{"paper_id":"a3816d97-e01c-4935-aac8-f09d09220f9f","body_text":"Optimisation of lytic herpes simplex virus infection in human induced pluripotent 1 \nstem cell derived cortical neurones 2 \nDaniel A. Nash1, Alex S. Nicholson2, Henry G. Barrow1, Viv Connor1, Colin M. Crump1, Janet E. 3 \nDeane2, Stephen C. Graham1  4 \n1Department of Pathology, University of Cambridge, Cambridge CB2 1QP , United Kingdom 5 \n2Cambridge Institute for Medical Research, Department of Clinical Neuroscience, University of 6 \nCambridge, Cambridge CB2 0XY , UK 7 \nCorresponding authors 8 \nJanet E. Deane jed55@cam.ac.uk; Stephen C. Graham scg34@cam.ac.uk  9 \nKeywords 10 \nNeurons, herpes simplex encephalitis (HSE), virus-host interactions, i3Neurons 11 \nAbstract 12 \nHerpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. 13 \nWhile we have substantial knowledge about the molecular virology of HSV-1 lytic infection in 14 \ncells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of 15 \nhuman neurones owing to the challenges of working with neuronal cell-based models. Here we 16 \ndemonstrate the use of a human induced pluripotent stem cell (iPSC)-derived cortical neurone 17 \nmodel (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and 18 \nefficiently differentiated into an isogenic population of cortical glutamatergic neurones. We 19 \nshow that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimised 20 \nprotocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at 21 \nnear-100% efficiency, and optimised fixation methods that preserves organelle and neurite 22 \nstructure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, 23 \nscalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic 24 \npathogens. 25 \nData summary 26 \nThe authors confirm all supporting data, code and protocols have been provided within the 27 \narticle or through supplementary data files.  28 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nIntroduction 29 \nNeuronal virus infections cause severe pathology. HSV-1 is the leading cause of viral 30 \nencephalitis [1], causing 70% mortality in untreated patients and up to 19% in patients treated 31 \nwith antivirals, with survivors often suffering severe neurological sequelae [2]. Similarly, 32 \ninfection with enteroviruses such as EV-A71 cause encephalitis and acute flaccid paralysis [3]; 33 \nZika virus and Oropouche virus infection in adults can cause Guillain-Barré syndrome [4, 5]; and 34 \ncongenital Zika virus infection can cause microcephaly, decreased brain tissue, plus ocular and 35 \nosteoskeletal abnormalities [6]. Furthermore, it is increasingly clear that infection with 36 \nneurotropic viruses like HSV is a risk factor for developing common neurodegenerative diseases 37 \n[7–9]. It is therefore important to identify robust and appropriate human cell-based systems to 38 \nstudy the molecular basis of neuronal infection by HSV-1 and other neurotropic pathogens. 39 \nMultiple different systems have been used for the study of neuronal HSV-1 infection, especially 40 \nin the context of latency (reviewed in [10]). Ex vivo infection of rat or mouse-derived ganglia that 41 \nrepresent the natural site of HSV-1 latency are widely used [11–13], although the number of 42 \nneurones that can be isolated even from a large number of animals is limited [14] and there are 43 \ndifferences in interactions between HSV-1 and mouse versus human immune responses [15, 44 \n16]. Ex vivo studies of human neurones is possible using post-mortem specimens [17, 18], but 45 \navailability and the capacity for genetic manipulation of the specimens is limited. Human 46 \nembryonic or induced pluripotent stem cells (iPSCs) allow interrogation of infection following 47 \ndifferentiation into neural stem cells, neurones or other glial cell types [19–23]. However, 48 \ndifferentiation timescales can be long and the procedures labour-intensive. Human stem-cell 49 \nderived organoids represent a powerful model for studying the functional consequences of 50 \nHSV-1 lytic and latent infection in human neural stem cells, neurones and glia [24–28]. While 51 \nexcellent for transcriptomic analysis [25, 28], these models are not well suited to high resolution 52 \nproteomics studies of infection such as quantitative temporal viromics [29], which require large 53 \nnumbers of homogenous cells (≥1×107) and high levels of synchronous infection (≥90%) [30, 54 \n31]. 55 \nScalable cancer-derived neuroblastoma cell lines like SH-SY5Y are widely used for HSV-1 56 \ninfection studies [21, 32, 33] but they have complex chromosomal aberrations [34, 35], are 57 \nhighly sensitive to the differentiation procedure used and yield mixed morphology populations 58 \n[36]. Lund human mesencephalic (LUHMES) cells have been developed as models to study 59 \nHSV-1 latency [37] and host shutoff during lytic infection [38]. Differentiated LUHMES resemble 60 \npost-mitotic dopaminergic neurones [39] and they represent a powerful homogenous cell-61 \nbased system for studies of neuronal infection. However, LUHMES cells are derived from the 62 \nmidbrain mesencephalon [40] whereas herpes simplex encephalitis (HSE) is generally localised 63 \nto the temporal lobes [41]. As we know that innate immune programs differ between different 64 \nclasses of neurone [42], there is a need for additional scalable systems for the study of HSV-1 65 \ninfection in the cerebral cortex. 66 \nDifferentiation of human iPSCs via the expression of integrated transcription factors represents 67 \na promising approach to rapidly obtain isogenic populations of differentiated neuronal and 68 \nother cell types [43]. For example, human iPSCs expressing the neuronal transcription factor 69 \nNeurogenin3 (NGN3) [43] can be differentiated into sensory human neurones that have been 70 \nused to characterise miRNAs and neuronal factors that regulate the efficiency of HSV-1 lytic 71 \nreplication or establishment of latency [44, 45].  Recently, a comprehensive analysis has shown 72 \nthat sensory neurones differentiated via NGN3 expression support synaptic firing plus lytic 73 \nreplication, latency and reactivation of HSV-1 [46]. However, given the clinical importance of 74 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nHSE it is also necessary to have scalable, tractable systems to probe lytic infection of cortical 75 \nneurones. This can be achieved via differentiation driven by Neurogenin2 [47]. Human iPSCs 76 \nwith doxycycline-inducible Ngn2 in a safe-harbour locus [48] can be differentiated in 14 days 77 \ninto cortical glutamatergic neurones with close to 100% efficiency using a simple two-step 78 \nprotocol [49]. These integrated, inducible, and isogenic iPSCs (i3Neurones) exhibit robust 79 \nsynchronous neuronal firing [50] and are amenable to genetic manipulation [50, 51], making 80 \nthem suitable for precisely targeted functional studies. The ability to generate a homogenous 81 \nisogenic population via activation of a stably-integrated master transcriptional regulator 82 \nreduces experimental variability that can confound the interpretation of genome- or proteome-83 \nwide screening experiments [48], making these i3Neurones a robust platform for molecular 84 \ndiscovery research. 85 \nHere we present optimisation and initial characterisation of lytic HSV-1 infection of i3Neurones, 86 \nexpanding the toolkit for biochemical and functional characterisation of neuronal HSV-1 87 \ninfection. 88 \nMethods 89 \nStem cell culture 90 \nHuman fibroblast-derived iPSCs containing a doxycycline-inducible Ngn2 transcription factor 91 \nand an inactivated (dead) Cas9 gene in a safe-harbour locus [52] were provided by Michael 92 \nWard (National Institutes of Health, USA) and cultured as per [49]. Briefly, iPSCs were 93 \nmaintained in a 5% CO2 humidified atmosphere at 37°C in dishes pre-coated with hESC-94 \nqualified Matrigel (Corning 354277) diluted 1:100 in Dulbecco’s Modified Eagle 95 \nMedium/Nutrient Mixture F-12 (DMEM/F-12; Gibco 11330032). Essential 8 medium (Gibco 96 \nA1517001) was used for 24 hr culture and Essential 8 Flex medium (Gibco A2858501) for 72 hr 97 \nculture, and colonies were subcultured by dissociation using 0.5 mM EDTA in PBS. iPSCs were 98 \ndissociated to single-cell suspension with StemPro Accutase Cell Dissociation Reagent (Gibco 99 \nA1110501) and seeded in medium supplemented with 50 nM Chroman1 (Rho-associated 100 \nprotein kinase (ROCK) inhibitor; Bio-Techne 7163/10).  101 \nNeuronal differentiation 102 \nDifferentiation of iPSCs into i3Neurones followed a two-step protocol of differentiation and 103 \nmaturation as outlined in [49]. In summary, 1.5–1.8×107 iPSCs were seeded following Accutase 104 \ndissociation in a Matrigel-coated 15 cm dish (Day 0) and incubated for three days in Induction 105 \nMedium (IM): DMEM/F-12, supplemented with 1× N2 supplement (Gibco 17502048), 1× non-106 \nessential amino acids (NEAA, Gibco 11140050), 1× L-glutamine (Gibco 25030081), plus 2 μg/mL 107 \ndoxycycline (Sigma Aldrich D3072) to induce Ngn2 expression. The medium was changed daily, 108 \nbeing supplemented with 50 nM Chroman 1 for the first day of differentiation. At day 3 the 109 \ni3Neurone precursor cells were dissociated with Accutase and frozen at -80°C in 110 \ncryopreservation media comprising 90% (v/v) KnockOut Serum Replacement (Gibco 10828010) 111 \nand 10% (v/v) DMSO before being stored in liquid nitrogen.  112 \nDay 3 i3Neurone precursor cells were cultured for a further 11 days (to day 14) in cortical 113 \nneurone (CN) culture medium comprising Neurobasal Plus Medium (Gibco A3582901) 114 \nsupplemented with 1× B27 supplement (Gibco A3582901), 10 ng/mL Brain Derived 115 \nNeurotrophic Factor (BDNF , PeproTech 450-02), 10 ng/mL Neurotrophin-3 (NT-3, PeproTech 450-116 \n03) and 1 μg/mL Laminin (Gibco 23017015). Frozen cells were thawed rapidly, diluted 10-fold in 117 \nDMEM/F12 medium, pelleted by centrifugation (300 × g, 5 min), resuspended in CN medium 118 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nsupplemented with 2 μg/mL doxycycline and counted using a Countess II FL automated cell 119 \ncounter (Invitrogen). i3Neurones were seeded on plates coated with 100 μg/mL poly-L-ornithine 120 \n(PLO) in half of the final culture volume of CN medium, incubated for 15 min at room 121 \ntemperature to allow cells to settle evenly, and then the remaining half of the medium was 122 \nadded before returning the cells to the incubator. When first seeding the cells the CN medium 123 \nwas supplemented with 2 μg/mL doxycycline, and half the volume of CN medium was replaced 124 \nevery three days until day 14, at which time the i3Neurones were used for experiments. At all 125 \nstages, cells were cultured in a 5% CO2 humidified atmosphere at 37°C, and final differentiated 126 \nneurones were confirmed as being free of mycoplasma. 127 \nNon-neuronal cell culture 128 \nVero (ATCC CRL-1586), U2OS (ATCC HTB-96) and U2OS pUL21-BirA*-HA cells (see below) were 129 \ngrown in DMEM supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) and 2 130 \nmM L-glutamine (complete DMEM) in a 5% CO2 humidified atmosphere at 37°C. All cells were 131 \nfrequently tested for mycoplasma and confirmed as mycoplasma-free.  132 \nU2OS cells expressing pUL21 tagged with an abortive biotin ligase (BirA*) [53] were generated by 133 \nco-transfection of Flp-In T-REx U2OS cells provided by Gopal Sapkota (University of Dundee, 134 \nUK) [54] with pOG44 (Invitrogen) and pcDNA5/FRT/TO (Invitrogen) encoding codon-optimised 135 \nHSV-1 pUL21 (UniProt F8RG07) [55] with a C-terminal BirA* plus HA epitope tag. At 72 h post-136 \ntransfection the culture medium was replaced with fresh medium containing 200 μg/mL 137 \nhygromycin B and 3 μg/mL blasticidin. Selection of hygromycin and blasticidin resistant cells 138 \nwas allowed to proceed for 19 days, with medium being refreshed every 2-3 days as required. 139 \npUL21-BirA*-HA expression was induced by addition of 2 µg/mL doxycycline 24 h prior to use. 140 \nAntibodies 141 \nAntibodies were used for immunofluorescence microscopy (IF), immunoblotting and virus 142 \nneutralisation. See Table 3.1 for full details of antibodies and dilutions used.  143 \nTable 1. Antibodies used in this study.  144 \nAntibody Source Dilution for \nimmunocytochemistry \nDilution for \nimmunoblotting \nMouse monoclonal anti-gD (LP2) [56] 1:9 – \nMouse monoclonal anti-gD (LP14) [56] – 1:20 \nMouse monoclonal anti-ICP4 ATCC 58S 1:9 – \nRabbit monoclonal anti-GOPC Abcam Ab133472 – 1:1000 \nMouse monoclonal anti-VP5 (DM165) [57] – 1:9 \nMouse monoclonal anti-GAPDH GeneTex GTX28245 – 1:5000 \nRabbit polyclonal anti-OCT4 NEB 2750s – 1:1000 \nRabbit polyclonal anti-βIII Tubulin Abcam Ab18207 – 1:5000 \nChicken polyclonal anti-βIII Tubulin NB100-1612 1:1000 – \nMouse monoclonal anti-TAU Abcam Ab80579 1:50000 1:50000 \nRabbit polyclonal anti-MAP2 Merck Ab5622 1:1000 – \nSheep polyclonal anti-TGN46 Bio-Rad AHP500G 1:200 – \nMouse monoclonal anti-pUL21 (1F10) [58] – 1:1 \nGoat anti-mouse 800 LI-COR 926-32210 – 1:10000 \nGoat anti-mouse 680 LI-COR 926-68020 – 1:10000 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nGoat anti-rabbit 800 LI-COR 926-32213 – 1:10000 \nGoat anti-rabbit 680 LI-COR 926-68023 – 1:10000 \nGoatanti-IgG2a 680 LI-COR 926-68051 – 1:10000 \nGoat anti-mouse 488 Invitrogen A32723 1:1000 to 1:2000 – \nDonkey anti-rabbit 568 Invitrogen A10042 1:1000 – \nDonkey anti-sheep 568 Invitrogen A21099 1:1000 – \nDonkey anti-mouse 568 Invitrogen A10037 1:1000 – \nGoat anti-rabbit 488 Invitrogen A11008 1:1000 – \nGoat anti-chicken 647 Invitrogen A21449 1:1000  \n 145 \nViruses 146 \nWild-type HSV-1 strain KOS (WT HSV-1) was derived from a bacterial artificial chromosome 147 \n(BAC) encoding the KOS genome [59], as was wild-type HSV-1 with EFYP-tagged ICP0 and 148 \nmCherry-tagged gC (WT timestamp HSV-1) [60]. A mutant timestamp HSV-1 lacking expression 149 \nof pUL21 (ΔpUL21 timestamp HSV-1) was generated by two-step red recombination [59] of the 150 \nWT timestamp BAC, introducing three stop codons into the pUL21 gene as described in [61]. 151 \nFollowing the initial transfection of purified BAC DNA and pGS403 (encoding Cre recombinase) 152 \ninto Vero cells, all subsequent propagation was performed in complementing U2OS pUL21-153 \nBirA*-HA cells to minimise the chance of virus adaptation [58]. HSV-1 strain 17 was from Stacey 154 \nEfstathiou (University of Cambridge, UK) and HSV-1 SC16 was from Tony Minson (University of 155 \nCambridge, UK). 156 \nVirus stocks were grown by infection of U2OS pUL21-BirA*-HA cells (ΔpUL21 timestamp HSV-1) 157 \nor Vero cells (all others) at low (0.01) multiplicity of infection (MOI) for 3–5 days, until 158 \nwidespread cytopathic effect was evident. For timestamp viruses, cells were scraped into 159 \nmedium, freeze-thawed and sonicated at 50% amplitude for 40 seconds in a cuphorn sonicator 160 \nbefore being clarified by centrifugation at 3,200×g for 5 min in a benchtop centrifuge. For all 161 \nother viruses, the culture medium was supplemented with 0.5 M NaCl and 100 μg/mL dextran 162 \nsulfate (7–20 kDa; SigmaAldrich 51227).  The following day the supernatant was harvested, 163 \nfiltered through a 0.8 μm cellulose nitrate membrane (Nalgene 450-0080), virions were pelleted 164 \nby centrifugation at 17,000 rpm in a Type 19 rotor for 45 min at 4°C, and viruses were 165 \nresuspended in PBS with 10% (v/v) glycerol. For all, virus stocks were aliquoted and stored at -166 \n70°C until required. 167 \nVirus titration 168 \nSamples serially diluted in 500 µL complete DMEM were used to infect 6-well plates containing 169 \nconfluent monolayers of Vero cells for 1 h at 37°C before being overlaid with 2 mL complete 170 \nDMEM containing 0.3% high viscosity carboxymethyl cellulose (CMC) and 0.3% low viscosity 171 \nCMC. After 3 days, cells were fixed with 3.7% (v/v) formal saline for 20 min before either being 172 \nimaged using an Incucyte SX5 (Sartorius) with a 20× long working distance objective (NA 0.45) 173 \nfor fluorescent (timestamp) viruses or stained with 0.1% toluidine blue. In both cases plaques 174 \nwere counted manually. 175 \nVirus infection 176 \nFor neuronal infections, day 3 i3Neurones were seeded at specified densities and allowed to 177 \nmature. After maturation (day 14), conditioned medium was reserved, i3Neurones were washed 178 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nwith PBS and then infected with the indicated viruses in fresh CN medium at the relevant MOI. 179 \nThe time of virus inoculation was assigned 0 h post infection (hpi). Inoculation volumes for 180 \nneurones per well were as follows: 100 µL for 96-well plates, 300 µL for 24-well plates, 600 µL 181 \nfor 6-well plates. Inoculated neurones were placed on a rocking platform at 37°C in a humidified 182 \n5% CO2 atmosphere. After 1 or 2 h incubation (as indicated), the inoculum was removed, cells 183 \nwere gently washed twice with PBS (unless stated otherwise), and washed cells were overlayed 184 \nwith a 1:1 mix of fresh and conditioned CN medium that had been clarified by centrifugation. 185 \nFinal volumes of overlay per well were as follows: 200 µL for 96-well plates, 1 mL for 24-well 186 \nplates and 2.5 mL for 6-well plates. Vero cells were infected as above but using complete DMEM 187 \nin place of CN medium. 188 \nImmunoblotting 189 \nDay 3 i3Neurones were seeded at 2×106 cells per well in a PLO-treated 6-well plate and allowed 190 \nto mature. Where indicated, were infected at day 13 cells with MOI 5 for 1 h with HSV-1 strains 191 \nKOS, strain 17 or SC16, or mock infected. For all, at day 14 cells plate were washed once with 192 \nroom temperature (RT) PBS and then ice-cold PBS containing 1% EDTA-free protease inhibitor 193 \ncocktail (SigmaAldrich P8849) was used to detach the neurone layer from the well, fully 194 \nsuspending it by quickly and repeatedly dispensing down the edge of the well. The cell 195 \nsuspension was transferred to a microcentrifuge tube and pelleted (5000 × g, 5 min, 4°C) to 196 \nremove the supernatant before lysis for 5 min using ice-cold RIPA buffer (50 mM Tris pH 8.0, 150 197 \nmM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% EDTA-free protease inhibitor cocktail) and 198 \ncentrifugation (9000 × g, 10 min, 4°C) to remove debris. Undifferentiated i3Neurone iPSCs were 199 \ngrown to ~80% confluence in Matrigel-coated 6-well plates before being washed with PBS, lysed 200 \nin situ, transferred to microcentrifuge tubes and the lysate clarified as above. For both, protein 201 \nconcentrations were analysed by BCA assay (Pierce) and normalised before samples were 202 \nboiled (95°C, 5 min) in Laemmli sample buffer and separated by SDS-PAGE. Separated proteins 203 \nwere transferred to Protran 0.45 µm nitrocellulose membranes (Cytiva) using the Mini-PROTEAN 204 \nsystem (Bio-Rad). Membranes were blocked using Tris-buffered saline (TBS; 50 mM Tris pH 7.6, 205 \n150 mM NaCl) supplemented with 5% (w/v) skim milk powder. Primary and secondary antibody 206 \nincubations were performed in TBS supplemented with 0.1% TWEEN (TBS-T) and 5% (w/v) skim 207 \nmilk powder. Immunoblots were imaged using an Odyssey CLx (LI-COR) and analysed using 208 \nImage Studio Lite (LI-COR). 209 \nImmunocytochemistry 210 \nFor confocal imaging, 13 mm #1.5 borosilicate glass coverslips were etched with 1 M nitric acid 211 \nfor 24 h before washing with ethanol and sterile PBS then coated with 100 μg/ml PLO for 24 h. 212 \nDay 3 i3Neurones were seeded on prepared coverslips at a density of 1×105 cells per coverslip 213 \nand were allowed to mature. After maturation (day 14), cells were washed with PBS and fixed 214 \nwith the 4% (v/v) EM-grade formaldehyde (Polysciences) in a 250 mM HEPES pH 7.5 buffer on 215 \nice for 10 min followed by an 8% (v/v) formaldehyde solution in 250 mM HEPES pH 7.5 buffer at 216 \nroom temperature (RT) for 20 min, unless otherwise stated. Fixed i3Neurones were washed 217 \nthrice with PBS and permeabilised at RT on a rocking platform using PBS supplemented with 218 \n0.2% Triton X-100 for 5 min or 0.1% saponin for 15 min. Cells were blocked for 30 min at RT using 219 \nblocking buffer comprising PBS supplemented with 5% (v/v) FBS alone (Triton X-100 220 \npermeabilisation) or 5% (v/v) FBS plus 0.01% saponin (saponin permeabilisation) and incubated 221 \nwith primary antibody in blocking buffer for 2 hr at RT. Coverslips were washed in blocking 222 \nbuffer, incubated with secondary antibodies in blocking buffer for 1 hr at RT in the dark, then 223 \nwashed in blocking buffer, PBS and then MQW. Cells were mounted on microscope slides using 224 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nMowiol 4-88 (Merck) supplemented with 200 nM 4′,6-diamidino-2-phenylindole (DAPI) and left 225 \nto dry overnight before being stored at 4°C. Slides were imaged using an EVOS M5000 226 \n(Invitrogen) using a 20× plan fluorite long working distance objective (NA 0.45) or a Zeiss 227 \nLSM700 confocal laser scanning microscopy system mounted on an AxioObserver.Z1 inverted 228 \nmicroscope with a 64× plan apochromat objective (NA 1.4). 229 \nFor monitoring percentage infection, cells grown in PLO-treated culture vessels were fixed in 230 \nsitu using 4% then 8% formaldehyde in HEPES buffer and stained for ICP4 as detailed above. 231 \nImmunostained cells were overlayed with propidium iodide (2.5 µg/mL in PBS + 0.02% sodium 232 \nazide) to stain the nuclei and were imaged via phase contrast and fluorescence using an 233 \nIncucyte SX5 (Sartorius) with a 20× long working distance objective (NA 0.45). Images were 234 \nquantified using the Basic Analyser algorithm in the Incucyte analysis software (Sartorius).  235 \nFor all, figures were generated using ImageJ [62, 63].  236 \nInoculation condition optimisation 237 \nDay 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 238 \nallowed to mature. Infection proceeded as described above with changes to the inoculation 239 \nconditions as follows: cells were inoculated for 60, 90 or 120 min being rocked either manually 240 \nevery 15 min or automatically at a low speed on a rocking platform. The inoculum was removed 241 \nand cells gently washed with PBS twice (unless stated otherwise), overlayed with a 1:1 mix of 242 \nfresh and clarified conditioned CN medium. Cells were fixed at 16 hpi and percentage infection 243 \nwas monitored by immunocytochemistry using 4 to 8% formaldehyde in HEPES buffer for 244 \nfixation and Triton X-100 for permeabilisation as described above. Data were analysed using a 245 \ntwo-way ANOVA and significance of differences to 60 min sample was assessed using Sidak’s 246 \ntest in Prism 7 (GraphPad). The equivalency of variance across all data points at MOI 5 for the 247 \nautomatic versus manual rocking was assessed using Levine’s test [64] in Prism 7 (GraphPad). 248 \nPercentage of infected i3Neurones at different MOIs  249 \nDay 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 250 \nallowed to mature. At day 13, Vero cells were seeded separately at 5×105 cells per well. The 251 \nfollowing day, both i3Neurones and Vero cells were infected with a 2-fold serial dilution of WT 252 \nHSV-1, with MOIs ranging from 10 to 0.0098. After 2 h the inoculum was removed and the cells 253 \ngently washed twice with PBS (unless stated otherwise) before being overlayed with a 1:1 mix of 254 \nfresh and clarified conditioned media. Cells were fixed at 16 hpi and percentage infection was 255 \nmonitored by immunocytochemistry as described above.  256 \nNeurone survival following infection 257 \nDay 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 258 \nallowed to mature before being infected at MOI 5 for 1 h with HSV-1 strains KOS, strain 17 or 259 \nSC16, or mock infected. Cells were washed twice with PBS, and then overlayed with a 1:1 mix of 260 \nfresh and clarified conditioned CN medium supplemented with 2.5 µg/mL propidium iodide. 261 \nCell viability, defined as exclusion of the propidium iodide, was monitored by capturing phase 262 \ncontrast and orange fluorescence images every 6 h using an Incucyte SX5 (Sartorius). Images 263 \nwere analysed using the Basic Analyser algorithm in the Incucyte analysis software (Sartorius). 264 \nHalf the volume of CN medium, supplemented with fresh 2.5 µg/mL propidium iodide, was 265 \nreplaced every 3 days. 266 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nInoculum inactivation  267 \nDay 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 268 \nallowed to mature. The i3Neurones were infected at MOI 5 for 1 hr before the inoculum was 269 \nremoved and cells were either: washed thrice immediately with PBS; incubated with 25 μg/mL 270 \nLP2 for 15 min at 37°C before three PBS washes; or washed once with citric acid (40 mM citric 271 \nacid pH 3.0, 135 mM NaCl, 10 mM KCl) for 1 min before three PBS washes. Cells were overlayed 272 \nwith a 1:1 mix of fresh and clarified conditioned CN medium. At 3 and 24 hpi, neurones were 273 \nharvested by thrice freezing the plate at -70°C and then thawing. Lysed cells were scraped into 274 \nthe medium and the virus concentration was assessed by plaque assay.  Data were analysed 275 \nusing a one-way ANOVA and significance was assessed using Tukey’s test in Prism 7 276 \n(GraphPad). 277 \nFixative Condition Tests 278 \nDay 3 i3Neurones were seeded at 1×105 cells per well on prepared coverslips in 24-well plates 279 \nand allowed to mature. The i3Neurones were infected with WT HSV-1 at MOI 3. At 16 hpi, 280 \nneurones were washed with PBS and fixed using one of the following four treatments: 4% (v/v) 281 \nEM-grade formaldehyde in a 250 mM HEPES pH 7.5 on ice for 10 min then an 8% (v/v) 282 \nformaldehyde solution in 250 mM HEPES pH 7.5 at RT for 20 min; cytoskeletal fixing buffer 283 \n(300 mM NaCl, 10 mM EDTA, 10 mM glucose, 10 mM MgCl2, 20 mM PIPES pH 6.8, 2% sucrose, 284 \n4% formaldehyde) on ice for 15 min; 100% methanol on ice for 10 min; or glyoxal buffer pH 4 285 \n(3% (v/v) glyoxal,  20% (v/v) ethanol, 0.75% acetic acid, pH adjusted with NaOH) on ice for 30 286 \nmin followed by a further 30 min at RT. Fixed neurones were washed, permeabilised, stained 287 \nand imaged as described above.  288 \nHSV-1 spread assay 289 \nDay 3 i3Neurones were seeded at 3×105 cells per well in a PLO-treated 24-well plate and 290 \nallowed to mature before being infected at MOI 0.1 for 2 h with timestamp viruses, washed 291 \ntwice with PBS, and then overlayed with a 1:1 mix of fresh and clarified conditioned CN medium 292 \nsupplemented with 25 µg/mL LP2 antibody. Virus spread was monitored by live-cell 293 \nfluorescence imaging using an Incucyte SX5 (Sartorius), recording phase contrast images, green 294 \nfluorescence and orange fluorescence every 3 h. Data were analysed using the Basic Analyser 295 \nalgorithm in the Incucyte analysis software (Sartorius). Half the volume of CN medium was 296 \nreplaced every 3 days. 297 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nResults 298 \nOptimisation of the i3Neurone differentiation protocol for infection studies 299 \nThe protocols for iPSC culture and i3Neurone differentiation were adapted from [49] to increase 300 \nthe efficiency of differentiation and promote survival during maturation (Fig. 1a). Specifically, 301 \nduration of the Accutase dissociation was decreased to reduce cell death. The plating 302 \nprocedure was modified by seeding cells in half the final culture volume of medium and 303 \nincubating the cells on the plate for 15 min at RT before adding the remaining medium. This 304 \nensured neurones evenly coated the well and reduced clumping of neurone cell bodies. To 305 \nensure iPSCs had correctly differentiated into neurones following these adaptations, 306 \nimmunoblotting (Fig. 1b) and immunofluorescence microscopy (Fig. 1c) of cultured iPSCs and 307 \ni3Neurones was performed. Immunoblotting confirmed the loss of pluripotency marker OCT4 308 \n \nFig. 1. \nDifferentiation of human iPSCs into cortical glutamatergic neurones (i3Neurones). (a) Schematic of the \ndifferentiation procedure. PLO, poly-L-ornithine; Ngn2, Neurogenin 2. (b) Validation of iPSC differentiation into \nneurones. Lysates of iPSCs and i3Neurones were immunoblotted for pluripotency marker OCT4 and neuronal \nmarkers TAU and βIII-tubulin. GAPDH is a loading control. (c) Confocal microscopy of differentiated i3Neurones, \nshowing neuron-like morphology. Neuronal markers TAU (green) and MAP2 (magenta) are shown, and the merge \nimage includes DAPI (blue). 20 µm scale bar. \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nand the gain of neuronal markers TAU and βIII tubulin following differentiation. The presence of 309 \nextensive neurites and neuronal cytoskeletal proteins MAP2 and TAU (Fig. 1c) confirmed 310 \nsuccessful differentiation.  311 \nOptimisation of i3Neurone infection protocol 312 \nInitial infection tests suffered from technical issues including cell death, cell layers peeling off 313 \nthe plate and lower levels of infection than expected given the titre of virus inoculum. Several 314 \nstrategies were employed to combat these issues, testing different inoculation conditions. The 315 \nfirst optimisation was to increase the volume of medium used for virus inoculation, as 316 \notherwise i3Neurones dried out and died during the inoculation step (Fig. S1a). Using 317 \napproximately 30–50% of the final overlay volume for inoculation prevented such cell death. 318 \nSecondly, to prevent peeling of the neurone layer (Fig. S1b) it was important to pipette liquids 319 \ndropwise directly onto the cell layer rather than pipetting liquid onto the walls of the culture 320 \nvessel [49]. 321 \nTo test if inoculation conditions could be optimised to increase the proportion of cells infected, 322 \nneurones were infected at an MOI of either 1 or 5 via incubation with inoculum for 60, 90 or 120 323 \nmin, either with continual rocking on a rocking platform or with manual rocking every 15 min 324 \n(Fig. 2a). Cells were fixed at 16 hpi and stained for ICP4, an immediate-early viral protein that 325 \nlocalises predominantly to the nucleus [65], plus propidium iodide to visualise nuclear DNA. 326 \nCells were imaged via automated microscopy to determine the percentage of infected cells (Fig. 327 \n2b). Automatic or manual rocking did not make a statistically significant difference to efficiency 328 \nof infection at either MOI (two-way ANOVA of three independent experiments, p = 0.718 [MOI 1] 329 \nor 0.280 [MOI 5]). At MOI 1 there was a significant increase in infection efficiency with increased 330 \nduration of inoculation (Fig. 2a), but even after 2 h incubation with inoculum the proportion of 331 \ninfected cells remained below the theoretical maximum of 63.2% expected if infections 332 \noccurred randomly. Extending the inoculum incubation time at MOI 5 did not alter the efficiency 333 \nof infection, but use of an automated rocker resulted in significantly less variability of infection 334 \nlevel across the replicates and time points (Levine’s test, p = 0.0346). 335 \nTo further investigate the efficiency of infection, a 2-fold dilution series of WT HSV-1 (from MOI 336 \n10 to 0.01) was used to inoculate i3Neurones and Vero cells in parallel (Fig. 2c). Below MOI 5 the 337 \nproportion of infected cells was consistently lower than theoretically expected. Interestingly, a 338 \nhigher proportion of Vero cells were infected than expected, suggesting that virus titration via 339 \nplaque assay may systematically underestimate the infectious titres. At high MOI, automated 340 \nmeasurement of infection in Vero cells suggested that >100% of cells were infected. This arose 341 \ndue to the non-homogenous distribution of ICP4 staining in nuclei, with some infected Vero cell 342 \nnuclei being counted twice by the Incucyte software (Fig. S2). Such double-counting may have 343 \nalso contributed to the greater-than-expected level of infection observed for Vero cells at 344 \ndifferent MOIs. Since double-counting of infected cells was not observed for i3Neurones, this 345 \nphenomenon was not investigated further. 346 \nFor temporally-resolved experiments like high MOI (single-step) growth curves that require a 347 \nsynchronous infection, it is necessary to inactivate any input virus particles that have not 348 \nentered cells after a fixed time. This inactivation is often achieved via low pH treatment [66, 67]. 349 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nBecause the neurones may be more sensitive to chemical treatment than other cell lines, 350 \ndifferent methods were used to assess their inactivation efficiency whilst preserving cell 351 \n \nFig. 2. \nOptimisation of i3Neurone infection by HSV-1. (a) i3Neurones were infected at MOI 5 or 1 and incubated for the listed \nduration with manual or automated rocking. Percent of infected cells as determined by automated microscopy (mean ± \nSD from three independent experiments) is shown. Two-way ANOVA (automated or manual rocker versus time) \nconfirmed no significant effect of rocking method but a significant effect of time for MOI 1 but not MOI 5. ns, no \nsignificance; * *,  p  <  0.005; *** p < 0.001. (b) Representative automated microscopy images of infected i3Neurones. \nObjects where ICP4 (green, left) and propidium iodide (orange, middle) signals overlap (cyan, right) are counted as \ninfected cells, expressed as a percentage of total propidium iodide objects (total cells). 50 μm scale bar. (c) Percentage \nof i3Neurones and Vero cells infected at different MOI (mean ± SD from three independent experiments). The \npercentage expected if infections occur randomly is shown. Scale bar represents 50 μm. (d) Optimisation of inoculum \ninactivation. Inoculum was removed at 1 hpi (MOI 5 HSV-1) and i3Neurones were either washed with PBS, incubated \nwith neutralising antibody (nAb LP2) for 15 min, or washed with citric acid pH 3.0. Cells were harvested at 3 or 24 hpi \nand virus titres were determined by plaque assay. Mean and data points from two independent experiments, each \nperformed in technical duplicate. One-way ANOVA confirms that the citrate wash yields a significant inactivation of \ninput virus (3 hpi) but no difference in virus production at 24 hpi. ns, no significance; * , p < 0.05. (e) Schematic diagram \nof the optimised workflow for infecting i3Neurones with HSV-1.  \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nviability. Differentiated i3Neurones were infected at MOI 5 using an automated plate rocker. 352 \nAfter 1 h the inoculum was removed and cells were washed with citric acid, incubated with a 353 \npotent neutralising antibody (LP2), or washed with PBS.  The cultures were harvested at 3 and 354 \n24 hpi to assess both inoculum inactivation and subsequent virus production, which would 355 \ndecrease dramatically if cell viability was affected (Fig. 2d). The citric acid wash was the most 356 \neffective treatment for inactivation, reducing the viral titre to below detection in some 357 \nreplicates.  The antibody incubation was marginally more effective than PBS washing alone at 358 \nremoving input virus, but both were inferior to a citrate wash. There was no significant difference 359 \nin virus yield at 24 hpi between any of the three conditions, demonstrating that none of these 360 \nprotocols adversely affected neurone viability. Based on these optimisations, refined protocols 361 \nfor low- and high-MOI infection of i3Neurones are summarised in Fig. 2e. 362 \nDuring the optimisation of the infection protocol, it was noted that neurones seemed highly 363 \ntolerant of infection, with reduced morphology changes and prolonged survival compared to 364 \nother cell types like Vero. The survival of i3Neurones following synchronous high MOI infection 365 \nwas thus investigated. Neurones were infected at MOI 5 with HSV-1 strains KOS, strain 17 or 366 \nSC16, or mock-infected. Neurones were then incubated in the presence of propidium iodide, a 367 \nDNA stain excluded from live cells, and imaged every 6 h via automated microscopy. By 5 days 368 \npost-infection the neurones displayed morphology changes, with changes in the appearance of 369 \nthe soma and, in the case of KOS, clustering of the soma that is potentially indicative of 370 \nsyncytium formation. However, the neurones remained alive for upwards of 8 (KOS) or 10 (strain 371 \n17 and SC16) days, with visibly axonal degradation occurring close to the time of cell death 372 \n(Fig. 3). 373 \n \nFig. 3. \nI3Neurones survive for over one week following HSV-1 infection. (a) Live-cell microscopy of i3Neurones infected at \nMOI 5 with HSV-1 strains KOS, strain 17 and SC16, at listed days post infection (dpi). Propidium iodide signal, which is \nexcluded from live cells, is shown in green. Scale bar 100 µm. (b) Quantification of neurone survival, with cell death \nmeasured as increased number of propidium iodide positive nuclei (dead nuclei / mm²). Data are representative of \nthree independent experiments. \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nOptimised fixation and permeabilization of i3Neurones for immunocytochemistry 374 \nNeurites often became visibly damaged during the fixation and staining procedures used for 375 \npreliminary infection quantification experiments, presumably due to their delicate nature. 376 \nDifferent fixation conditions were thus tested for i3Neurones grown on coverslips that had been 377 \nacid-etched to improve neurone adhesion [68]. Two conditions commonly used for 378 \nimmunocytochemistry of epithelial cells were tested (4% followed by 8% formaldehyde in 250 379 \nmM HEPES pH 7.4, or 100% methanol), as were two identified in the literature as being more 380 \neffective for neurones or for preserving cytoskeletal structures (3% glyoxal pH 4.0 in 20% 381 \nethanol, or 4% formaldehyde in a cytoskeletal preservation solution)[69, 70]. Two different 382 \npermeabilisation solutions were trialled, either 0.2% Triton X-100 for 5 min or 0.1% saponin for 383 \n15 min. i3Neurones were stained for the neuronal cytoskeletal protein β-III tubulin, the trans-384 \nGolgi network protein TGN46, the nuclear marker of infection ICP4, and for DNA using DAPI. The 385 \nblocking and staining protocols used for each fixation/permeabilisation condition was identical, 386 \nexcept for the inclusion of 0.01% saponin in the blocking buffer for cells permeabilised with 387 \nsaponin. Wide-field microscopy of coverslips fixed using the different protocols and 388 \npermeabilised with Triton X-100 or saponin are shown in Figs S3 and S4, respectively. 389 \nFixation using 4% then 8% formaldehyde in 250 mM HEPES yielded the best preservation of 390 \ncells morphology, with excellent preservation of neurites. Triton permeabilization yielded visibly 391 \nbrighter signal for TGN46 and similar staining for the other markers, both in wide field (Figs S3 392 \nand S4) and confocal (Fig. 4) microscopy. Acquiring confocal Z-stacks (7–14 nm) proved the 393 \nmost reliable method for imaging both the thin neurites and the thicker cell bodies. It is notable 394 \nthat at 16 hpi there is no apparent change in overall cell morphology (contrast Fig. 1c and Fig. 4). 395 \n 396 \n \nFig. 4. \nConfocal microscopy of HSV-1 infected (MOI 5) i3Neurons, fixed 16 hpi using 4% then 8% formaldehyde in 250 mM \nHEPES buffer and permeabilised using either 0.2% Triton X-100 for 5 min or 0.1% Saponin for 15 min. Cells were \nstained for ICP4 (green), TGN46 (yellow), βIII tubulin (magenta) and DNA (DAPI, blue) and maximum-intensity \nprojections of 7 nm (Triton X-100) or 14 nm (saponin)  Z stacks are shown. Triton X-100 permeabilisation yields stronger \ncytoskeletal (βIII tubulin) and organelle (TGN46) staining. 20 µm scale bar. \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\ni3Neurones as a model for HSV-1 lytic infection of cortical neurones 397 \nHaving optimised the infection procedure, the utility of i3Neurones for monitoring HSV-1 spread 398 \nwas assessed. Plaque assays, which monitor the spread of virus to adjacent cells following 399 \ninfection of a single cell, are a well-established technique for measuring HSV-1 cell-to-cell 400 \nspread in cells of the periphery like fibroblasts [30, 71]. However, the sparsity of neuronal cell 401 \nbodies and potential for long-distance spread via intracellular transport of virions along neurites 402 \nconfounds the use of plaque assays to measure HSV-1 spread in i3Neurones. Therefore, virus 403 \nneurone-to-neurone spread was monitored by infecting i3Neurones at low MOI (0.1) with HSV-1 404 \nstrain KOS expressing the early protein ICP0 tagged with EYFP and the late protein gC tagged 405 \nwith mCherry [60]. Neutralising antibody (LP2) was included in the culture medium to inhibit 406 \ncell-free spread and the spread of fluorescence, a proxy for infection spread, was monitored 407 \nevery 3 h by automated microscopy. While no signal was visible for ICP0-EYFP, a robust gC-408 \nmCherry signal was evident and this signal spread throughout the culture over the course of 409 \n96 h (Fig. 5a), confirming productive neurone-to-neurone HSV-1 spread. The infection appeared 410 \nto spread most rapidly between the soma of adjacent cells but by 48 hpi infection was also 411 \nevident in the soma of distant cells, consistent with spread of the infection via axonal transport 412 \nof newly produced HSV-1 virions. 413 \nMany proteins present within the tegument layer of HSV-1 are known to contribute to efficient 414 \ncell-to-cell spread in fibroblasts or keratinocytes [61, 72]. However, the contributions these 415 \nproteins make to neurone-to-neurone spread is less clear. The role of tegument protein pUL21 is 416 \nof particular interest, as its effect upon virus spread is known to vary by HSV strain and by cell 417 \ntype [73]. To assess its contribution to neurone-to-neurone spread, a mutant virus lacking 418 \nexpression of pUL21 was generated in the timestamp background (timestamp ΔpUL21, Fig. S5). 419 \nThe spread of timestamp ΔpUL21 infection was monitored via automated microscopy following 420 \nlow MOI (0.1) infection of i3Neurones (Fig. 5a). The spread of timestamp ΔpUL21 was 421 \nsubstantially delayed when compared to the wild-type timestamp virus (Fig. 5b). However, there 422 \nwas still evidence of spread to soma of neurons distal to the initial site of infection, suggesting 423 \nthat transport of virions along neurites had not been completely impaired. Taken together, this 424 \n \nFig. 5. \nHSV-1 neurone-to-neurone spread. (a) Live-cell microscopy of i3Neurones infected at low MOI (0.1) with WT or ΔpUL21 \ntimestamp HSV-1. gC-mCherry signal is shown in red or orange for WT and ΔpUL21 HSV-1, respectively. Scale bar 200 \nµm. (b) Quantification of timestamp virus spread, measured as increase area of gC-mCherry fluorescence (μm² / \nimage) for 4 days post infection. Mean ± SD for two independent experiments performed in technical triplicate are \nshown.  \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nexperiment demonstrates how i3Neurones can be combined with fluorescently tagged HSV-1 to 425 \nmeasure the effects of HSV-1 proteins on viral neurone-to-neurone spread. 426 \nIn addition to monitoring virus spread, it is often desirable to monitor the abundance of cellular 427 \nor viral proteins in synchronous populations of infected cells. While immunoblotting is a 428 \nconvenient technique for monitoring protein abundance, it can be difficult to obtain enough 429 \ninfected-cell lysate for immunoblotting when working with organoids or primary neurones. The 430 \nscalability of i3Neurones [49], combined with the ability to perform efficient synchronous 431 \ninfection (Fig. 2), overcomes this limitation. To assess the feasibility of using immunoblots to 432 \nmonitor changes in host protein abundance, single wells of a 6-well dish containing 2×106 433 \ni3Neurones were synchronously infected (MOI 5) with HSV-1 strains KOS, strain 17 and SC16. At 434 \n24 hpi cells were harvested, lysed and subjected to immunoblot analysis. For all three strains 435 \nthe viral capsid protein VP5 could be detected, confirming successful infection and late gene 436 \nexpression (Fig. 6). Additionally, compared to the mock-infected sample, all three infected 437 \nlysates showed lower abundance of the cellular protein GOPC, a known target of HSV pUL56-438 \nmediated degradation [30]. This confirms that the i3Neurone system is suitable for biochemical 439 \nanalysis of HSV-1 neuronal infection. 440 \nDiscussion 441 \nHere we present optimised protocols for the differentiation of human iPSC-derived cortical 442 \nglutamatergic neurones (i3Neurones) and their infection with HSV-1. The i3Neurone system is 443 \nhighly scalable, allowing production of >107 differentiated neurones with ease, and these 444 \nneurones can be synchronously infected with high (>90%) efficiency (Fig. 2). These neurons 445 \nsurvive for upwards of 8 days following infection (Fig. 3), consistent with previous reports of 446 \nsympathetic mouse neurones surviving for up to 30 days following lytic infection with HSV-1 447 \n[74]. We show that i3Neurones are suitable for biochemical analysis of lytic HSV-1 infection 448 \n(Fig. 6) and i3Neurones thus show strong potential for use in high resolution infection 449 \nproteomics analysis [29, 30]. We have previously shown that i3Neurones can be infected with 450 \nZika virus [75], human astroviruses [76] and human enteroviruses [77]. i3Neurones thus 451 \nrepresent a promising platform for advanced biochemical analysis of many neurotropic virus 452 \ninfections. 453 \n \nFig. 6. \nValidation of viral gene expression and function in i3Neurones by immunoblot. i3Neurones were infected at MOI 5 with \nindicated HSV-1 strains and lysed 24 hpi. Samples were immunoblotted for infection marker VP5, the cellular protein \nGOPC that is a target of pUL56-mediated degradation, and the cellular loading control GAPDH.  \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nIn addition to biochemical analyses, the reproducibility of i3Neurone differentiation [48] and 454 \ntheir amenability to gene overexpression [51] or knockdown via the integrated dead Cas9 [50–455 \n52] make them a powerful platform for functional analysis. We show here that i3Neurones can 456 \nbe combined with fluorescent virus strains of HSV-1 to monitor neurone-to-neurone spread of 457 \nHSV-1. While we observed a signal for ICP0-EYFP in i3Neurones when imaged using a wide-field 458 \nmicroscope, consistent with prior studies using timestamp HSV-1 [60, 78], we could not 459 \nvisualise ICP0-EYFP using the Incucyte SX5 automated microscope. This difference is likely to 460 \narise from a combination of lower ICP0 expression in i3Neurones, the use of a long working 461 \ndistance objective with low numerical aperture, plus suboptimal matching of the excitation 462 \n(453–485 nm) and emission (494–533 nm) filters on our automated microscope to the EYFP 463 \nfluorophore (peak excitation 515 nm and emission 530 nm). In the presence of neutralising 464 \nantibody, we show that HSV-1 strain KOS spreads to the soma of neurones far from the initial 465 \nsite of infection within 48 hpi, consistent with intracellular transport of virions along neurites. It 466 \nis unclear whether this spread represents virus particles budding from the soma of an infected 467 \ncell, entering a neurite and undergoing retrograde transport to the nucleus, or whether it 468 \nrepresents anterograde transport of newly assembled virions to neurite termini where they then 469 \nbud to infect other neurones. Since HSV-1 strain KOS lacks a functional pUS9 protein [79], 470 \nknown to be important for both anterograde axonal transport and virus assembly at axon termini 471 \n[80], it seems likely that the observed long-distance spread represents retrograde transport 472 \nfollowing infection of neurites. This could be confirmed in future studies using directional 473 \ninfection of soma or neurites in compartmentalised culture systems [81]. 474 \nIn summary, using HSV-1 as a model we have demonstrated the i3Neurone system to be a 475 \nrobust tool for measuring the replication and spread of viruses in cortical neurones. We 476 \nanticipate that optimised neurone culture, infection and analysis protocols presented here will 477 \naccelerate research into a broad range of clinically important neurotropic infections. 478 \nAuthor contributions 479 \nConceptualisation: JED, SCG; Funding Acquisition: JED, SCG; Investigation: DAN, ASN, HGB, 480 \nVC; Project Administration: JED, SCG; Resources: CMC, JED; Supervision: JED, SCG; 481 \nVisualisation: DAN; Writing – Original Draft Preparation: DAN, SCG; Writing – Review & Editing: 482 \nDAN, HGB, AN, JED, SCG 483 \nConflicts of interest 484 \nThe authors declare no competing interests. 485 \nFunding information 486 \nDAN was supported by a Department of Pathology studentship funded by the Gwynaeth Pretty 487 \nFund. HGB was supported by a Wellcome Trust PhD studentship. This work was supported by a 488 \nWellcome Trust Senior Research Fellowship (219447/Z/19/Z) to JED. The funders had no role in 489 \nstudy design, data collection and analysis, decision to publish, or preparation of the 490 \nmanuscript. 491 \nAcknowledgements 492 \nWe thank Dr Michael Ward for the i3Neurones, Dr Gopal Sapkota for the Flp-In T-REx U2OS 493 \ncells, Profs Stacey Efstathiou and Tony Minson for HSV-1 isolates, and the Cambridge 494 \nMicroscopy Bioscience Platform for their support and assistance in this work.  495 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint \n\nReferences 496 \n1. Matthews E, Beckham JD, Piquet AL, Tyler KL, Chauhan L, et al. Herpesvirus-Associated 497 \nEncephalitis: an Update. Curr Trop Med Rep 2022;9:92–100. 498 \n2. 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Methods 705 \nMol Biol 2022;2431:181–206. 706 \n 707 \n.CC-BY 4.0 International licensemade available under a \n(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 \nThe copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.22.671689doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}