Biology subjects

Kinchington, P. R.

Publications and source records attributed to Kinchington, P. R..

5 recordsLinked to original sources

bioRxiv2026

Fixed Nonsynonymous vOka Mutations in IE62 Drive Varicella-Zoster Virus Attenuation in Human Skin

The live, attenuated vaccine for Varicella-Zoster Virus (VZV) protects against chickenpox in children and adults. However, the vaccine strain Oka (vOka) establishes latency and may reactivate. The molecular basis of vOka attenuation remains unknown. We investigated whether three high-frequency single-nucleotide polymorphisms (SNPs) that differentiate vOka from parental strain Oka (pOka) are responsible: S628G and R958G in the immediate-early regulatory protein IE62, and *130R in the membrane protein ORF0. We evaluated how these fixed SNPs mediate the attenuated growth phenotype of VZV and diminish skin pathology by creating recombinant pOka VZV carrying these SNPs in the wild-type background. These were assessed in cells and human skin organ cultures (SOCs). In epithelial and fibroblast cell lines, the individual IE62 mutations slightly delayed viral growth at 8 - 24 hours post-infection but not in human epidermal keratinocytes (htert-HEK). The fixed ORF62 SNPs also affected ORF62 transcription, IE62 protein abundance at early times, and delayed IE62 accumulation in the cytoplasm later during infection. The *130R mutation in ORF0 conferred a growth advantage in culture, suggesting it is an adaptation to cell culture. While there was donor-to-donor variability and differences in overall virus spread in SOC, the R958G and *130R conferred an attenuated growth phenotype similar to that of vOka in SOC. Histopathologic analysis of VZV-infected skin sections revealed that, similar to vOka, S628G and R958G in IE62 limited the spread of VZV skin lesions. Our findings underscore the critical roles of conserved vOka SNPs in understanding the attenuation of the live vaccine strain vOka.

microbiology↗

bioRxiv2025

A human sensory neuron model for varicella-zoster virus latency and reactivation in vitro.

Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that establishes lifelong latency in sensory ganglionic neurons. Subsequent viral reactivation causes herpes zoster, a morbid disease often complicated by neuropathic pain. Mechanisms underlying VZV latency and reactivation are not understood, mostly due to the lack of permissive animal models and challenges of current in vitro latency modelling. Here, we evaluated HD10.6 cells, a simplified and easily expandable human sensory neuron line to model VZV latency and reactivation. Mature HD10.6 (mHD10.6) differentiated neurons supported productive VZV infection, viral DNA replication, production of infectious progeny, and viral spread in cultures. VZV infection was associated with limited cytopathic effects and ultrastructural changes. Infecting mHD10.6 neurons with cell-free VZV in the presence of antivirals resulted in a quiescent-persistent state, characterized by persistent VZV genomes with restricted VZV gene expression and absence of infectious virus. Importantly, VZV could be reactivated by treatment with capsaicin, as evidenced by increased lytic viral transcription and virus spread. In conclusion, this study establishes human HD10.6 neurons as a novel and scalable in vitro model for studying VZV latency and reactivation to identify virus and host factors governing latency that may serve as therapeutic targets to restrict VZV reactivation. ImportanceMost individuals carry latent varicella-zoster virus (VZV) in their dorsal root ganglia (DRG), which can reactivate to cause shingles and chronic pain. The mechanisms by which VZV establishes latency and triggers of reactivation are incompletely understood. Current platforms for the study of VZV latency do not easily support functional experiments (ganglia) or are difficult to expand and complicated by mixed neuronal populations (stem cell-derived neurons). Here, we demonstrate that matured HD10.6 (mHD10.6) cells derived from immortalized human DRG-derived neurons provide a clonal, scalable, and easy-to-expand platform for studying lytic, latent, and reactivated VZV infection in sensory neurons. We propose that the HD10.6 platform could provide the basis to conduct studies on the viral latent state that have hitherto not been possible.

microbiology↗

bioRxiv2023

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3 scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.

microbiology↗

bioRxiv2023

Viral modulation of type II interferon increases T cell adhesion and virus spread

During primary infection, varicella zoster virus (VZV) infects epithelial cells in the respiratory lymphoid organs and mucosa. Subsequent infection of lymphocytes, T cells in particular, causes primary viremia allowing systemic spread throughout the host, including the skin. This results in the expression of cytokines, including interferons (IFNs) which partly limit primary infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. How VZV infects lymphocytes from epithelial cells while evading the cytokine response has not been fully established. Here, we show that VZV glycoprotein C (gC) binds IFN-{gamma} and modifies its activity. Transcriptomic analysis revealed that gC in combination with IFN-{gamma} increased the expression of a small subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), as well as several chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of epithelial cells resulted in lymphocyte function-associated antigen 1 (LFA-1)-dependent T cell adhesion. This gC activity required a stable interaction with IFN-{gamma} and signalling through the IFN-{gamma} receptor. Finally, the presence of gC during infection increased VZV spread from epithelial cells to peripheral blood mononuclear cells. This constitutes the discovery of a novel strategy to modulate the activity of IFN-{gamma}, inducing the expression of a subset of ISGs, leading to enhanced T cell adhesion and virus spread.

microbiology↗

bioRxiv2020

Stem Cell Transplantation Rescued A Primary Open-Angle Glaucoma Mouse Model

Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if exogenous stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.

developmental biology↗