Biology subjects

Breuer, J.

Publications and source records attributed to Breuer, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Comparative analysis of varicella-zoster virus and herpes simplex virus 1 interaction with epidermal terminal differentiation in primary human keratinocytes models of differentiation

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and herpes zoster, while herpes simplex virus 1 (HSV-1) causes oral and genital herpes. Both infections manifest with skin blisters from which the viruses are transmitted to new hosts either via aerosol (VZV) or skin microabrasions (HSV-1). VZV reaches the skin through the blood route, and in the skin epidermis it first infects undifferentiated keratinocytes of the basal layer. Conflicting evidence exists for HSV-1, making it unclear whether HSV-1 infects undifferentiated or differentiated keratinocytes. Here, we developed in vitro models of primary human epidermal keratinocytes differentiation to recapitulate infection of distinct layers of the epidermis by VZV and HSV-1. Our data show that replication of both viruses is restricted, VZV more than HSV-1, if initial infection occurs in differentiated keratinocytes, but not if initial infection occurs in basal undifferentiated keratinocytes. Like VZV, HSV-1 downregulates expression of proteins associated with keratinocyte differentiation, such as the suprabasal keratin K10. However, whereas in VZV downregulation of K10 occurs soon after VZV infection and appears to be independent from viral replication, HSV-1-mediated K10 downregulation requires full viral replication. These observations provide insights into the potential for VZV and HSV-1 interactions with epidermal differentiation to yield strategies for developing host- and pathogen-directed antiviral agents.

microbiology↗

bioRxiv2026

Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, against respiratory syncytial virus (RSV) and human parainfluenza (hPIV) using monotherapy or dual-drug combinations with epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir-favipiravir, remdesivir-molnupiravir and favipiravir-molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Importantly, antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/699296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1eb00dforg.highwire.dtl.DTLVardef@186358forg.highwire.dtl.DTLVardef@265181org.highwire.dtl.DTLVardef@1b10d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Castanet: a pipeline for rapid analysis of targeted multi-pathogen genomic data

MotivationTarget enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present the Castanet pipeline: an analysis pipeline for end-to-end processing and consensus sequence generation for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. It is packaged with usability features, including graphical interface and installer script. ResultsIn addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard laptop computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in < 2 h. Availability and ImplementationSource code freely available under GPL-3 license at https://github.com/MultipathogenGenomics/castanet, implemented in Python 3.10 and supported in Ubuntu Linux 22.04 and other Bash-like environments. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB77004.

bioinformatics↗

bioRxiv2023

Genomic insights into local scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovar A-C, is the leading infectious cause of blindness worldwide. Its impact is substantial, with approximately 1.9 million individuals suffering from visual impairment. Africa bears the highest burden, accounting for over 86% of global trachoma cases. We investigated Ct serovar A (SvA) and serovar B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swab samples across nine Gambian villages during a six-month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted Whole-Genome Sequencing (WGS) on a limited number (n=43) of Ct positive samples. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had a highest identity to Ct strain A/HAR13 (NC_007429) and 30 had a highest identity to Ct strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains; Ct A/HAR13 and Ct B/Jali20 indicated a single nucleotide polymorphism accumulation rate of 2.4 {xi} 10^-5/site/year for the Gambian SvA and 1.3 {xi} 10^-5/site/year for SvB variants (p<0.0001). Variant calling resulted in a total of 1,371 single nucleotide variants (SNVs) with a frequency > 25% in SvA sequences, and 438 SNVs in SvB sequences, from which 740 (62.8%), and 241 (66.4%) were non-synonymous, respectively. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/ repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP), and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies. Impact StatementChlamydia trachomatis (Ct) is a globally significant pathogen. It is the leading infectious cause of blindness--a disease called trachoma. In addition, Ct causes the majority of bacterial sexually transmitted infections. Current control measures for trachoma are based on the "SAFE" strategy; Surgery for trichiasis (S), Antibiotics (A), Facial cleanliness (F) and Environmental improvement (E). Whilst this strategy has achieved remarkable success, the target date for the global elimination of blinding trachoma as a public health problem has been pushed back from 2020 to 2030. Prior studies demonstrated evidence indicating variations in infection loads and severity among different ocular Ct serovars. However, there remains a significant knowledge gap regarding the specific genes and mechanisms responsible for these variations. We generated genetic data from two main serovars of Ct that infect human eyes: Serovar A (SvA) and Serovar B (SvB) variants collected from four villages in two different administrative regions on opposing sides of the river Gambia to elucidate (i) the factors driving the diversification of ocular Ct strains; (ii) disparities in mutation frequency/accumulation profiles; (iii) selective pressures between serovar A and B; and (iv) the dynamics of mutation accumulation within the Gambian ocular Ct positive population over a short timeframe. Our findings suggest a different variation accumulation pattern in SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

genomics↗

bioRxiv2023

Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in {Delta}rtt109 and {Delta}asf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent MMS (methylmethane sulfonate), but not HU (hydroxyurea). In contrast, chk2 mutants are fertile and resistant to MMS, but not HU. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response. Article summaryIn the filamentous fungus Sordaria macrospora, the conserved histone chaperone ASF1, which interacts with histones H3 and H4, was previously shown to be required for multicellular development and DNA damage response. Here, we have analyzed two additional chromatin-associated proteins. rtt109 encodes a histone acetyltransferase, and deletion of the gene in S. macrospora results in a phenotype similar to that of a {Delta}asf1 mutant, whereas chk2 is involved in different aspects of the DNA damage response, but not in development.

molecular biology↗

bioRxiv2023

Histone binding of ASF1 is required for fruiting body development, but not for genome stability in the filamentous fungus Sordaria macrospora

The highly conserved eukaryotic histone chaperone ASF1 is involved in the assembly and disassembly of nucleosomes during transcription, DNA replication and repair. It was the first chaperone discovered to be involved in all three of these processes. The filamentous fungus Sordaria macrospora is one of only two multicellular organisms where asf1 deletions are viable, which makes it useful for in vivo analysis of this central regulator of eukaryotic chromatin structure. Deletion of asf1 in S. macrospora leads to sterility, a reduction of DNA methylation, and upregulation of genes that are usually weakly expressed in the wild type. Here, we focused on the functions of the highly conserved core and the divergent C-terminal tail of ASF1, studied the effects of ASF1 on histone modifications and tested its relevance for genomic stability. By Co-IP and complementation analysis we showed that substitutions of amino acid V94 or truncations of the C-terminal tail abolish histone binding and do not complement the sterile mutant phenotype. {Delta}asf1 is sensitive to the DNA damaging agent MMS, while complementation strains, even those with non-histone-binding variants, regain wild type-like resistance. To aid in subsequent ChIP-seq and Hi-C analyses, we generated a chromosome-resolved genome assembly of S. macrospora. ChIP-Seq analysis revealed a global increase of H3K27me3 in {Delta}asf1, accompanied by a global decrease of H3K56ac. By using Hi-C we detected a tandem duplication of around 600 kb on chromosome 2 in the mutant. Crossing experiments indicated linkage between the viability of {Delta}asf1 strains and the presence of the duplication. ImportanceHistone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the {Delta}asf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the {Delta}asf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

molecular biology↗

bioRxiv2023

Genetic consequences of effective and suboptimal dosing with mutagenic drugs in a hamster model of SARS-CoV-2 infection

Mutagenic antiviral drugs have shown promising results against multiple viruses, yet concerns have been raised about whether their use might promote the emergence of new and harmful viral variants. Here, we examine the genetic consequences of effective and suboptimal dosing of favipiravir and molnupiravir in the treatment of SARS-CoV-2 infection in a hamster model. We identify a dose-dependent effect upon the mutational load in a viral population, with molnupiravir having a greater potency than favipiravir per mg/kg of treatment. The emergence of de novo variants was largely driven by stochastic processes, with evidence of compensatory adaptation but not of the emergence of drug resistance or novel immune phenotypes. Effective doses for favipiravir and molunpiravir correspond to similar levels of mutational load. Combining both drugs had an increased impact on both efficacy and mutational load. Our results suggest the potential for mutational load to provide a marker for clinical efficacy.

evolutionary biology↗

bioRxiv2023

The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (<11y), adult (30- 50y) and elderly (>70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hi progenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/524211v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@dab12aorg.highwire.dtl.DTLVardef@1a57334org.highwire.dtl.DTLVardef@12e7983org.highwire.dtl.DTLVardef@2bbe6e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

AYUKA: A toolkit for fast viral genotyping using whole genome sequencing.

Technological advances enabled the frequent use of whole genome sequencing in the clinical microbiology laboratory. While generating data is now easier than ever, the computational resources and expertise required for analysis are still a challenge for clinical applications. Since it is not always possible to collect clinical specimens at the peak viral load, sequencing results are also not always amenable for analysis with bioinformatics pipelines that always require high quality data. Here we present a fast and reliable method, we named AYUKA, for analysis of viral sequencing data that does not require data pre-processing and provides quality control metrics including estimates for sequencing depth and genome coverage, as well as identifying the viral genotypes in a sample and distinguishing mixed infection from recombinants. This method can be applied to any virus where a classification by genotype is employed and determining it is relevant. We generated a validation dataset composed of cultured and sequenced reference adenoviruses from distinct species, that we compared with the gold standard clinical processing pipeline currently implemented to demonstrate reliability. The validation shows better sensitivity than mapping and perfect specificity in detecting the correct genotypes and in a wide range of adenovirus species. Run time was consistently under one minute per sample on a standard laptop, allowing the analysis of more than 100 samples per hour. This open-source method is available at https://github.com/afonsoguerra/AYUKA and precomputed databases are available at https://zenodo.org/record/6521576 allowing analysis of raw data straight from the sequencer within minutes on a standard computer, with minimum setup or expertise required to perform the analysis. The information contained within the AYUKA report can be of use for both the clinical team that collected the sample, but also for guiding the bioinformatics analysis team in the in-depth downstream analyses and genetic epidemiology investigations.

bioinformatics↗

bioRxiv2022

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

evolutionary biology↗

bioRxiv2021

WeightedLD: The Application of Sequence Weights to Linkage Disequilibrium

Sequence-weighting methods are commonly employed to account for biases in sequence datasets. We use a weighting scheme which considers the observed distinctiveness of sequences and apply it to calculations of linkage disequilibrium. Each sequence now contributes a weighted score to linkage disequilibrium measurements of pairwise loci. We demonstrate that this reduces the effect of uneven sampling, as underrepresented groups of sequences will each contribute more individually than redundant, similar sequences. AvailabilitySource code for a python and rust implementation are freely available at under an MIT license at github.com/ojcharles/WeightedLD. Contactr.goldstein@ucl.ac.uk or oscar.charles.18@ucl.ac.uk

genetics↗

bioRxiv2020

Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5 rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

microbiology↗

bioRxiv2020

Genomics of Ocular Chlamydia trachomatis after 5 years of SAFE interventions for trachoma in Amhara, Ethiopia

BackgroundTo eliminate trachoma as a public health problem, the WHO recommends the SAFE strategy. As part of the SAFE strategy in the Amhara Region, Ethiopia, the Trachoma Control Program distributed over 124 million doses of antibiotic between 2007 and 2015. Despite these interventions, trachoma remained hyperendemic in many districts and a considerable level of Chlamydia trachomatis (Ct) infection was evident. MethodsWe utilised residual material from Abbott m2000 Ct diagnostic tests to sequence 99 ocular Ct samples from Amhara and investigated the role of Ct genomic variation in the continued transmission of Ct following 5 years of SAFE. FindingsSequences were typical of ocular Ct, at the whole-genome level and in tissue tropism-associated genes. There was no evidence of macrolide-resistance in this Ct population. Polymorphism in a region around ompA gene was associated with village-level TF prevalence. Additionally, greater ompA diversity at the district-level was associated with increased Ct infection prevalence. InterpretationWe found no evidence for Ct genomic variation contributing to continued transmission of Ct after treatment, adding to previous evidence that azithromycin does not drive acquisition of macrolide resistance alleles in Ct. Increased Ct infection in villages and in districts with more ompA variants requires longitudinal investigation to understand what impact this may have on treatment success and host immunity. FundingEuropean Commission; Neglected Tropical Disease Support Center; International Trachoma Initiative

genomics↗

bioRxiv2020

Decoding the architecture of the varicella-zoster virus transcriptome

Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, non-canonical ORFs, and ORF fusions, as well as putative non-coding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated as immediate-early, were expressed with late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection.

microbiology↗

bioRxiv2020

cmvdrg - An R package for Human Cytomegalovirus antiviral Drug Resistance Genotyping

The prevention and treatment of many herpesvirus associated diseases is based on the utilization of antiviral therapies, however therapeutic success is limited by the development of drug resistance. A comprehensive point of truth of resistance conferring mutations has been missing but would be important to aid the development of antiviral drugs and in management of infections. We therefore developed HerpesDRG, a drug resistance mutation database for all the known important genes and current treatment options, built from a systematic review of available genotype to phenotype literature. The database is released along with an R package to provide a low barrier of entry to variant resistance annotation and clinical implication analysis from common sanger and NGS sequence data. This represents the first openly available and community maintainable knowledgebase of drug resistance mutations for the human herpes viruses (HHV), developed for the community of researchers and clinicians tackling HHV drug resistance. AvailabilityThe HerpesDRG database is available at github.com/ojcharles/herpesdrg-db. The R package for resistance genotyping data is available at github.com/ojcharles/herpesdrg. A user-friendly webserver is available at cmv-resistance.ucl.ac.uk/herpesdrg

bioinformatics↗

bioRxiv2020

SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms

Genomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.

molecular biology↗

bioRxiv2020

A large effective population size for within-host influenza virus infection

Strains of the influenza virus form coherent global populations, yet exist at the level of single infections in individual hosts. The relationship between these scales is a critical topic for understanding viral evolution. Here we investigate the within-host relationship between selection and the stochastic effects of genetic drift, estimating an effective population size of infection Ne for influenza infection. Examining whole-genome sequence data describing a chronic case of influenza B in a severely immunocompromised child we infer an Ne of 2.5 x 107 (95% confidence range 1.0 x 107 to 9.0 x 107) suggesting the importance of genetic drift to be minimal. Our result, supported by the analysis of data from influenza A infection, suggests that positive selection during within-host infection is primarily limited by the typically short period of infection. Atypically long infections may have a disproportionate influence upon global patterns of viral evolution.

evolutionary biology↗

bioRxiv2020

β-L-1- uracil (L-BHDU) effectiveness against varicella-zoster virus and herpes simplex virus type 1 depends on thymidine kinase activity

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 {micro}M in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TKG22R-GST and TKR130Q- GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDUR virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.

microbiology↗