**2. Methods**

### *2.1. Virus and Cells*

Tula virus (TULV) strain Moravia/5302v/95 was obtained from The National Collection of Pathogenic Viruses, PHE, UK. Stocks were shown to be mycoplasma free using MycoAlert (Lonza group AG, Basel, Switzerland). Vero E6 cells are a clone of Vero 76 cells isolated from *Cercopithecus aethiops* (African green monkey) kidney cells, and were obtained from the European Collection of Authenticated Cell Cultures (ECACC 85020206). These cells were selected as previous findings show that TULV-infection allows the establishment of a persistently infected state [23]. Virus titres were calculated by indirect staining using the anti-NP antibody in conjunction with an Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, Waltham, MA USA) as previously described [23].

### *2.2. Antibody Production and Validation*

Antisera specific for TULV NP were generated in house as previously described [23], using bacterially expressed SEOV NP core domain as an antigen, purified as previously described, and used to immunize a sheep (AltaBiosciences, Redditch, UK). Antibodies were validated by Western blot analysis.

### *2.3. TULV Infections of Vero E6 Cells*

Briefly, Vero E6 cells were plated out at 1 × 10<sup>6</sup> cells per 25 cm<sup>2</sup> flask or at 1 × 10<sup>5</sup> cells/well of a 12-well plate onto glass coverslips, sterilised by submerging into 100% ethanol and washing with sterile PBS. Cells were maintained in a humidified incubator at 37 ◦C with 5% CO2. TULV infections were carried out at a multiplicity of infection (MOI) of 0.1–0.5 in serum-free DMEM. Virus was adsorbed for 90 min before the addition of DMEM supplemented with 2% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. For long-term infections, TULV infections were carried out in 25 cm<sup>2</sup> flask and seeded onto coverslips 24 h prior to fixation. Fixation was carried out using 4% paraformaldehyde at 36 h post infection, 7 days post infection and 30 days post infection to represent early, peak and persistent infections.

### *2.4. Indirect Immunofluorescence*

Mock-infected (media alone) or TULV-infected monolayers on coverslips were permeabilized in ice-cold methanol before washing in 1X PBS and blocked in 5% BSA in 1×PBS for 30 min. Primary staining was carried out using the following antibodies and reagents: in house anti-TULV NP [23], Golgi (Santa Cruz Biotechnology Inc, Dallas, TX AE6, USA), ER (Thermo Fisher Scientific Concanavalin A), β-actin (New England Biolabs (NEB), Ipswich, MA, USA D6A8), β-tubulin (NEB 9F3), vimentin (NEB D21H3), Rabs 5, 7 and 11 (NEB 2143, D95F2, D4F5), LAMP1 (NEB D2D11), clathrin heavy chain (NEB 4796), TIA-1 (Santa Cruz G-3) and DCP1a (Santa Cruz 56-Y). Incubations were carried out for 2 h at room temperature, followed by thorough washing in 1X PBS. Secondary antibody staining was carried out using donkey anti-goat Alexa Fluor 488 (Thermo Fisher Scientific A-11055), donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific A-31572), donkey anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific A-31570), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific A-31573), donkey anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific A-31571) at a 1:500 dilution for 1 h. Nuclear staining was carried out by incubating with 300 nM DAPI for 3–5 min at room temperature. Cells to be visualised by confocal microscopy were mounted onto slides using Vectashield (Vector laboratories, Burlingame, CA, USA) hard set anti-fade mounting media and cured for 15 min before

storing at 4 ◦C overnight. Cells to be further analysed by RNA-FISH were fixed in 4% formaldehyde for 20 min and stored in 1×PBS supplemented with 0.1 % diethylprocarbonate (DEPC).

### *2.5. TEM Preparation of TULV-Infected Vero E6 Cells*

Vero E6 cells were fixed by adding 5% glutaraldehyde (*v*/*v* in 0.1 M phosphate bu ffer (PB) Agar Scientific, Stansted, UK) to an equal amount of cell media in the tissue culture flask, resulting in a final glutaraldehyde concentration of 2.5% (*v*/*v*). Cells were left for 2.5 h to fix at room temperature. After fixation, the cells were removed from the tissue culture flask by scraping and washed in 0.1 M PB for 30 min, twice. The cells were then post-fixed in 0.1 M PB with 1% osmium tetroxide ( *w*/*v*) (Agar Scientific) for 1 h followed by four washes in 0.1 M PB. Subsequently, the post-fixed cells were dehydrated using an ascending ethanol series (40%, 60%, 80% and 2×100%) for 20 min, and then twice in propylene oxide (Agar Scientific) for 20 min. Dehydrated cells were incubated with a 1:1 mixture of propylene oxide and araldite epoxy resin (araldite CY212, dodecenyl succinic anhydride, and tri-dimethylaminomethyl phenol (Agar Scientific)) overnight, after which the mixture was replaced by pure araldite and incubated for 8 h. To ensure adequate embedding, new resin was added four times, after which the cells were polymerized at 60 ◦C for 24 to 48 h.

Embedded cells were then sectioned in the ultra-thin range (80 to 100 nm) using a EM UC7 Ultramicrotome (Leica, Wetzlar, Germany). Ultra-thin sections were then placed upon 200 mesh carbon-coated copper-formvar grids and stained with lead citrate and uranyl acetate (UA). Sectioned cells were stained in 8% saturated ( *w*/*v*) UA (Agar Scientific) for 1 h. UA-stained grids were then washed in dH2O for 5 min (×5). Afterwards, the sections were stained with lead citrate (Agar Scientific) for 10 min and washed in 0.02 M NaOH for 1 min (×3), and in dH2O for 1 min (×5). Sections were then imaged at 120 kV using a FEI Tecnai G2-Spirit electron microscope (Thermo Fisher Scientific) with a Gatan US4000/SP 4k × 4k CCD camera (Gatan, Pleasanton, CA, USA).

### *2.6. Fluorescent In-Situ Hybridisation (FISH) Probes*

Fluorescent in-situ hybridisation DNA probes were designed against the sense and anti-sense TULV S segmen<sup>t</sup> ORF sequence using the Stellaris Probe Designer version 4.2 (LGC Biosearch Technologies, Novato, CA, USA). The Stellaris Probe Designer provided 48 oligonucleotide probe sequences that were 20 nucleotides in length with a minimum spacing of 2 nucleotides. Target TULV S segmen<sup>t</sup> ORF sequence was obtained from sequencing viral RNA isolated from TULV-infected cell culture medium (Genewiz Inc, South Plainfield, NJ, USA). Probes were assessed for cross-hybridization using NCBI Nucleotide Blast against *C. aethiops* (taxid:9534) mRNAs and against the TULV M and L segmen<sup>t</sup> sense and anti-sense sequences (strain: Tula/Moravia/5302v/95). Any probes with more than 15 consecutive complimentary nucleotides were discarded. TULV S segmen<sup>t</sup> sense and anti-sense probes were labelled with Quasar670, and all probe sequences are shown Table S1. Strand sets were kept separate throughout this study.

### *2.7. Fluorescent In-Situ Hybridisation (FISH)*

Probe hybridisation was carried out according to manufacturers' instructions. Briefly, each set of strand-specific FISH probes were reconstituted to 12.5 mM in RNase free 1× TE bu ffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0 (G Biosciences) diluted in UltraPure DNase/RNase-Free Distilled water (Thermo Fisher Scientific) and stored separately at −20 ◦C. For each coverslip, 1 μL probe stock was used per 100 μL hybridisation bu ffer (Stellaris RNA FISH hybridisation bu ffer diluted with deionised formamide (Severn Biotech Ltd, Kidderminster, UK) in a 9:1 ratio).

Cells were incubated in Stellaris RNA FISH wash bu ffer A diluted with deionized formamide and UltraPure DNase/RNase-Free Distilled water in a 2:7:1 ratio for 5 min at room temperature. Additionally, 100 μL hybridisation bu ffer with probes was dispensed into a droplet onto parafilm in a humidified chamber. Coverslips were incubated cell-side down onto probe mix for 4 h at 37 ◦C, protected from light. Cells were incubated in 1 mL Stellaris RNA FISH wash bu ffer A for 30 min at 37 ◦C, protected from light and then incubated in 1 mL Stellaris RNA FISH wash bu ffer A with 300 nM DAPI for 5 min at room temperature. The bu ffer was removed and the cells were incubated in 1 mL Stellaris RNA FISH wash bu ffer B for 5 min before mounting onto glass microscope slides using Vectashield hard-set antifade mounting media (Vector laboratories). Slides were cured for 45 min at room temperature, protected from light, and imaged within 24 h.
