Biology subjects

Filipe, A. D. S.

Publications and source records attributed to Filipe, A. D. S..

7 recordsLinked to original sources

bioRxiv2025

Quantifying virus load and characterising virus diversity in wildlife samples with target enrichment sequencing

Metagenomics is a powerful tool for characterising viruses, with broad applications across diverse disciplines, from understanding the ecology and evolutionary history of viruses to identifying causative agents of emerging outbreaks with unknown aetiology. Additionally, metagenomic data contains valuable information about the amount of virus present within samples. However, we have yet to leverage metagenomics to assess viral load, which is a key epidemiological parameter. To effectively use sequencing outputs to inform transmission, we need to understand the relationship between read depth and viral load across a diverse set of viruses. Here, using target enrichment sequencing, we investigated the detection and recovery of virus genomes by spiking known concentrations of DNA and RNA viruses into wild rodent faecal samples. In total, 15 experimental replicates were sequenced with target enrichment sequencing and compared to shotgun sequencing of the same background samples. Target enriched sequencing recovered all spike-in viruses at every concentration (102, 103, and 105 {+/-} 1 log genome copies) and showed a log-linear relationship between spike-in concentration and mean read depth. Background viruses (including Kobuvirus and Cardiovirus) were recovered consistently across all biological and technical replicates, but genome coverage was variable between virus genera and likely reflected the composition of target enrichment probe panel. Overall, our study highlights the strengths and weaknesses of using commercially available panels to quantify and characterise wildlife viromes, and underscores the importance of probe panel design for accurately interpreting coverage and read depth. To advance the use of metagenomics for understanding virus transmission, further research will be needed to elucidate how sequencing strategy (e.g. library depth, pooling), virome composition, and probe design influence viral read counts and genome coverage.

genomics↗

bioRxiv2024

POLYGENIC DETERMINANTS OF H5N1 ADAPTATION TO BOVINE CELLS

Avian influenza H5N1 clade 2.3.4.4b viruses caused a global panzootic and, unexpectedly, widespread outbreaks in dairy cattle, therefore representing a pandemic threat. To inform effective control strategies, it is critical to determine whether the potential to adapt to bovine cells is a generalised feature of H5N1 viruses, or is specific to clade 2.3.4.4b, or even more restricted to specific genotypes within this clade (e.g., B3.13 and D1.1). Using a large panel of H5N1 viruses representing >60 years of their natural history and other IAV for comparative purposes, we demonstrate that virus adaptation to bovine cells is: (i) highly variable across 2.3.4.4b genotypes, (ii) limited in viruses predating the global expansion of this clade, (iii) determined by the viral internal gene cassette, and (iv) not restricted to udder epithelial cells. Mutations in the PB2 polymerase subunit, particularly M631L, emerge as key determinants of adaptation, although their phenotypic effects are context dependent and have limited enhanced viral polymerase activity in human cells. Bovine B3.13 and some avian genotypes also exhibit enhanced modulation of bovine interferon-induced antiviral responses, determined by at least the viral PB2, nucleoprotein, and the non-structural protein NS1. Our results highlight the polygenic nature of IAV host range and reveal that the potential to cross the species barrier varies during the evolutionary trajectory of H5N1, with some avian viruses more predisposed to spillover than others.

microbiology↗

bioRxiv2024

A Machine Learning Framework to Identify the Correlates of Disease Severity in Acute Arbovirus Infection

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013 and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to identify the key viral and host processes associated with disease pathogenesis. We identified five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, our study using an agnostic machine learning approach, can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection.

microbiology↗

bioRxiv2023

Mosquito virus diversity in Western and North-western Uganda

IntroductionSurveillance for mosquito borne arboviruses in Sub Saharan Africa has largely focussed on known viruses including Yellow fever virus (YFV), Rift Valley fever virus (RVFV), Chikungunya virus (CHIKV), West Nile virus (WNV) and Dengue viruses (DENV). Routine surveillance and outbreak investigations traditionally rely on serology, PCR and cell culture. Although such methods are useful, they are do not detect novel or unexpected viruses. MethodsThis study employed unbiased metagenomic next generation sequencing (MNGS) to characterise viruses circulating in mosquitoes of Arua and Kasese districts of Uganda collected systematically as part the ArboViral Infection (AVI) study. Adult mosquito sampling was carried out from multiple sites using light traps baited with solid carbon dioxide (indoors) and pyrethrum spray (outdoors). 10,026 mosquitoes were identified using appropriate morphological identification keys and separated into 96 pools by species and location of collection. Viral RNA extracted from homogenised mosquitoes was reverse transcribed to complimentary DNA and sequenced on the flow cell of an Illumina NextSeq platform. Bioinformatic analysis was performed using a customized in-house metagenomics pipeline. Downstream analyses were carried out in R version 4.2.1. Results97 viruses from 24 families and 31 genera were detected in 96 mosquito pools from Arua and Kasese districts. The abundance of viruses in different families was in the order Rhabdoviridae (33), Flaviviridae (28), Orthomyxoviridae (13), Mesoniviridae (9), Piconarviridae (9), Peribunyaviridae (9), Phasmaviridae (9), Iflaviridae (8), Phenuiviridae (7), Nodaviridae (5), Xinmoviridae (4), Reoviridae (4), Virusidae (2), Nairoviridae (2), Qinviridae (2), Togaviridae (2), Alphatetraviridae (2), Picornaviralesidae (2), Iridoviridae, (1), Nudiviridae (1), Parvoviridae (1), Permutetraviridae (1) and 22 viruses from different families remain unclassified. The Flaviviridae, Togaviridae, Rhabdoviridae, Peribunyaviridae, Phenuiviridae, Nairoviridae, Nodaviridae and Orthomyxoviridae harbor viruses that cause disease in humans and other mammals. Viruses from insect-specific virus families that were detected included the Chuviridae, Iflaviridae, Phasmaviridae and Alphatetraviridae. 92/173 (53%) of the viral genomes had full opening reading frames (ORFs) and diverged by >30% nucleotide pairwise distance from the nearest reference genome. ConclusionThe majority of viruses detected were novel species described for the first time with unknown potential to cause disease. The diversity of virus species in mosquitoes from Uganda, a hotspot for emerging arboviruses has been only partially characterized. This study, carried out in Western and North West Uganda illustrates the scale of richness and virus diversity in the region and the need to further characterise the virome in mosquitoes, especially those with a propensity to feed from human and animal hosts.

genomics↗

bioRxiv2023

Temperature elevation synergises with and enhances the type-I IFN-mediated restriction of MPXV

Fever is an evolutionary conserved host pro-inflammatory immune response that governs the regulation of multiple biological processes to control the outcome of infection. In January 2022, the World Health Organization (WHO) reported a global outbreak in mpox cases with a high incidence of human-to-human transmission. A frequent prodromal symptom of monkeypox virus (MPXV) infection is fever, with a febrile temperature range of 38.3 to 40.5 {degrees}C. However, the outcome of temperature elevation on MPXV infection remains poorly defined. Here, we isolated a circulating strain of MPXV from a patient who presented with fever (38.5 {degrees}C) and rash from the 2022 outbreak. Genomic sequencing identified this isolate to belong to the epidemic Clade IIb.B1. Transcriptomic analysis of infected cells demonstrated this virus to induce a strong IL6 pro-inflammatory immune response, consistent with a role for this pyrogen in the regulation of fever. We identify host-cell temperature at both physiological skin (33 {degrees}C) and clinical febrile temperatures (38.5 and 40 {degrees}C) to be a key determinant in the outcome of infection through the differential regulation of MPXV transcription and associated amplitude of host cytokine response to infection. Incubation of infected cells at 38.5 or 40 {degrees}C led to a restriction or ablation in MPXV replication, respectively. Importantly, this thermal inhibition was reversible upon temperature downshift to 37 {degrees}C without detriment to viral replication fitness. Co-stimulation of the type-I interferon (IFN) response led to a dose- and temperature-dependent inhibition in MPXV replication that restricted the re-establishment of infection upon temperature downshift and withdrawal of IFN as an immune stimulus. Our data identify febrile temperatures associated with mpox disease to be a critical component of the host pro-inflammatory immune response to infection which can synergise with the type-I IFN response to enhance the host-cell mediated restriction of MPXV.

microbiology↗

bioRxiv2023

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning

SARS-CoV-2 has continued to evolve throughout the COVID-19 pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2 and BA.2.75. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

microbiology↗

bioRxiv2023

Ticks; a reservoir for virus emergence at the human-livestock interface in Uganda

BackgroundUganda is one of the most biodiverse regions on the planet and a hotspot for virus emergence. In particular, the warm-humid lowlands favour tick population growth with the associated risk of tick-borne disease. The prevalent tick species Rhipicephalus appendiculatus, R. evertsi evertsi and Amblyomma variegatum harbour a diverse range of viruses, from harmless to highly pathogenic. Notably, the orthonairoviruses cause human outbreaks of Crimean-Congo haemorrhagic fever (CCHF) regularly within the cattle corridor of Uganda, a region spanning from the south-west to the north-east of the country. MethodsIn the ArboViral Infection (AVI) study, the first to explore the virome of ticks in Uganda using next generation sequencing (NGS), we collected ticks from three geographically diverse areas and subjected these to target-enrichment (TE) NGS. Viral genomes were detected by de novo assembly, mapping and BLASTn. ResultsWe analyzed a total of 2,754 ticks collected from 31 livestock farms in the districts of Arua, Nakaseke and Lyantonde. These were combined into 219 pools by site of collection and tick species, including R. appendiculatus, R. evertsi evertsi, A. variegatum and Hyalomma rufipes. We detected partial or near-complete viral genomes in 163 tick pools; 110 (67%) of which were from Arua, 39 (24%) from Nakaseke and 12 (7%) from Lyantonde districts. 2 pools (2%) were from Arua/Lyantonde. These included 22 species of virus, representing 15 genera and 9 families, including the Nairoviridae, Retroviridae, Orthomyxoviridae, Chuviridae, Rhabdoviridae, Phenuiviridae, Parvoviridae, Poxviridae and Flaviviridae. There were 8 viral species known to be pathogens of humans or animals and 5 highly divergent genomes detected, representing novel virus species. A high abundance of orthonairoviruses was notable, including CCHFV, Dugbe virus and a novel Orthonairovirus species that we have named Macira virus. InterpretationTicks in Uganda are an important reservoir of diverse virus species, many of which remain uncharacterised and of unknown pathogenic potential. Author SummaryTicks are parasitic arachnids that may transmit a spectrum of viral diseases to humans and animals. Uganda is a hotspot for such tick-borne diseases. In this study, we sequenced ticks collected from three geographically diverse regions of Uganda using a semi-agnostic next- generation sequencing method in order to detect viruses from all known virus families. We collected and analyzed 2,754 ticks from 31 farms across the country. Within these ticks, we detected 22 species of virus from 15 genera and 9 viral families, including 8 animal or human pathogens and 5 new novel virus species. Notably, orthonairoviruses, including the highly pathogenic Crimean-Congo haemorrhagic fever virus, were highly prevalent in the ticks. The researchers suggest that ticks in Uganda serve as an important reservoir for diverse viruses, many of which have significant pathogenic potential. This information will inform public health efforts to prevent and control tick-borne diseases in Uganda and other similar regions.

microbiology↗