Oscar Enrique Torres Montaguth

SauUSI is a dimeric, ATP-dependent type IV restriction enzyme that protects Staphylococcus aureus by cleaving nonself DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. Using biophysical, single-molecule and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). In half of events, the partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, looping is more frequent and both motors can engage in bidirectional loop translocation. Cleavage occurs stochastically throughout translocation but events that increase dwell time such as rebinding methylated sites elevate the chance of cleavage. Interactions with other SauUSI dimers or protein roadblocks influence cleavage distributions but are not required for nuclease activation. While the first cleavages principally generate 1-nt 3ʹ overhangs or blunt ends, multiple binding–translocation–cleavage cycles by individual enzymes ultimately fragment the modified nonself DNA, neutralizing its threat.

Hybrid capture approaches can detect a broad range of pathogens directly from clinical samples with greater sensitivity than untargeted approaches. Clinical adoption has been limited by long turnaround times, complex workflows, and perceived high costs. We iteratively developed and validated a rapid hybrid capture workflow based on Illumina’s RPIP protocol (standard workflow). Rapid-RPIP v1 reduced wet-lab time from ∼11.4 to ∼7.3 h, for a turnaround time (TAT) of <15 h. Rapid-RPIP v2 further reduced wet-lab time by ∼0.7 h and used Illumina Turbo Sequencing shortening the sequencing time from ∼5.5 to ∼2.5 h thus achieving a TAT of <10 h without compromising sensitivity and yielding higher RPKMs for RNA viruses than the standard workflow. This approach could be readily adapted to a broad range of clinical and public health applications.

This multicentre study investigated the utility of next‐generation sequencing (NGS) to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load (from 0.2 to 6207 IU/mL). 23 HBV DNA‐positive plasma samples of genotypes A‐E and one HBV‐negative control sample were assayed blindly via 9 established NGS methods from 6 European laboratories. Methods included untargeted metagenomics, pre‐enrichment by probe‐capture followed by Illumina sequencing, and HBV‐specific PCR pre‐amplification followed by sequencing with Nanopore or Illumina. Full HBV genomes were obtained only from samples with viral loads > 1000 IU/mL using probe‐capture methods, > 200 IU/mL using PCR‐Illumina methods, > 10 IU/mL using PCR‐Nanopore methods, and in no samples using metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads in PCR‐based methods. Probe‐capture and metagenomic methods detected additional viruses not routinely screened in blood donations, including polyomaviruses and herpesviruses; positive results were confirmed by PCR. In conclusion, NGS may delineate whole‐genome sequences at low viral loads if supported by a PCR pre‐amplification step. Probe‐capture methods also reliably detect HBV without pre‐amplification but show limited genome coverage for samples with low viral loads; they may additionally detect a wide range of blood‐borne viruses.

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.

SauUSI is a dimeric, ATP-dependent Type IV restriction enzyme that protects Staphylococcus aureus by cleaving non-self DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. Using biophysical, single-molecule, and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). The partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, both subunits engage in bidirectional loop translocation. Cleavage is triggered by binding a distal methylated site, by preferred DNA sequences, or upon reaching a DNA end. Interactions with other SauUSI dimers or protein roadblocks affect cleavage site distributions but are not required for nuclease activation. While the first cleavages principally generate blunt-ends or one-nucleotide 3′ overhangs, multiple binding-translocation cycles by individual enzymes ultimately shred the modified non-self DNA, neutralizing its threat.

Metagenomics is becoming more widely used for diagnosis of viral infections and surveillance of viruses. Its pathogen-agnostic approach makes metagenomics useful for unknown and novel infection diagnosis, outbreak investigation, and new and emerging pathogen surveillance. New metagenomics methods, such as the use of rapid sequencing technologies and approaches that can selectively enrich for a wide range of viruses, are expanding the range of clinical and public health scenarios in which metagenomics can be used. Following the COVID-19 pandemic, there is increasing interest in viral surveillance worldwide, using clinical samples, potential zoonotic reservoirs and environmental sources, such as wastewater. Validation and accreditation of metagenomics protocols to ensure quality, together with further innovation in methods, will be necessary to bring metagenomics into routine service in clinical and public health laboratories.

Objectives: This multicentre study investigated the utility of next-generation sequencing (NGS) to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load (from 0.2 to 6207 IU/ml). Methods: 23 HBV DNA positive plasma samples of genotypes A-E and one HBV-negative control sample were assayed blindly via 9 established NGS methods from 6 European laboratories. Methods included untargeted metagenomics, pre-enrichment by probe-capture followed by Illumina sequencing, and HBV-specific PCR pre-amplification followed by sequencing with Nanopore or Illumina. Results: Full HBV genomes were obtained only from samples with viral loads >1000 IU/ml using probe-capture methods, >200 IU/ml using PCR-Illumina methods, >10 IU/ml using PCR-Nanopore methods, and in no samples using metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads in all PCR-based methods. Probe-capture and metagenomic methods detected additional viruses not routinely screened in blood donations, including polyomaviruses and herpesviruses; positive results were confirmed by PCR. Conclusions: NGS may delineate whole-genome sequences at low viral loads if supported by a PCR pre-amplification step. Probe-capture methods also reliably detect HBV without pre-amplification but achieve limited genome characterisation at low viral loads; they may additionally detect a wide range of blood-borne viruses.

Hybridization capture approaches can significantly increase the sensitivity of metagenomics. However, their implementation with Oxford Nanopore Technologies (ONT) sequencing has been limited due to the lack of dedicated workflows and the need for an extra ligation-based library preparation step. We developed a universal four-primer PCR approach that allows the implementation of hybridization capture workflows with ONT without additional ligation-based steps. Our method allowed the fast conversion of a targeted metagenomics workflow, increasing ONT sensitivity by 10 to 100-fold compared to untargeted approaches and providing both rapid detection and whole-genome sequences where the pathogen was abundant and identification where viral loads were low.

Adeno-associated viruses (AAVs) are the most used vectors in gene therapy but can frequently cause liver complications in patients. The mechanisms underlying AAV-related liver toxicity remain poorly understood, posing challenges for effective prevention and intervention. We undertook long and short read metagenomic sequencing of liver tissue from a child with spinal muscular atrophy type 1 experiencing significant hepatitis after receiving onasemnogene abeparvovec. We identified manufacturing plasmid sequences, with evidence of complex structures and recombination. Vector genomes had extensive disruption and concatemerisation. We also identified the presence of human betaherpesvirus 6B in the liver. It is possible that presence of the manufacturing plasmid sequences or helper viruses allow replication of the vector within cells, contributing to the development of complex concatemeric structures and associated hepatitis.

Background: Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity. Methods: We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. Results: Capture with the Twist CVRP increased sensitivity by at least 10–100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600–6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. Conclusions: Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.