Biology subjects

Donovan-Banfield, I.

Publications and source records attributed to Donovan-Banfield, I..

7 recordsLinked to original sources

bioRxiv2025

A targeted amplicon sequencing panel for cost-effective high-throughput genotyping of Aedes aegypti

The mosquito Aedes aegypti is the primary vector for several medically significant arboviruses, including dengue, Zika, chikungunya, and yellow fever. Studying the genetic diversity of Ae. aegypti is crucial for understanding its evolutionary history, population dynamics, and the effectiveness of vector control methods. Due to the large genome size of Ae. aegypti, whole-genome sequencing (WGS) is often cost-prohibitive for large-scale studies. Recent advances in reduced-representation methods, aiming to reduce costs by sequencing only a small portion of the genome, provide cost-effective alternatives. However, a standardized set of genome-wide markers specifically designed for population genetic studies of Ae. aegypti remains unavailable. Here, we present a targeted amplicon sequencing panel designed for cost-effective, high-throughput genotyping across 291 loci distributed throughout the Ae. aegypti genome. Our in silico analyses indicate that this amplicon panel effectively replicates population structure analyses typically derived from WGS data. We demonstrate that the amplicon panel accurately discriminates between diverse laboratory colonies of Ae. aegypti and consistently measures individual genetic admixture to a degree comparable with WGS. By enabling high-throughput genotyping at a reduced cost, we anticipate that our targeted amplicon sequencing panel will facilitate large-scale genotyping studies of Ae. aegypti for vector surveillance and population structure analyses, especially in resource-limited settings.

genomics↗

bioRxiv2025

A direct RNA-seq-based EBV Latency Transcriptome Offers Insights into the Biogenesis of EBV Gene Products.

Epstein-Barr virus (EBV) ubiquitously infects humans, establishing lifelong persistence in B cells. In vitro, EBV-infected B cells can establish a lymphoblastoid cell line (LCL). EBVs transcripts in LCLs (Latency III) produce six nuclear proteins (EBNAs), two latency membrane proteins (LMPs) and various microRNAs and putative long non-coding RNAs (BARTs). The BART and EBNA transcription units are characterised by extensive alternative splicing. We generated LCLs with B95-8 EBV-BACs, including one engineered with "barcodes" in the first and last repeat of internal repeat 1 (IR1), and analysed their EBV transcriptomes using long-read nanopore direct RNA-seq. Our pipeline ensures appropriate mapping of the W promoter (Wp) 5 exon, and corrects W1-W2 exon counts that misalign to IR1. This suggests that splicing across IR1 largely includes all W exons, and that Wp-derived transcripts more frequently encode the EBNA-LP start codon than Cp transcripts. Analysis identified a short variant of exon W2 and a novel polyA site before EBNA2, provided insights into BHRF1 miRNA processing and suggested co-ordination between polyA and splice site usage, although improved read depth and integrity are required to confirm this. The BAC region disrupts the integrity of BART transcripts through premature polyadenylation and cryptic splice sites in the hygromycin expression cassette. Finally, a few transcripts extended across established gene boundaries, running from EBNA, to BART to LMP2 gene regions, sometimes including novel exons between EBNA1 and the BART promoter. We have produced an EBV annotation based on these findings to help others better characterise EBV transcriptomes in future. Data SummaryScripts (and the shell scripts used to combine commands into the pipeline), and guidance in their usage are available on Github (github.com/robertewhite/ebv-transcriptomics-tools). All of the RNA-seq raw data and analyses relevant to this study are available from the EMBL Nucleotide Archive Study accession PRJEB83447 (read files ERR14129300-303), or from the authors website, ebv.org.uk. Processed and analysed data is presented in Excel format in Supplementary tables ST1-8, and the intermediate data processing conducted in excel is linked from the front page of ebv.org.uk, alongside the scripts, raw reads and updated B95-8-BAC and prototype EBV transcriptome annotation (gff3) files. Impact statementThis article showcases the potential of direct RNA-seq to characterise complex transcriptomes like Epstein-Barr virus, highlights specific challenges of interpreting RNA-seq data, and presents tools to solve specific challenges of mapping RNA-seq reads to EBV exons. Biologically, the data analysis identifies several new aspects of Epstein-Barr virus transcription, offering insights into miRNA proteins, and identifies idiosyncrasies of the transcriptome of the widely used B95-8 BAC that will inform studies using this system. Finally we provide an improved annotation for EBV RNA-seq studies.

microbiology↗

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↗

bioRxiv2023

Recombinant SARS-CoV-2 lacking initiating and internal methionine codons within ORF10 is attenuated in vivo

SARS-CoV-2 has been proposed to encode ORF10 as the 3 terminal gene in the viral genome. However, the potential role and even existence of a functional ORF10 product has been the subject of debate. There are significant structural features in the viral genomic RNA that could, by themselves, explain the retention of the ORF10 nucleotide sequences without the need for a functional protein product. To explore this question further we made two recombinant viruses, firstly a control virus (WT) based on the genome sequence of the original Wuhan isolate and with the inclusion of the early D614G mutation in the Spike protein. We also made a second virus, identical to WT except for two additional changes that replaced the initiating ORF10 start codon and an internal methionine codon for stop codons (ORF10KO). Here we show that the two viruses have apparently identical growth kinetics in a VeroE6 cell line that over expresses TMPRSS2 (VTN cells). However, in A549 cells over expressing ACE2 and TMPRSS2 (A549-AT cells) the ORF10KO virus appears to have a small growth rate advantage. Growth competition experiments were used whereby the two viruses were mixed, passaged in either VTN or A549-AT cells and the resulting output virus was sequenced. We found that in VTN cells the WT virus quickly dominated whereas in the A549-AT cells the ORF10KO virus dominated. We then used a hamster model of SARS-CoV-2 infection and determined that the ORF10KO virus has attenuated pathogenicity (as measured by weight loss). We found an almost 10-fold reduction in viral titre in the lower respiratory tract for ORF10KO vs WT. In contrast, the WT and ORF10KO viruses had similar titres in the upper respiratory tract. Sequencing of viral RNA in the lungs of hamsters infected with ORF10KO virus revealed that this virus frequently reverts to WT. Our data suggests that the retention of a functional ORF10 sequence is highly desirable for SARS-CoV-2 infection of hamsters and affects the viruss ability to propagate in the lower respiratory tract.

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↗

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↗