. the culture supernatant was collected to determine infectious viral titers. (C) On day 6 p.i., the muscle strips were fixed, and RABV replication was visualized by staining for the viral N-protein (green); cell nuclei were stained with Hoechst (blue). Results of a representative example of 3 independent experiments are shown. Scale bar: 1 mm.