Biology subjects

Goldswain, H.

Publications and source records attributed to Goldswain, H..

7 recordsLinked to original sources

bioRxiv2026

Characterisation of naturally occurring MERS-CoV Spike mutations and their impact on entry and neutralisation.

In this study the phenotypic consequences of naturally occurring single nucleotide polymorphisms (SNPs) in the MERS-CoV Spike protein were investigated. The impact of Spike mutations on virus entry and neutralisation of contemporary MERS-CoV strains is not currently well understood. Naturally occurring mutations were identified by aligning 584 MERS-CoV Spike sequences from either human clinical isolates collected between 2012 - 2024 or from viruses passaged in human cells. Fifteen SNPs of interest occurring in the NTD, RBD and adjacent to the S1/S2 cleavage site were selected for further characterisation based on their location in the Spike protein, frequency and identification in previous studies. A representative clade B, lineage 5 wildtype Spike sequence, which reflected those carried by MERS-CoV viruses circulating in the Middle East, was used in this study. The mutations of interest were introduced to the wildtype backbone to generate Spike variants. A lentiviral-based pseudotyping system was then used to investigate the impact of these Spike mutations on entry and neutralisation. I529T, E536K and L745F were shown to improve MERS-CoV entry. L411F, T424I, L506F, L745F and T746K were found to increase resistance to neutralisation by pooled patient sera. This study has identified novel naturally occurring Spike mutations that resulted in phenotypic differences in virus entry and neutralisation of contemporary MERS-CoV strains. Continued investigation of the phenotypic consequences of naturally occurring MERS-CoV Spike mutations is essential for assessing the risk to public health, especially given the pandemic potential of this virus. ImportanceThe main aim of this study was to investigate the impact of naturally occurring MERS-CoV Spike mutations on virus entry and neutralisation. The phenotypic consequences of mutations occurring in the Spike protein of contemporary MERS-CoV strains are not currently well understood. Improving our understanding is of particular importance due to MERS-CoV continuing to pose a public health risk, with frequent spillover events and mounting evidence of human-to-human transmission since the virus emerged in 2012. A major concern is that as MERS-CoV continues to evolve, it may become more infectious, resulting in increased transmission between humans. To add to this, surveillance is limited and there are currently no specific medical countermeasures available to treat MERS-CoV disease. The MERS-CoV Spike pseudotyping system developed in this study is a useful tool that could be used alongside surveillance systems to rapidly assess novel Spike mutations in functional assays. This MERS-CoV pseudotyping system could also be used to aid the development of medical countermeasures such as vaccines, antivirals and antibody therapies.

microbiology↗

bioRxiv2023

Inefficient transmission of African swine fever virus to sentinel pigs from environmental contamination under experimental conditions

Knowledge about African swine fever virus (ASFV) transmission and its survival in the environment is mandatory to develop rational control strategies and combat this serious disease in pigs. In this study, the risk that environmental contamination poses for infection of naive pigs was investigated. Naive pigs were introduced as sentinels into contaminated pens either on the same day or up to three days after ASFV-infected pigs were removed. Three experiments were carried out in which four to six pigs per pen were inoculated with virulent ASFV isolates OURT88/1 (genotype I), Georgia 2007/1 or POL/2015/Podlaskie (genotype II), respectively. The majority of the inoculated pigs developed acute disease but with no evident haemorrhagic lesions or haemorrhagic diarrhoea and were culled at the predefined humane endpoint. The levels of ASFV DNA detected in the blood of the infected animals reached 107-9 genome copies/ml before euthanasia. Environmental swabs were taken from different surfaces in the animal rooms, as well as from faeces and urine, close to the time of introduction of the naive animals. Relatively low quantities of virus DNA were detected in the environmental samples, in the order of 103-7 genome copies. Neither clinical signs nor virus genomes were detected in the blood of any of the sentinel pigs over a period of two to three weeks after exposure, indicating that transmission from the ASFV-contaminated environment did not occur. Interestingly, viral DNA was detected in nasal and oral swabs from some of the sentinel animals at early days of exposure (ranging between 103.7-5.8 genome copies), though none of them developed ASF. The results indicate a relatively low risk of ASFV transmission from a contaminated environment in the absence of blood from infected animals.

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↗