*2.8. Quantitative RT-PCR*

TULV RNA was isolated from infected cells or supernatant using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), and the quantitative reverse transcriptase real-time PCR was carried out using the 1-step RT-qPCR kit (Promega, Madison, WI, USA), both as previously described [23]. For each qRT-PCR experiment, in vitro transcribed TULV S segmen<sup>t</sup> RNAs were diluted to generate 108, 106, 104, 10<sup>2</sup> genome copies/5 μL and used to build a standard curve from which the sample relative genome copies per mL could be calculated, as previously described [23].

### *2.9. Inhibition of Intermediate Filaments*

Vero E6 were infected with TULV at a MOI of 0.5 in a 6-well plate. Infected cells were harvested for immunofluorescence and q-RT PCR analysis at 30 days post infection. Prior to harvesting, the cells were treated with the following cytoskeletal inhibitors; 17 μM nocodazole (Sigma-Aldrich, St. Louis, MO, USA) for 60 min and 400 nM okadaic acid (OA) (Sigma-Aldrich) for 30, 60 or 90 min. Infected Vero E6 cells treated with OA were also recovered by removing the inhibitor and incubating overnight with fresh media before harvesting. Mock treatments were carried out using an equal volume of solvent (dimethyl sulfoxide (DMSO)/ethanol).

### *2.10. Laser Scanning Confocal Microscopy*

Laser scanning confocal imaging was carried out using the Zeiss LSM700 confocal microscope using the 40×/1.3 Oil DIC Plan Apochromat, M27 objective lens with the diode 405 nm, 488 nm, 55 nm and 639 nm lasers or the Zeiss LSM880 + Airyscan upright confocal microscope using the Plan-Apochromat 40×/1.4 Oil DIC objective lens with the diode 405 nm, argon 488 nm, DPSS 561 nm and HeNe 633 nm lasers. Image analysis was carried out in Fiji Is Just ImageJ (FIJI).
