Optimisation of lytic herpes simplex virus infection in human induced pluripotent stem cell derived cortical neurones

10 Neurons, herpes simplex encephalitis (HSE), virus-host interactions, i3Neurons 11

12 Herpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. 13 While we have substantial knowledge about the molecular virology of HSV-1 lytic infection in 14 cells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of 15 human neurones owing to the challenges of working with neuronal cell-based models. Here we 16 demonstrate the use of a human induced pluripotent stem cell (iPSC)-derived cortical neurone 17 model (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and 18 efficiently differentiated into an isogenic population of cortical glutamatergic neurones. We 19 show that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimised 20 protocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at 21 near-100% efficiency, and optimised fixation methods that preserves organelle and neurite 22 structure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, 23 scalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic 24 pathogens. 25 Data summary 26 The authors confirm all supporting data, code and protocols have been provided within the 27 article or through supplementary data files. 28 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint

29 Neuronal virus infections cause severe pathology. HSV-1 is the leading cause of viral 30 encephalitis [1], causing 70% mortality in untreated patients and up to 19% in patients treated 31 with antivirals, with survivors often suffering severe neurological sequelae [2]. Similarly, 32 infection with enteroviruses such as EV-A71 cause encephalitis and acute flaccid paralysis [3]; 33 Zika virus and Oropouche virus infection in adults can cause Guillain-Barré syndrome [4, 5]; and 34 congenital Zika virus infection can cause microcephaly, decreased brain tissue, plus ocular and 35 osteoskeletal abnormalities [6]. Furthermore, it is increasingly clear that infection with 36 neurotropic viruses like HSV is a risk factor for developing common neurodegenerative diseases 37 [7–9]. It is therefore important to identify robust and appropriate human cell-based systems to 38 study the molecular basis of neuronal infection by HSV-1 and other neurotropic pathogens. 39 Multiple different systems have been used for the study of neuronal HSV-1 infection, especially 40 in the context of latency (reviewed in [10]). Ex vivo infection of rat or mouse-derived ganglia that 41 represent the natural site of HSV-1 latency are widely used [11–13], although the number of 42 neurones that can be isolated even from a large number of animals is limited [14] and there are 43 differences in interactions between HSV-1 and mouse versus human immune responses [15, 44 16]. Ex vivo studies of human neurones is possible using post-mortem specimens [17, 18], but 45 availability and the capacity for genetic manipulation of the specimens is limited. Human 46 embryonic or induced pluripotent stem cells (iPSCs) allow interrogation of infection following 47 differentiation into neural stem cells, neurones or other glial cell types [19–23]. However, 48 differentiation timescales can be long and the procedures labour-intensive. Human stem-cell 49 derived organoids represent a powerful model for studying the functional consequences of 50 HSV-1 lytic and latent infection in human neural stem cells, neurones and glia [24–28]. While 51 excellent for transcriptomic analysis [25, 28], these models are not well suited to high resolution 52 proteomics studies of infection such as quantitative temporal viromics [29], which require large 53 numbers of homogenous cells (≥1×107) and high levels of synchronous infection (≥90%) [30, 54 31]. 55 Scalable cancer-derived neuroblastoma cell lines like SH-SY5Y are widely used for HSV-1 56 infection studies [21, 32, 33] but they have complex chromosomal aberrations [34, 35], are 57 highly sensitive to the differentiation procedure used and yield mixed morphology populations 58 [36]. Lund human mesencephalic (LUHMES) cells have been developed as models to study 59 HSV-1 latency [37] and host shutoff during lytic infection [38]. Differentiated LUHMES resemble 60 post-mitotic dopaminergic neurones [39] and they represent a powerful homogenous cell-61 based system for studies of neuronal infection. However, LUHMES cells are derived from the 62 midbrain mesencephalon [40] whereas herpes simplex encephalitis (HSE) is generally localised 63 to the temporal lobes [41]. As we know that innate immune programs differ between different 64 classes of neurone [42], there is a need for additional scalable systems for the study of HSV-1 65 infection in the cerebral cortex. 66 Differentiation of human iPSCs via the expression of integrated transcription factors represents 67 a promising approach to rapidly obtain isogenic populations of differentiated neuronal and 68 other cell types [43]. For example, human iPSCs expressing the neuronal transcription factor 69 Neurogenin3 (NGN3) [43] can be differentiated into sensory human neurones that have been 70 used to characterise miRNAs and neuronal factors that regulate the efficiency of HSV-1 lytic 71 replication or establishment of latency [44, 45]. Recently, a comprehensive analysis has shown 72 that sensory neurones differentiated via NGN3 expression support synaptic firing plus lytic 73 replication, latency and reactivation of HSV-1 [46]. However, given the clinical importance of 74 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint HSE it is also necessary to have scalable, tractable systems to probe lytic infection of cortical 75 neurones. This can be achieved via differentiation driven by Neurogenin2 [47]. Human iPSCs 76 with doxycycline-inducible Ngn2 in a safe-harbour locus [48] can be differentiated in 14 days 77 into cortical glutamatergic neurones with close to 100% efficiency using a simple two-step 78 protocol [49]. These integrated, inducible, and isogenic iPSCs (i3Neurones) exhibit robust 79 synchronous neuronal firing [50] and are amenable to genetic manipulation [50, 51], making 80 them suitable for precisely targeted functional studies. The ability to generate a homogenous 81 isogenic population via activation of a stably-integrated master transcriptional regulator 82 reduces experimental variability that can confound the interpretation of genome- or proteome-83 wide screening experiments [48], making these i3Neurones a robust platform for molecular 84 discovery research. 85 Here we present optimisation and initial characterisation of lytic HSV-1 infection of i3Neurones, 86 expanding the toolkit for biochemical and functional characterisation of neuronal HSV-1 87 infection. 88

89 Stem cell culture 90 Human fibroblast-derived iPSCs containing a doxycycline-inducible Ngn2 transcription factor 91 and an inactivated (dead) Cas9 gene in a safe-harbour locus [52] were provided by Michael 92 Ward (National Institutes of Health, USA) and cultured as per [49]. Briefly, iPSCs were 93 maintained in a 5% CO2 humidified atmosphere at 37°C in dishes pre-coated with hESC-94 qualified Matrigel (Corning 354277) diluted 1:100 in Dulbecco’s Modified Eagle 95 Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco 11330032). Essential 8 medium (Gibco 96 A1517001) was used for 24 hr culture and Essential 8 Flex medium (Gibco A2858501) for 72 hr 97 culture, and colonies were subcultured by dissociation using 0.5 mM EDTA in PBS. iPSCs were 98 dissociated to single-cell suspension with StemPro Accutase Cell Dissociation Reagent (Gibco 99 A1110501) and seeded in medium supplemented with 50 nM Chroman1 (Rho-associated 100 protein kinase (ROCK) inhibitor; Bio-Techne 7163/10). 101 Neuronal differentiation 102 Differentiation of iPSCs into i3Neurones followed a two-step protocol of differentiation and 103 maturation as outlined in [49]. In summary, 1.5–1.8×107 iPSCs were seeded following Accutase 104 dissociation in a Matrigel-coated 15 cm dish (Day 0) and incubated for three days in Induction 105 Medium (IM): DMEM/F-12, supplemented with 1× N2 supplement (Gibco 17502048), 1× non-106 essential amino acids (NEAA, Gibco 11140050), 1× L-glutamine (Gibco 25030081), plus 2 μg/mL 107 doxycycline (Sigma Aldrich D3072) to induce Ngn2 expression. The medium was changed daily, 108 being supplemented with 50 nM Chroman 1 for the first day of differentiation. At day 3 the 109 i3Neurone precursor cells were dissociated with Accutase and frozen at -80°C in 110 cryopreservation media comprising 90% (v/v) KnockOut Serum Replacement (Gibco 10828010) 111 and 10% (v/v) DMSO before being stored in liquid nitrogen. 112 Day 3 i3Neurone precursor cells were cultured for a further 11 days (to day 14) in cortical 113 neurone (CN) culture medium comprising Neurobasal Plus Medium (Gibco A3582901) 114 supplemented with 1× B27 supplement (Gibco A3582901), 10 ng/mL Brain Derived 115 Neurotrophic Factor (BDNF , PeproTech 450-02), 10 ng/mL Neurotrophin-3 (NT-3, PeproTech 450-116 03) and 1 μg/mL Laminin (Gibco 23017015). Frozen cells were thawed rapidly, diluted 10-fold in 117 DMEM/F12 medium, pelleted by centrifugation (300 × g, 5 min), resuspended in CN medium 118 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint supplemented with 2 μg/mL doxycycline and counted using a Countess II FL automated cell 119 counter (Invitrogen). i3Neurones were seeded on plates coated with 100 μg/mL poly-L-ornithine 120 (PLO) in half of the final culture volume of CN medium, incubated for 15 min at room 121 temperature to allow cells to settle evenly, and then the remaining half of the medium was 122 added before returning the cells to the incubator. When first seeding the cells the CN medium 123 was supplemented with 2 μg/mL doxycycline, and half the volume of CN medium was replaced 124 every three days until day 14, at which time the i3Neurones were used for experiments. At all 125 stages, cells were cultured in a 5% CO2 humidified atmosphere at 37°C, and final differentiated 126 neurones were confirmed as being free of mycoplasma. 127 Non-neuronal cell culture 128 Vero (ATCC CRL-1586), U2OS (ATCC HTB-96) and U2OS pUL21-BirA*-HA cells (see below) were 129 grown in DMEM supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) and 2 130 mM L-glutamine (complete DMEM) in a 5% CO2 humidified atmosphere at 37°C. All cells were 131 frequently tested for mycoplasma and confirmed as mycoplasma-free. 132 U2OS cells expressing pUL21 tagged with an abortive biotin ligase (BirA*) [53] were generated by 133 co-transfection of Flp-In T-REx U2OS cells provided by Gopal Sapkota (University of Dundee, 134 UK) [54] with pOG44 (Invitrogen) and pcDNA5/FRT/TO (Invitrogen) encoding codon-optimised 135 HSV-1 pUL21 (UniProt F8RG07) [55] with a C-terminal BirA* plus HA epitope tag. At 72 h post-136 transfection the culture medium was replaced with fresh medium containing 200 μg/mL 137 hygromycin B and 3 μg/mL blasticidin. Selection of hygromycin and blasticidin resistant cells 138 was allowed to proceed for 19 days, with medium being refreshed every 2-3 days as required. 139 pUL21-BirA*-HA expression was induced by addition of 2 µg/mL doxycycline 24 h prior to use. 140 Antibodies 141 Antibodies were used for immunofluorescence microscopy (IF), immunoblotting and virus 142 neutralisation. See Table 3.1 for full details of antibodies and dilutions used. 143 Table 1. Antibodies used in this study. 144 Antibody Source Dilution for immunocytochemistry Dilution for immunoblotting Mouse monoclonal anti-gD (LP2) [56] 1:9 – Mouse monoclonal anti-gD (LP14) [56] – 1:20 Mouse monoclonal anti-ICP4 ATCC 58S 1:9 – Rabbit monoclonal anti-GOPC Abcam Ab133472 – 1:1000 Mouse monoclonal anti-VP5 (DM165) [57] – 1:9 Mouse monoclonal anti-GAPDH GeneTex GTX28245 – 1:5000 Rabbit polyclonal anti-OCT4 NEB 2750s – 1:1000 Rabbit polyclonal anti-βIII Tubulin Abcam Ab18207 – 1:5000 Chicken polyclonal anti-βIII Tubulin NB100-1612 1:1000 – Mouse monoclonal anti-TAU Abcam Ab80579 1:50000 1:50000 Rabbit polyclonal anti-MAP2 Merck Ab5622 1:1000 – Sheep polyclonal anti-TGN46 Bio-Rad AHP500G 1:200 – Mouse monoclonal anti-pUL21 (1F10) [58] – 1:1 Goat anti-mouse 800 LI-COR 926-32210 – 1:10000 Goat anti-mouse 680 LI-COR 926-68020 – 1:10000 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint Goat anti-rabbit 800 LI-COR 926-32213 – 1:10000 Goat anti-rabbit 680 LI-COR 926-68023 – 1:10000 Goatanti-IgG2a 680 LI-COR 926-68051 – 1:10000 Goat anti-mouse 488 Invitrogen A32723 1:1000 to 1:2000 – Donkey anti-rabbit 568 Invitrogen A10042 1:1000 – Donkey anti-sheep 568 Invitrogen A21099 1:1000 – Donkey anti-mouse 568 Invitrogen A10037 1:1000 – Goat anti-rabbit 488 Invitrogen A11008 1:1000 – Goat anti-chicken 647 Invitrogen A21449 1:1000 145 Viruses 146 Wild-type HSV-1 strain KOS (WT HSV-1) was derived from a bacterial artificial chromosome 147 (BAC) encoding the KOS genome [59], as was wild-type HSV-1 with EFYP-tagged ICP0 and 148 mCherry-tagged gC (WT timestamp HSV-1) [60]. A mutant timestamp HSV-1 lacking expression 149 of pUL21 (ΔpUL21 timestamp HSV-1) was generated by two-step red recombination [59] of the 150 WT timestamp BAC, introducing three stop codons into the pUL21 gene as described in [61]. 151 Following the initial transfection of purified BAC DNA and pGS403 (encoding Cre recombinase) 152 into Vero cells, all subsequent propagation was performed in complementing U2OS pUL21-153 BirA*-HA cells to minimise the chance of virus adaptation [58]. HSV-1 strain 17 was from Stacey 154 Efstathiou (University of Cambridge, UK) and HSV-1 SC16 was from Tony Minson (University of 155 Cambridge, UK). 156 Virus stocks were grown by infection of U2OS pUL21-BirA*-HA cells (ΔpUL21 timestamp HSV-1) 157 or Vero cells (all others) at low (0.01) multiplicity of infection (MOI) for 3–5 days, until 158 widespread cytopathic effect was evident. For timestamp viruses, cells were scraped into 159 medium, freeze-thawed and sonicated at 50% amplitude for 40 seconds in a cuphorn sonicator 160 before being clarified by centrifugation at 3,200×g for 5 min in a benchtop centrifuge. For all 161 other viruses, the culture medium was supplemented with 0.5 M NaCl and 100 μg/mL dextran 162 sulfate (7–20 kDa; SigmaAldrich 51227). The following day the supernatant was harvested, 163 filtered through a 0.8 μm cellulose nitrate membrane (Nalgene 450-0080), virions were pelleted 164 by centrifugation at 17,000 rpm in a Type 19 rotor for 45 min at 4°C, and viruses were 165 resuspended in PBS with 10% (v/v) glycerol. For all, virus stocks were aliquoted and stored at -166 70°C until required. 167 Virus titration 168 Samples serially diluted in 500 µL complete DMEM were used to infect 6-well plates containing 169 confluent monolayers of Vero cells for 1 h at 37°C before being overlaid with 2 mL complete 170 DMEM containing 0.3% high viscosity carboxymethyl cellulose (CMC) and 0.3% low viscosity 171 CMC. After 3 days, cells were fixed with 3.7% (v/v) formal saline for 20 min before either being 172 imaged using an Incucyte SX5 (Sartorius) with a 20× long working distance objective (NA 0.45) 173 for fluorescent (timestamp) viruses or stained with 0.1% toluidine blue. In both cases plaques 174 were counted manually. 175 Virus infection 176 For neuronal infections, day 3 i3Neurones were seeded at specified densities and allowed to 177 mature. After maturation (day 14), conditioned medium was reserved, i3Neurones were washed 178 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint with PBS and then infected with the indicated viruses in fresh CN medium at the relevant MOI. 179 The time of virus inoculation was assigned 0 h post infection (hpi). Inoculation volumes for 180 neurones per well were as follows: 100 µL for 96-well plates, 300 µL for 24-well plates, 600 µL 181 for 6-well plates. Inoculated neurones were placed on a rocking platform at 37°C in a humidified 182 5% CO2 atmosphere. After 1 or 2 h incubation (as indicated), the inoculum was removed, cells 183 were gently washed twice with PBS (unless stated otherwise), and washed cells were overlayed 184 with a 1:1 mix of fresh and conditioned CN medium that had been clarified by centrifugation. 185 Final volumes of overlay per well were as follows: 200 µL for 96-well plates, 1 mL for 24-well 186 plates and 2.5 mL for 6-well plates. Vero cells were infected as above but using complete DMEM 187 in place of CN medium. 188 Immunoblotting 189 Day 3 i3Neurones were seeded at 2×106 cells per well in a PLO-treated 6-well plate and allowed 190 to mature. Where indicated, were infected at day 13 cells with MOI 5 for 1 h with HSV-1 strains 191 KOS, strain 17 or SC16, or mock infected. For all, at day 14 cells plate were washed once with 192 room temperature (RT) PBS and then ice-cold PBS containing 1% EDTA-free protease inhibitor 193 cocktail (SigmaAldrich P8849) was used to detach the neurone layer from the well, fully 194 suspending it by quickly and repeatedly dispensing down the edge of the well. The cell 195 suspension was transferred to a microcentrifuge tube and pelleted (5000 × g, 5 min, 4°C) to 196 remove the supernatant before lysis for 5 min using ice-cold RIPA buffer (50 mM Tris pH 8.0, 150 197 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% EDTA-free protease inhibitor cocktail) and 198 centrifugation (9000 × g, 10 min, 4°C) to remove debris. Undifferentiated i3Neurone iPSCs were 199 grown to ~80% confluence in Matrigel-coated 6-well plates before being washed with PBS, lysed 200 in situ, transferred to microcentrifuge tubes and the lysate clarified as above. For both, protein 201 concentrations were analysed by BCA assay (Pierce) and normalised before samples were 202 boiled (95°C, 5 min) in Laemmli sample buffer and separated by SDS-PAGE. Separated proteins 203 were transferred to Protran 0.45 µm nitrocellulose membranes (Cytiva) using the Mini-PROTEAN 204 system (Bio-Rad). Membranes were blocked using Tris-buffered saline (TBS; 50 mM Tris pH 7.6, 205 150 mM NaCl) supplemented with 5% (w/v) skim milk powder. Primary and secondary antibody 206 incubations were performed in TBS supplemented with 0.1% TWEEN (TBS-T) and 5% (w/v) skim 207 milk powder. Immunoblots were imaged using an Odyssey CLx (LI-COR) and analysed using 208 Image Studio Lite (LI-COR). 209 Immunocytochemistry 210 For confocal imaging, 13 mm #1.5 borosilicate glass coverslips were etched with 1 M nitric acid 211 for 24 h before washing with ethanol and sterile PBS then coated with 100 μg/ml PLO for 24 h. 212 Day 3 i3Neurones were seeded on prepared coverslips at a density of 1×105 cells per coverslip 213 and were allowed to mature. After maturation (day 14), cells were washed with PBS and fixed 214 with the 4% (v/v) EM-grade formaldehyde (Polysciences) in a 250 mM HEPES pH 7.5 buffer on 215 ice for 10 min followed by an 8% (v/v) formaldehyde solution in 250 mM HEPES pH 7.5 buffer at 216 room temperature (RT) for 20 min, unless otherwise stated. Fixed i3Neurones were washed 217 thrice with PBS and permeabilised at RT on a rocking platform using PBS supplemented with 218 0.2% Triton X-100 for 5 min or 0.1% saponin for 15 min. Cells were blocked for 30 min at RT using 219 blocking buffer comprising PBS supplemented with 5% (v/v) FBS alone (Triton X-100 220 permeabilisation) or 5% (v/v) FBS plus 0.01% saponin (saponin permeabilisation) and incubated 221 with primary antibody in blocking buffer for 2 hr at RT. Coverslips were washed in blocking 222 buffer, incubated with secondary antibodies in blocking buffer for 1 hr at RT in the dark, then 223 washed in blocking buffer, PBS and then MQW. Cells were mounted on microscope slides using 224 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint Mowiol 4-88 (Merck) supplemented with 200 nM 4′,6-diamidino-2-phenylindole (DAPI) and left 225 to dry overnight before being stored at 4°C. Slides were imaged using an EVOS M5000 226 (Invitrogen) using a 20× plan fluorite long working distance objective (NA 0.45) or a Zeiss 227 LSM700 confocal laser scanning microscopy system mounted on an AxioObserver.Z1 inverted 228 microscope with a 64× plan apochromat objective (NA 1.4). 229 For monitoring percentage infection, cells grown in PLO-treated culture vessels were fixed in 230 situ using 4% then 8% formaldehyde in HEPES buffer and stained for ICP4 as detailed above. 231 Immunostained cells were overlayed with propidium iodide (2.5 µg/mL in PBS + 0.02% sodium 232 azide) to stain the nuclei and were imaged via phase contrast and fluorescence using an 233 Incucyte SX5 (Sartorius) with a 20× long working distance objective (NA 0.45). Images were 234 quantified using the Basic Analyser algorithm in the Incucyte analysis software (Sartorius). 235 For all, figures were generated using ImageJ [62, 63]. 236 Inoculation condition optimisation 237 Day 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 238 allowed to mature. Infection proceeded as described above with changes to the inoculation 239 conditions as follows: cells were inoculated for 60, 90 or 120 min being rocked either manually 240 every 15 min or automatically at a low speed on a rocking platform. The inoculum was removed 241 and cells gently washed with PBS twice (unless stated otherwise), overlayed with a 1:1 mix of 242 fresh and clarified conditioned CN medium. Cells were fixed at 16 hpi and percentage infection 243 was monitored by immunocytochemistry using 4 to 8% formaldehyde in HEPES buffer for 244 fixation and Triton X-100 for permeabilisation as described above. Data were analysed using a 245 two-way ANOVA and significance of differences to 60 min sample was assessed using Sidak’s 246 test in Prism 7 (GraphPad). The equivalency of variance across all data points at MOI 5 for the 247 automatic versus manual rocking was assessed using Levine’s test [64] in Prism 7 (GraphPad). 248 Percentage of infected i3Neurones at different MOIs 249 Day 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 250 allowed to mature. At day 13, Vero cells were seeded separately at 5×105 cells per well. The 251 following day, both i3Neurones and Vero cells were infected with a 2-fold serial dilution of WT 252 HSV-1, with MOIs ranging from 10 to 0.0098. After 2 h the inoculum was removed and the cells 253 gently washed twice with PBS (unless stated otherwise) before being overlayed with a 1:1 mix of 254 fresh and clarified conditioned media. Cells were fixed at 16 hpi and percentage infection was 255 monitored by immunocytochemistry as described above. 256 Neurone survival following infection 257 Day 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 258 allowed to mature before being infected at MOI 5 for 1 h with HSV-1 strains KOS, strain 17 or 259 SC16, or mock infected. Cells were washed twice with PBS, and then overlayed with a 1:1 mix of 260 fresh and clarified conditioned CN medium supplemented with 2.5 µg/mL propidium iodide. 261 Cell viability, defined as exclusion of the propidium iodide, was monitored by capturing phase 262 contrast and orange fluorescence images every 6 h using an Incucyte SX5 (Sartorius). Images 263 were analysed using the Basic Analyser algorithm in the Incucyte analysis software (Sartorius). 264 Half the volume of CN medium, supplemented with fresh 2.5 µg/mL propidium iodide, was 265 replaced every 3 days. 266 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint Inoculum inactivation 267 Day 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 268 allowed to mature. The i3Neurones were infected at MOI 5 for 1 hr before the inoculum was 269 removed and cells were either: washed thrice immediately with PBS; incubated with 25 μg/mL 270 LP2 for 15 min at 37°C before three PBS washes; or washed once with citric acid (40 mM citric 271 acid pH 3.0, 135 mM NaCl, 10 mM KCl) for 1 min before three PBS washes. Cells were overlayed 272 with a 1:1 mix of fresh and clarified conditioned CN medium. At 3 and 24 hpi, neurones were 273 harvested by thrice freezing the plate at -70°C and then thawing. Lysed cells were scraped into 274 the medium and the virus concentration was assessed by plaque assay. Data were analysed 275 using a one-way ANOVA and significance was assessed using Tukey’s test in Prism 7 276 (GraphPad). 277 Fixative Condition Tests 278 Day 3 i3Neurones were seeded at 1×105 cells per well on prepared coverslips in 24-well plates 279 and allowed to mature. The i3Neurones were infected with WT HSV-1 at MOI 3. At 16 hpi, 280 neurones were washed with PBS and fixed using one of the following four treatments: 4% (v/v) 281 EM-grade formaldehyde in a 250 mM HEPES pH 7.5 on ice for 10 min then an 8% (v/v) 282 formaldehyde solution in 250 mM HEPES pH 7.5 at RT for 20 min; cytoskeletal fixing buffer 283 (300 mM NaCl, 10 mM EDTA, 10 mM glucose, 10 mM MgCl2, 20 mM PIPES pH 6.8, 2% sucrose, 284 4% formaldehyde) on ice for 15 min; 100% methanol on ice for 10 min; or glyoxal buffer pH 4 285 (3% (v/v) glyoxal, 20% (v/v) ethanol, 0.75% acetic acid, pH adjusted with NaOH) on ice for 30 286 min followed by a further 30 min at RT. Fixed neurones were washed, permeabilised, stained 287 and imaged as described above. 288 HSV-1 spread assay 289 Day 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 290 allowed to mature before being infected at MOI 0.1 for 2 h with timestamp viruses, washed 291 twice with PBS, and then overlayed with a 1:1 mix of fresh and clarified conditioned CN medium 292 supplemented with 25 µg/mL LP2 antibody. Virus spread was monitored by live-cell 293 fluorescence imaging using an Incucyte SX5 (Sartorius), recording phase contrast images, green 294 fluorescence and orange fluorescence every 3 h. Data were analysed using the Basic Analyser 295 algorithm in the Incucyte analysis software (Sartorius). Half the volume of CN medium was 296 replaced every 3 days. 297 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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298 Optimisation of the i3Neurone differentiation protocol for infection studies 299 The protocols for iPSC culture and i3Neurone differentiation were adapted from [49] to increase 300 the efficiency of differentiation and promote survival during maturation (Fig. 1a). Specifically, 301 duration of the Accutase dissociation was decreased to reduce cell death. The plating 302 procedure was modified by seeding cells in half the final culture volume of medium and 303 incubating the cells on the plate for 15 min at RT before adding the remaining medium. This 304 ensured neurones evenly coated the well and reduced clumping of neurone cell bodies. To 305 ensure iPSCs had correctly differentiated into neurones following these adaptations, 306 immunoblotting (Fig. 1b) and immunofluorescence microscopy (Fig. 1c) of cultured iPSCs and 307 i3Neurones was performed. Immunoblotting confirmed the loss of pluripotency marker OCT4 308 Fig. 1. Differentiation of human iPSCs into cortical glutamatergic neurones (i3Neurones). (a) Schematic of the differentiation procedure. PLO, poly-L-ornithine; Ngn2, Neurogenin 2. (b) Validation of iPSC differentiation into neurones. Lysates of iPSCs and i3Neurones were immunoblotted for pluripotency marker OCT4 and neuronal markers TAU and βIII-tubulin. GAPDH is a loading control. (c) Confocal microscopy of differentiated i3Neurones, showing neuron-like morphology. Neuronal markers TAU (green) and MAP2 (magenta) are shown, and the merge image includes DAPI (blue). 20 µm scale bar. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint and the gain of neuronal markers TAU and βIII tubulin following differentiation. The presence of 309 extensive neurites and neuronal cytoskeletal proteins MAP2 and TAU (Fig. 1c) confirmed 310 successful differentiation. 311 Optimisation of i3Neurone infection protocol 312 Initial infection tests suffered from technical issues including cell death, cell layers peeling off 313 the plate and lower levels of infection than expected given the titre of virus inoculum. Several 314 strategies were employed to combat these issues, testing different inoculation conditions. The 315 first optimisation was to increase the volume of medium used for virus inoculation, as 316 otherwise i3Neurones dried out and died during the inoculation step (Fig. S1a). Using 317 approximately 30–50% of the final overlay volume for inoculation prevented such cell death. 318 Secondly, to prevent peeling of the neurone layer (Fig. S1b) it was important to pipette liquids 319 dropwise directly onto the cell layer rather than pipetting liquid onto the walls of the culture 320 vessel [49]. 321 To test if inoculation conditions could be optimised to increase the proportion of cells infected, 322 neurones were infected at an MOI of either 1 or 5 via incubation with inoculum for 60, 90 or 120 323 min, either with continual rocking on a rocking platform or with manual rocking every 15 min 324 (Fig. 2a). Cells were fixed at 16 hpi and stained for ICP4, an immediate-early viral protein that 325 localises predominantly to the nucleus [65], plus propidium iodide to visualise nuclear DNA. 326 Cells were imaged via automated microscopy to determine the percentage of infected cells (Fig. 327 2b). Automatic or manual rocking did not make a statistically significant difference to efficiency 328 of infection at either MOI (two-way ANOVA of three independent experiments, p = 0.718 [MOI 1] 329 or 0.280 [MOI 5]). At MOI 1 there was a significant increase in infection efficiency with increased 330 duration of inoculation (Fig. 2a), but even after 2 h incubation with inoculum the proportion of 331 infected cells remained below the theoretical maximum of 63.2% expected if infections 332 occurred randomly. Extending the inoculum incubation time at MOI 5 did not alter the efficiency 333 of infection, but use of an automated rocker resulted in significantly less variability of infection 334 level across the replicates and time points (Levine’s test, p = 0.0346). 335 To further investigate the efficiency of infection, a 2-fold dilution series of WT HSV-1 (from MOI 336 10 to 0.01) was used to inoculate i3Neurones and Vero cells in parallel (Fig. 2c). Below MOI 5 the 337 proportion of infected cells was consistently lower than theoretically expected. Interestingly, a 338 higher proportion of Vero cells were infected than expected, suggesting that virus titration via 339 plaque assay may systematically underestimate the infectious titres. At high MOI, automated 340 measurement of infection in Vero cells suggested that >100% of cells were infected. This arose 341 due to the non-homogenous distribution of ICP4 staining in nuclei, with some infected Vero cell 342 nuclei being counted twice by the Incucyte software (Fig. S2). Such double-counting may have 343 also contributed to the greater-than-expected level of infection observed for Vero cells at 344 different MOIs. Since double-counting of infected cells was not observed for i3Neurones, this 345 phenomenon was not investigated further. 346 For temporally-resolved experiments like high MOI (single-step) growth curves that require a 347 synchronous infection, it is necessary to inactivate any input virus particles that have not 348 entered cells after a fixed time. This inactivation is often achieved via low pH treatment [66, 67]. 349 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint Because the neurones may be more sensitive to chemical treatment than other cell lines, 350 different methods were used to assess their inactivation efficiency whilst preserving cell 351 Fig. 2. Optimisation of i3Neurone infection by HSV-1. (a) i3Neurones were infected at MOI 5 or 1 and incubated for the listed duration with manual or automated rocking. Percent of infected cells as determined by automated microscopy (mean ± SD from three independent experiments) is shown. Two-way ANOVA (automated or manual rocker versus time) confirmed no significant effect of rocking method but a significant effect of time for MOI 1 but not MOI 5. ns, no significance; * *, p < 0.005; *** p < 0.001. (b) Representative automated microscopy images of infected i3Neurones. Objects where ICP4 (green, left) and propidium iodide (orange, middle) signals overlap (cyan, right) are counted as infected cells, expressed as a percentage of total propidium iodide objects (total cells). 50 μm scale bar. (c) Percentage of i3Neurones and Vero cells infected at different MOI (mean ± SD from three independent experiments). The percentage expected if infections occur randomly is shown. Scale bar represents 50 μm. (d) Optimisation of inoculum inactivation. Inoculum was removed at 1 hpi (MOI 5 HSV-1) and i3Neurones were either washed with PBS, incubated with neutralising antibody (nAb LP2) for 15 min, or washed with citric acid pH 3.0. Cells were harvested at 3 or 24 hpi and virus titres were determined by plaque assay. Mean and data points from two independent experiments, each performed in technical duplicate. One-way ANOVA confirms that the citrate wash yields a significant inactivation of input virus (3 hpi) but no difference in virus production at 24 hpi. ns, no significance; * , p < 0.05. (e) Schematic diagram of the optimised workflow for infecting i3Neurones with HSV-1. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint viability. Differentiated i3Neurones were infected at MOI 5 using an automated plate rocker. 352 After 1 h the inoculum was removed and cells were washed with citric acid, incubated with a 353 potent neutralising antibody (LP2), or washed with PBS. The cultures were harvested at 3 and 354 24 hpi to assess both inoculum inactivation and subsequent virus production, which would 355 decrease dramatically if cell viability was affected (Fig. 2d). The citric acid wash was the most 356 effective treatment for inactivation, reducing the viral titre to below detection in some 357 replicates. The antibody incubation was marginally more effective than PBS washing alone at 358 removing input virus, but both were inferior to a citrate wash. There was no significant difference 359 in virus yield at 24 hpi between any of the three conditions, demonstrating that none of these 360 protocols adversely affected neurone viability. Based on these optimisations, refined protocols 361 for low- and high-MOI infection of i3Neurones are summarised in Fig. 2e. 362 During the optimisation of the infection protocol, it was noted that neurones seemed highly 363 tolerant of infection, with reduced morphology changes and prolonged survival compared to 364 other cell types like Vero. The survival of i3Neurones following synchronous high MOI infection 365 was thus investigated. Neurones were infected at MOI 5 with HSV-1 strains KOS, strain 17 or 366 SC16, or mock-infected. Neurones were then incubated in the presence of propidium iodide, a 367 DNA stain excluded from live cells, and imaged every 6 h via automated microscopy. By 5 days 368 post-infection the neurones displayed morphology changes, with changes in the appearance of 369 the soma and, in the case of KOS, clustering of the soma that is potentially indicative of 370 syncytium formation. However, the neurones remained alive for upwards of 8 (KOS) or 10 (strain 371 17 and SC16) days, with visibly axonal degradation occurring close to the time of cell death 372 (Fig. 3). 373 Fig. 3. I3Neurones survive for over one week following HSV-1 infection. (a) Live-cell microscopy of i3Neurones infected at MOI 5 with HSV-1 strains KOS, strain 17 and SC16, at listed days post infection (dpi). Propidium iodide signal, which is excluded from live cells, is shown in green. Scale bar 100 µm. (b) Quantification of neurone survival, with cell death measured as increased number of propidium iodide positive nuclei (dead nuclei / mm²). Data are representative of three independent experiments. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint Optimised fixation and permeabilization of i3Neurones for immunocytochemistry 374 Neurites often became visibly damaged during the fixation and staining procedures used for 375 preliminary infection quantification experiments, presumably due to their delicate nature. 376 Different fixation conditions were thus tested for i3Neurones grown on coverslips that had been 377 acid-etched to improve neurone adhesion [68]. Two conditions commonly used for 378 immunocytochemistry of epithelial cells were tested (4% followed by 8% formaldehyde in 250 379 mM HEPES pH 7.4, or 100% methanol), as were two identified in the literature as being more 380 effective for neurones or for preserving cytoskeletal structures (3% glyoxal pH 4.0 in 20% 381 ethanol, or 4% formaldehyde in a cytoskeletal preservation solution)[69, 70]. Two different 382 permeabilisation solutions were trialled, either 0.2% Triton X-100 for 5 min or 0.1% saponin for 383 15 min. i3Neurones were stained for the neuronal cytoskeletal protein β-III tubulin, the trans-384 Golgi network protein TGN46, the nuclear marker of infection ICP4, and for DNA using DAPI. The 385 blocking and staining protocols used for each fixation/permeabilisation condition was identical, 386 except for the inclusion of 0.01% saponin in the blocking buffer for cells permeabilised with 387 saponin. Wide-field microscopy of coverslips fixed using the different protocols and 388 permeabilised with Triton X-100 or saponin are shown in Figs S3 and S4, respectively. 389 Fixation using 4% then 8% formaldehyde in 250 mM HEPES yielded the best preservation of 390 cells morphology, with excellent preservation of neurites. Triton permeabilization yielded visibly 391 brighter signal for TGN46 and similar staining for the other markers, both in wide field (Figs S3 392 and S4) and confocal (Fig. 4) microscopy. Acquiring confocal Z-stacks (7–14 nm) proved the 393 most reliable method for imaging both the thin neurites and the thicker cell bodies. It is notable 394 that at 16 hpi there is no apparent change in overall cell morphology (contrast Fig. 1c and Fig. 4). 395 396 Fig. 4. Confocal microscopy of HSV-1 infected (MOI 5) i3Neurons, fixed 16 hpi using 4% then 8% formaldehyde in 250 mM HEPES buffer and permeabilised using either 0.2% Triton X-100 for 5 min or 0.1% Saponin for 15 min. Cells were stained for ICP4 (green), TGN46 (yellow), βIII tubulin (magenta) and DNA (DAPI, blue) and maximum-intensity projections of 7 nm (Triton X-100) or 14 nm (saponin) Z stacks are shown. Triton X-100 permeabilisation yields stronger cytoskeletal (βIII tubulin) and organelle (TGN46) staining. 20 µm scale bar. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint i3Neurones as a model for HSV-1 lytic infection of cortical neurones 397 Having optimised the infection procedure, the utility of i3Neurones for monitoring HSV-1 spread 398 was assessed. Plaque assays, which monitor the spread of virus to adjacent cells following 399 infection of a single cell, are a well-established technique for measuring HSV-1 cell-to-cell 400 spread in cells of the periphery like fibroblasts [30, 71]. However, the sparsity of neuronal cell 401 bodies and potential for long-distance spread via intracellular transport of virions along neurites 402 confounds the use of plaque assays to measure HSV-1 spread in i3Neurones. Therefore, virus 403 neurone-to-neurone spread was monitored by infecting i3Neurones at low MOI (0.1) with HSV-1 404 strain KOS expressing the early protein ICP0 tagged with EYFP and the late protein gC tagged 405 with mCherry [60]. Neutralising antibody (LP2) was included in the culture medium to inhibit 406 cell-free spread and the spread of fluorescence, a proxy for infection spread, was monitored 407 every 3 h by automated microscopy. While no signal was visible for ICP0-EYFP, a robust gC-408 mCherry signal was evident and this signal spread throughout the culture over the course of 409 96 h (Fig. 5a), confirming productive neurone-to-neurone HSV-1 spread. The infection appeared 410 to spread most rapidly between the soma of adjacent cells but by 48 hpi infection was also 411 evident in the soma of distant cells, consistent with spread of the infection via axonal transport 412 of newly produced HSV-1 virions. 413 Many proteins present within the tegument layer of HSV-1 are known to contribute to efficient 414 cell-to-cell spread in fibroblasts or keratinocytes [61, 72]. However, the contributions these 415 proteins make to neurone-to-neurone spread is less clear. The role of tegument protein pUL21 is 416 of particular interest, as its effect upon virus spread is known to vary by HSV strain and by cell 417 type [73]. To assess its contribution to neurone-to-neurone spread, a mutant virus lacking 418 expression of pUL21 was generated in the timestamp background (timestamp ΔpUL21, Fig. S5). 419 The spread of timestamp ΔpUL21 infection was monitored via automated microscopy following 420 low MOI (0.1) infection of i3Neurones (Fig. 5a). The spread of timestamp ΔpUL21 was 421 substantially delayed when compared to the wild-type timestamp virus (Fig. 5b). However, there 422 was still evidence of spread to soma of neurons distal to the initial site of infection, suggesting 423 that transport of virions along neurites had not been completely impaired. Taken together, this 424 Fig. 5. HSV-1 neurone-to-neurone spread. (a) Live-cell microscopy of i3Neurones infected at low MOI (0.1) with WT or ΔpUL21 timestamp HSV-1. gC-mCherry signal is shown in red or orange for WT and ΔpUL21 HSV-1, respectively. Scale bar 200 µm. (b) Quantification of timestamp virus spread, measured as increase area of gC-mCherry fluorescence (μm² / image) for 4 days post infection. Mean ± SD for two independent experiments performed in technical triplicate are shown. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint experiment demonstrates how i3Neurones can be combined with fluorescently tagged HSV-1 to 425 measure the effects of HSV-1 proteins on viral neurone-to-neurone spread. 426 In addition to monitoring virus spread, it is often desirable to monitor the abundance of cellular 427 or viral proteins in synchronous populations of infected cells. While immunoblotting is a 428 convenient technique for monitoring protein abundance, it can be difficult to obtain enough 429 infected-cell lysate for immunoblotting when working with organoids or primary neurones. The 430 scalability of i3Neurones [49], combined with the ability to perform efficient synchronous 431 infection (Fig. 2), overcomes this limitation. To assess the feasibility of using immunoblots to 432 monitor changes in host protein abundance, single wells of a 6-well dish containing 2×106 433 i3Neurones were synchronously infected (MOI 5) with HSV-1 strains KOS, strain 17 and SC16. At 434 24 hpi cells were harvested, lysed and subjected to immunoblot analysis. For all three strains 435 the viral capsid protein VP5 could be detected, confirming successful infection and late gene 436 expression (Fig. 6). Additionally, compared to the mock-infected sample, all three infected 437 lysates showed lower abundance of the cellular protein GOPC, a known target of HSV pUL56-438 mediated degradation [30]. This confirms that the i3Neurone system is suitable for biochemical 439 analysis of HSV-1 neuronal infection. 440

441 Here we present optimised protocols for the differentiation of human iPSC-derived cortical 442 glutamatergic neurones (i3Neurones) and their infection with HSV-1. The i3Neurone system is 443 highly scalable, allowing production of >107 differentiated neurones with ease, and these 444 neurones can be synchronously infected with high (>90%) efficiency (Fig. 2). These neurons 445 survive for upwards of 8 days following infection (Fig. 3), consistent with previous reports of 446 sympathetic mouse neurones surviving for up to 30 days following lytic infection with HSV-1 447 [74]. We show that i3Neurones are suitable for biochemical analysis of lytic HSV-1 infection 448 (Fig. 6) and i3Neurones thus show strong potential for use in high resolution infection 449 proteomics analysis [29, 30]. We have previously shown that i3Neurones can be infected with 450 Zika virus [75], human astroviruses [76] and human enteroviruses [77]. i3Neurones thus 451 represent a promising platform for advanced biochemical analysis of many neurotropic virus 452 infections. 453 Fig. 6. Validation of viral gene expression and function in i3Neurones by immunoblot. i3Neurones were infected at MOI 5 with indicated HSV-1 strains and lysed 24 hpi. Samples were immunoblotted for infection marker VP5, the cellular protein GOPC that is a target of pUL56-mediated degradation, and the cellular loading control GAPDH. .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint In addition to biochemical analyses, the reproducibility of i3Neurone differentiation [48] and 454 their amenability to gene overexpression [51] or knockdown via the integrated dead Cas9 [50–455 52] make them a powerful platform for functional analysis. We show here that i3Neurones can 456 be combined with fluorescent virus strains of HSV-1 to monitor neurone-to-neurone spread of 457 HSV-1. While we observed a signal for ICP0-EYFP in i3Neurones when imaged using a wide-field 458 microscope, consistent with prior studies using timestamp HSV-1 [60, 78], we could not 459 visualise ICP0-EYFP using the Incucyte SX5 automated microscope. This difference is likely to 460 arise from a combination of lower ICP0 expression in i3Neurones, the use of a long working 461 distance objective with low numerical aperture, plus suboptimal matching of the excitation 462 (453–485 nm) and emission (494–533 nm) filters on our automated microscope to the EYFP 463 fluorophore (peak excitation 515 nm and emission 530 nm). In the presence of neutralising 464 antibody, we show that HSV-1 strain KOS spreads to the soma of neurones far from the initial 465 site of infection within 48 hpi, consistent with intracellular transport of virions along neurites. It 466 is unclear whether this spread represents virus particles budding from the soma of an infected 467 cell, entering a neurite and undergoing retrograde transport to the nucleus, or whether it 468 represents anterograde transport of newly assembled virions to neurite termini where they then 469 bud to infect other neurones. Since HSV-1 strain KOS lacks a functional pUS9 protein [79], 470 known to be important for both anterograde axonal transport and virus assembly at axon termini 471 [80], it seems likely that the observed long-distance spread represents retrograde transport 472 following infection of neurites. This could be confirmed in future studies using directional 473 infection of soma or neurites in compartmentalised culture systems [81]. 474 In summary, using HSV-1 as a model we have demonstrated the i3Neurone system to be a 475 robust tool for measuring the replication and spread of viruses in cortical neurones. We 476 anticipate that optimised neurone culture, infection and analysis protocols presented here will 477 accelerate research into a broad range of clinically important neurotropic infections. 478 Author contributions 479 Conceptualisation: JED, SCG; Funding Acquisition: JED, SCG; Investigation: DAN, ASN, HGB, 480 VC; Project Administration: JED, SCG; Resources: CMC, JED; Supervision: JED, SCG; 481 Visualisation: DAN; Writing – Original Draft Preparation: DAN, SCG; Writing – Review & Editing: 482 DAN, HGB, AN, JED, SCG 483 Conflicts of interest 484 The authors declare no competing interests. 485 Funding information 486 DAN was supported by a Department of Pathology studentship funded by the Gwynaeth Pretty 487 Fund. HGB was supported by a Wellcome Trust PhD studentship. This work was supported by a 488 Wellcome Trust Senior Research Fellowship (219447/Z/19/Z) to JED. The funders had no role in 489 study design, data collection and analysis, decision to publish, or preparation of the 490 manuscript. 491

492 We thank Dr Michael Ward for the i3Neurones, Dr Gopal Sapkota for the Flp-In T-REx U2OS 493 cells, Profs Stacey Efstathiou and Tony Minson for HSV-1 isolates, and the Cambridge 494 Microscopy Bioscience Platform for their support and assistance in this work. 495 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint

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