Biology subjects

Lloyd, M. G.

Publications and source records attributed to Lloyd, M. G..

3 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↗

bioRxiv2022

An Acyclic Phosphonate Prodrug of HPMPC is Effective Against VZV in Skin Organ Culture and Mice

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 M, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.

microbiology↗

bioRxiv2018

Blocking the alternative sigma factor RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

Multidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment. P. aeruginosa has virulence factors that are regulated by sigma factors in response to the tissue microenvironment. The alternative sigma factor, RpoN ({sigma}54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a \"molecular roadblock\", binding RpoN consensus promoters at the -24 site and blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains both in vitro and in vivo, but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa - C. elegans infection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strain P. aeruginosa PA19660 Xen5. Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

microbiology↗