Biology subjects

Penrice-Randal, R.

Publications and source records attributed to Penrice-Randal, R..

14 recordsLinked to original sources

bioRxiv2026

The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis, with an estimated 20 million human infections annually. Its diverse host range presents a significant zoonotic threat, yet the molecular mechanisms underpinning HEV-host interactions remain poorly characterised. Here, we used transposon-based insertion screening of the HEV pORF1 polyprotein to identify a novel region within the viral PCP domain that permits HA-epitope insertion, enabling the first epitope tagging of this domain. Combined with V5 tagging of the hypervariable region (HVR), this approach enabled the generation of a replication-competent stable cell line expressing dual-tagged ORF1. Co-immunoprecipitation and TMT-based LC-MS proteomics revealed that pORF1 interacts with host proteins involved in endoplasmic reticulum-associated degradation (ERAD) and the 26S proteasome. Pharmacological inhibition demonstrated that both pathways are required for efficient HEV replication. Treatment with the FDA-approved proteasome inhibitor bortezomib altered pORF1 precursor abundance and led to loss of full-length pORF1 through enhanced proteolysis. Importantly, the kinetics of ORF1 degradation following proteasome inhibition correlated with immediate loss of HEV replication, demonstrating a functional link between pORF1 proteostasis and viral replication. Collectively, our findings identify the ERAD network as a previously unrecognised requirement for HEV replication and establish a role for the 26S proteasome in maintaining pORF1 proteostasis.

microbiology↗

bioRxiv2026

The biodistribution and effect of post-exposure neutralising monoclonal antibody treatment in a mouse model of SARS-CoV-2 infection with viral spread to the brain

Ronapreve, a combination of two neutralising monoclonal antibodies, casirivimab and imdevimab, was amongst the authorised treatments against SARS-CoV-2 early in the COVID-19 pandemic. Ronapreve has lost some of its efficiency with the rise of new virus variants, however, it remains a valuable tool for experimental studies to gain insights into the mechanisms and effects of anti-viral drugs. In this study we combined morphological, pharmacokinetic and molecular approaches (including multiomics) to investigate the biodistribution of Ronapreve in the K18-hACE2 murine model of SARS-CoV-2 neuroinvasion, as well as possible consequences for the brain. We also investigated the effect of the treatment on the infection status. Our results showed that after intraperitoneal injection, Ronapreve accumulates in the serum and is unable to cross the blood-brain barrier, thus not reaching the brain parenchyma; treatment has only a minimal effect on the brain transcriptome, with no significant changes in the brain lipidome or metabolome. Nonetheless, post-exposure Ronapreve treatment resulted in reduced viral loads in the lung and, in particular, the brain, with markedly reduced tissue response in the brain, as shown by the transcriptomic analysis. The results suggest a peripheral mode of action of Ronapreve to block brain infection, possibly by lowering viral replication in the nasal epithelium, reducing a subsequent spread to the brain.

pathology↗

bioRxiv2026

Efficient plasmid-based rescue of T7 RNA polymerase-driven calicivirus reverse genetics systems in mammalian cells using vaccinia virus RNA capping enzymes

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

microbiology↗

bioRxiv2025

Hypoxia inducible factors regulate Pneumovirus replication by enhancing innate immune sensing

The immune mechanisms responsible for protection and pathogenesis in pneumoviral infection are not well defined. We demonstrated that pharmacological activation of the hypoxic inducible factor (HIF) signalling axis using Daprodustat limited viral replication through enhanced immune signalling. Transcriptomic analysis revealed HIF augmented activation of innate immune response genes, including interferon-stimulated gene 15 (Isg15), in the lung and spleen of mice infected with pneumonia virus of mice (PVM). In human respiratory syncytial virus (hRSV) infected airway epithelial cells, Daprodustat inhibited viral replication and enhanced ISG15 expression in a HIF-dependent manner. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. Moreover, Daprodustat increased interferon signalling in response to viral RNA, suggesting that HIF inhibits pneumovirus replication through enhancing viral RNA sensing. Mechanistically, Daprodustat reduced N6-methyladenosine modification of viral RNA through upregulation of RNA demethylases, promoting detection by innate immune sensors. This study highlights the intricate interplay between hypoxia and antiviral immunity and offers valuable insights into pneumovirus-host interactions and potential therapeutic interventions. SIGNIFICANCE STATEMENTThis study explores the role of the hypoxic inducible factor (HIF) signalling pathway in limiting pneumoviral infections, such as respiratory syncytial virus (hRSV) and pneumonia virus of mice (PVM). Using Daprodustat, a clinically approved activator of HIF signalling we demonstrated that stimulation of this pathway enhanced innate immune signalling and limited viral replication. Transcriptomic analysis showed that HIF promoted innate immune response genes in mice infected with PVM and human airway epithelial cells infected with hRSV. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. We uncovered a role for HIF to regulate the levels of N6-methyladenosine modification of viral RNA transcripts, resulting in the increased activation of nucleic acid sensing.

microbiology↗

bioRxiv2025

SARS-CoV-2 infection of the brain: the K18-hACE2 mouse model to illustrate the role and response of the vasculature in neurotropic viral infection

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) primarily affects the respiratory tract and lungs; however, the associated disease, coronavirus disease 2019 (COVID-19), can involve the central nervous system (CNS) in both its acute and long-term (Long COVID) clinical manifestation. The pathomechanisms underlying neurological impairments in COVID-19 are not yet fully understood, hence experimental studies to clarify the direct effect of SARS-CoV-2 in the brain can provide further insight. In the present study we used the K18-hACE2 model, intranasally challenged with SARS-CoV-2 ancestral and Delta isolates at low or medium doses, to address the hypothesis that the inflammatory response raised in the brain of infected mice is secondary to neuronal infection. Our data confirmed that the virus reaches the brain even after low dose (102 PFU/mouse, Delta isolate) infection where it targets the neurons without overt neuropathic effect, sparing the blood vessels. In situ investigation of the resulting inflammatory response showed the recruitment of leukocytes via postcapillary venules, with their accumulation in the perivascular space and occasional migration into the neuroparenchyma, without targeting and/or damage to the vessel wall. These changes were reflected in the brain transcriptome and proteome which showed positive enrichment of pathways and up-regulation of genes involved in the inflammatory response including the recruitment (including adhesion and migration) and activity of leukocytes. Additionally, morphological and transcriptome/proteome changes suggest minimal associated blood-brain barrier dysfunction. The brain metabolome and lipidome showed minimal changes; these were consistent with oxidative stress and inflammatory and immune/antiviral responses. The results obtained from our model indicate that SARS-CoV-2 infection of the neurons can result in limited neuroinflammation. These data can help to understand more fully the reaction of the CNS in COVID-19 patients, and neurotropic virus infections in general.

pathology↗

bioRxiv2024

The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in infected and immune suppressed mice

SynopsisO_ST_ABSObjectivesC_ST_ABSImmunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. MethodsMice were immunosuppressed using cyclophosphamide and infected with a B daughter lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed. ResultsTreatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of G>A and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir. Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs). ConclusionsFurther investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.

pharmacology and toxicology↗

bioRxiv2021

SARS-CoV-2 Omicron-B.1.1.529 Variant leads to less severe disease than Pango B and Delta variants strains in a mouse model of severe COVID-19

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The B.1.1.529 Omicron variant is rapidly emerging and has been designated a Variant of Concern (VOC). The variant is highly transmissible and partially or fully evades a spectrum of neutralising antibodies due to a high number of substitutions in the spike glycoprotein. A major question is the relative severity of disease caused by the Omicron variant compared with previous and currently circulating variants of SARS-CoV-2. To address this, a mouse model of infection that recapitulates severe disease in humans, K18-hACE2 mice, were infected with either a Pango B, Delta or Omicron variant of SARS-CoV-2 and their relative pathogenesis compared. In contrast to mice infected with Pango B and Delta variant viruses, those infected with the Omicron variant had less severe clinical signs (weight loss), showed recovery and had a lower virus load in both the lower and upper respiratory tract. This is also reflected by less extensive inflammatory processes in the lungs. Although T cell epitopes may be conserved, the antigenic diversity of Omicron from previous variants would suggest that a change in vaccine may be required to mitigate against the higher transmissibility and global disease burden. However, the lead time to develop such a response may be too late to mitigate the spread and effects of Omicron. These animal model data suggest the clinical consequences of infection with the Omicron variant may be less severe but the higher transmissibility could still place huge burden upon healthcare systems even if a lower proportion of infected patients are hospitalised.

microbiology↗

bioRxiv2021

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

microbiology↗

bioRxiv2021

Antiviral activity of molnupiravir precursor NHC against Variants of Concern (VOCs) and its therapeutic window in a human lung cell model

Several regulatory agencies have either licensed or given emergency use approval for treatment of patients at risk of developing severe COVID-19 with the anti-viral drug, Molnupiravir. Recent trials involving Molnupiravir suggested the drug was not as efficacious as earlier studies suggested. This study aimed to: (i) determine the effectiveness of the Molnupiravir active metabolite (NHC) against different SARS-CoV-2 Variants of Concern (VoCs), (ii) establish the therapeutic window of NHC in a human lung cell model, and (iii) and evaluate the genetic barrier to resistance. Dose response assays were performed in parallel to determine the IC50 (the concentration required to inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were treated with NHC at different time points either before, during or after infection with SARS-CoV-2. Multiple passaging in the presence or absence of drug was used to evaluate whether resistance occurred. To obtain genomic information, virus was sequenced at regular intervals. After 20 passages in the presence of the drug, dose response assays and sequencing showed the virus did not appear to have developed resistance. The drug had equivalent activity against four VOCs ranging from 0.04 to 0.16M IC50. The efficacy of the drug diminished when applied after 24 hours post-infection. Our results suggest that earlier administration in patients, perhaps pre- or post-exposure rather than symptom onset, would be a more effective treatment option.

microbiology↗

bioRxiv2021

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

microbiology↗

bioRxiv2021

Sequence analysis of SARS-CoV-2 in nasopharyngeal samples from patients with COVID-19 illustrates population variation and diverse phenotypes, placing the in vitro growth properties of B.1.1.7 and B.1.351 lineage viruses in context.

New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK Kent variant) and B.1.351 (the South African variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants. ImportanceSARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.

microbiology↗

bioRxiv2021

Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and aberrant pattens in humans

IntroductionSARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5 sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. ResultsHigh resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. DiscussionLeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.

microbiology↗

bioRxiv2020

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified - which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome.

molecular biology↗

bioRxiv2020

Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that rapidly caused a pandemic. Coalescence of this virus with seasonal respiratory viruses, particularly influenza virus is a global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, they exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by infection with IAV is responsible for the observed inhibition of SARS-CoV-2, however, infection with attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest that the concept of twinfection is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

microbiology↗