key: cord-0297505-x7c5nf4t
authors: Wang, Xinyu; Terrie, Lisanne; Wu, Guanghui; Van Damme, Els; Thorrez, Lieven; Fooks, Anthony R; Banyard, Ashley C; Neyts, Johan; Jochmans, Dirk
title: The Urtica Dioica Agglutinin Prevents Rabies Virus Infection in a Muscle Explant Model
date: 2021-10-20
journal: bioRxiv
DOI: 10.1101/2021.05.21.441744
sha: 5d91954a9c0bd4027569db0277e05ccfecd5a531
doc_id: 297505
cord_uid: x7c5nf4t

Infection with the rabies virus (RABV) causes fatal encephalitis and paralysis in humans and animals. Post-exposure prophylaxis (PEP) consists of vaccination and the injection of anti-rabies immunoglobulins (RIGs) around the (bite) wound. This is 100% effective in preventing disease if administered in a timely manner. However, the costs, the required cold chain for storage and transport and the limited availability of RIGs makes the treatment challenging. Cheaper and easier to produce alternatives are urgently needed. To aid in the discovery and development of such alternative therapeutics, we developed a physiologically relevant infection model. Strips of freshly dissected swine skeletal muscle were placed under tension in culture medium and infected with RABV. Viral antigens were produced in the muscle explants and the virus production increased significantly over time, indicating that RABV infects and replicates in the muscle explants. Subsequently, in a search for inhibitors of RABV entry in muscle cells, we first screened a panel of 34 different lectins in a RABV / BHK-21J cell assay. The Urtica dioica (stinging nettle) Agglutinin (UDA; a N-acetyl-D-glucosamine specific agglutinin) was found to be able to completely inhibit infection of cells with the RABV (EC50 8.2 μg/mL) by preventing binding of the virus to the host cell. When the infection of the muscle strips was carried out in the presence of UDA, infection of the tissue was completely prevented. We thus developed a physiologically relevant RABV muscle infection model and identified an easy to produce component that (i) may serve as a reference for further studies and (ii) holds promise as an alternative for RIGs in PEP.

Rabies virus (RABV), a member of the Lyssavirus genus, is a neurotropic virus that causes an 47 fluorescence was quantified (SPARK, Tecan, Belgium) ® . 109

Pre-exposure of BHK to lectins prior to infection 110 BHK cells were pre-incubated with lectins, final concentration 25 μg/mL, at 37 °C for 2 hours. 111

Then, virus was added (MOI=1) and incubated with the cells at 37 °C for 2 hours. After 112 incubation, cells were washed 3 times with PBS. At day 5 post-infection, the mCherry 113 fluorescence was quantified. 114

Swine skeletal muscle explant culture and antiviral assay 115 The biceps femoris muscle was dissected from freshly euthanized pigs (3 months, female), 116 obtained as residual tissue from ORSI Academy (Melle, Belgium). Next, the muscle biopsy was 117 dissected, parallel to the muscle fibers, in 2 cm long 2 mm thick strips using sterile scalpels. 118

Tissue strips were maintained under tension ex vivo using minutia pins (Entosphinx, stainless 119 steel pins, diameter 0.25 mm) on sterile sylgard coated wells of a 6-well plate. Tissue strips 120 were submerged in DMEM with 10% FBS, P/S (100 U/mL), and in 2% FBS DMEM with P/S 121 during antiviral experiments at 37°C incubator with 5% CO2. UDA was diluted to a final 122 concentration of 25 μg/mL in the supernatant and incubated with muscle explants at 37 °C for 123 2h. Then the muscle explants were inoculated with 2 × 10^4 TCID50 RABV. After 4 h incubation, 124 muscle explants were washed 5 times with PBS, and maintained with 25 μg/mL UDA for 6 125 days. A sample of 300 μL of the culture SN was collected every day for RT-qPCR or titration. 126 Germany). All pictures were obtained with a 10 x objective and z position between 1820 µm to 141 1950 µm. 142 Statistics 153 Statistical analysis in this study was done by GraphPad Prism 9. A P-value less than 0.05 was 154 considered as statistically significant. P values associated with each graph are indicated as: *, 155 P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001. 156 158 We tested the potential effect of a panel of 34 different lectins on infection of BHK cells with 159 RABV using a reporter virus. Several lectins with selective antiviral activity against RABV 160 were identified (Table 1) bind with viral glycans of the enveloped viruses, such as HIV and coronavirus (Hansen et al., 213 1989; Balzarini, 2006; Keyaerts et al., 2007; Lam and Ng, 2011) . We screened a library of 214 lectins and identified UDA among the most active. We demonstrated that UDA interacts with 215

virus receptor binding. Typically, when people are exposed to RABV infection, HRIG provides 217 passive protection by neutralizing the virus in the wound and muscles before vaccine-induced 218 antibodies appear (Warrell, 2014) . We demonstrate that UDA can protect muscle cells from 219 rabies infection indicating that lectins may be an alternative for the RIGs. 

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 229 We are grateful to T. Van Buyten for excellent technical assistance. 230