Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing

Shipley, Mackenzie M.; Rathbun, Molly M.; Szpara, Moriah L.

doi:10.1007/978-1-4939-9814-2_11

Cited by 6 publications

(7 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Swabs selected based on the criteria above were processed for target enrichment and sequencing according to our published protocol [ 56 ]. Briefly, samples were processed through a phenol:chloroform separation followed by ethanol precipitation for DNA extraction, with subsequent quantification of total DNA by Qubit and HSV genome copies by qPCR for the gB gene (UL27) [ 55 ].…”

Section: Methodsmentioning

confidence: 99%

“…Samples were then sheared into approximately 500–1,000 base pair fragments and processed for library preparation with the KAPA Biosystems HyperPrep Library Kit according to the manufacturer’s protocol (with 14 cycles of amplification). Custom HSV-specific oligonucleotide probes (myBaits) from Arbor Biosciences were used with the Arbor Biosciences myBaits Target Capture Kit to enrich for HSV-1 DNA material according to the manufacturer’s protocol, followed by a second round of amplification (14 cycles) using the KAPA HiFi HotStart Library Amplification Kit [ 56 ]. A final round of quantification by Qubit and gB-specific qPCR was performed and used to adjust each sample to the appropriate concentration for sequencing with an Illumina MiSeq, using version 3 chemistry and 300 × 300-bp paired-end reads.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection

et al. 2022

Self Cite

View full text Add to dashboard Cite

Herpes simplex virus (HSV) causes chronic infection in the human host, characterized by self-limited episodes of mucosal shedding and lesional disease, with latent infection of neuronal ganglia. The epidemiology of genital herpes has undergone a significant transformation over the past two decades, with the emergence of HSV-1 as a leading cause of first-episode genital herpes in many countries. Though dsDNA viruses are not expected to mutate quickly, it is not yet known to what degree the HSV-1 viral population in a natural host adapts over time, or how often viral population variants are transmitted between hosts. This study provides a comparative genomics analysis for 33 temporally-sampled oral and genital HSV-1 genomes derived from five adult sexual transmission pairs. We found that transmission pairs harbored consensus-level viral genomes with near-complete conservation of nucleotide identity. Examination of within-host minor variants in the viral population revealed both shared and unique patterns of genetic diversity between partners, and between anatomical niches. Additionally, genetic drift was detected from spatiotemporally separated samples in as little as three days. These data expand our prior understanding of the complex interaction between HSV-1 genomics and population dynamics after transmission to new infected persons.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…HSV-1 genomic DNA was isolated according to the protocol described in [ 59 ], with minor modifications. Briefly, 300 μL of HSV-1 particle concentrate was mixed with 300 μL of extraction buffer (10 mM Tris-HCl (pH 8), 10 mM EDTA, 100 mM NaCl, 1% SDS) and 20 μL of proteinase K (20 mg/mL).…”

Section: Methodsmentioning

confidence: 99%

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems

Карпов

Demidova

Kulagin

et al. 2022

IJMS

View full text Add to dashboard Cite

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

show abstract

“…There are no standards for how many times a viral sample can be passaged in culture before it is no longer considered a "clinical isolate", nor are there standards as to whether a viral population should be plaque purified before calling it a strain. The introduction of sensitive DNA isolation and amplification techniques has also enabled whole viral genomes to be collected and sequenced directly from hosts, avoiding cell culture entirely (Greninger et al, 2018;Depledge et al, 2011;Watson et al, 2013;Johnston et al, 2017a;Shipley et al, 2018Shipley et al, , 2020. It is important that researchers (Breuer et al, 2010).…”

Section: Clear Nomenclature To Document Viral Strains and Genome Sourcesmentioning

confidence: 99%

“…However, growing viruses in cell culture is inherently different from how the virus would replicate in vivo, with the potential to introduce unexpected genetic bottlenecks or selective pressures (Parsons et al, 2015;Wilkinson et al, 2015;Kuny et al, 2020). The potential for cell cultureinduced bias in sequencing results has led to the development of oligonucleotide-based enrichment methods, with the goal of sequencing uncultured samples to the same quality as cultured stocks (Greninger et al, 2018;Depledge et al, 2011;Johnston et al, 2017a;Shipley et al, 2018Shipley et al, , 2020. This "oligo-enrichment" approach uses synthetic RNA or DNA probes, also known as baits, that are designed to hybridize with sparse amounts of the targeted viral genome(s) using sequence complementarity.…”

Section: The Path To Obtaining a Viral Genomementioning

confidence: 99%

Alphaherpesvirus Genomics: Past, Present and Future

Kuny¹,

Szpara²

2022

Current Issues in Molecular Biology

Self Cite

View full text Add to dashboard Cite

Alphaherpesviruses, as large double-stranded DNA viruses, were long considered to be genetically stable and to exist in a homogeneous state.Recently, the proliferation of high-throughput sequencing (HTS) and bioinformatics analysis has expanded our understanding of herpesvirus genomes and the variations found therein. Recent data indicate that herpesviruses exist as diverse populations, both in culture and in vivo, in a manner reminiscent of RNA viruses. In this review, we discuss the past, present, and potential future of alphaherpesvirus genomics, including the technical challenges that face the field. We also review how recent data has enabled genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures, including those introduced by cell culture.While we focus on the human alphaherpesviruses, we draw key insights from related veterinary species and from the beta-and gamma-subfamilies of herpesviruses. Promising technologies and potential future directions for herpesvirus genomics are highlighted as well, including the potential to link viral genetic differences to phenotypic and disease outcomes. Curr. Issues Mol. Biol. 42: 41-80. caister.com/cimb caister.com/cimb 41 Curr. Issues Mol. Biol. Vol. 42

show abstract

Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing

Cited by 6 publications

References 20 publications

Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection

Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems

Alphaherpesvirus Genomics: Past, Present and Future

Contact Info

Product

Resources

About