Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research.

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas http://www.peptideatlas.org/builds/borrelia/. Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.

A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness.

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Interrogating the Human Diplome: Computational Methods, Emerging Applications, and Challenges.

Human DNA sequencing protocols have revolutionized human biology, biomedical science, and clinical practice, but still have very important limitations. One limitation is that most protocols do not separate or assemble (i.e., "phase") the nucleotide content of each of the maternally and paternally derived chromosomal homologs making up the 22 autosomal pairs and the chromosomal pair making up the pseudo-autosomal region of the sex chromosomes. This has led to a dearth of studies and a consequent underappreciation of many phenomena of fundamental importance to basic and clinical genomic science. We discuss a few protocols for obtaining phase information as well as their limitations, including those that could be used in tumor phasing settings. We then describe a number of biological and clinical phenomena that require phase information. These include phenomena that require precise knowledge of the nucleotide sequence in a chromosomal segment from germline or somatic cells, such as DNA binding events, and insight into unique cis vs. trans-acting functionally impactful variant combinations-for example, variants implicated in a phenotype governed by compound heterozygosity. In addition, we also comment on the need for reliable and consensus-based diploid-context computational workflows for variant identification as well as the need for laboratory-based functional verification strategies for validating cis vs. trans effects of variant combinations. We also briefly describe available resources, example studies, as well as areas of further research, and ultimately argue that the science behind the study of human diploidy, referred to as "diplomics," which will be enabled by nucleotide-level resolution of phased genomes, is a logical next step in the analysis of human genome biology.

Comparative transcriptomics reveals circadian and pluripotency networks as two pillars of longevity regulation.

Mammals differ more than 100-fold in maximum lifespan. Here, we conducted comparative transcriptomics on 26 species with diverse lifespans. We identified thousands of genes with expression levels negatively or positively correlated with a species' maximum lifespan (Neg- or Pos-MLS genes). Neg-MLS genes are primarily involved in energy metabolism and inflammation. Pos-MLS genes show enrichment in DNA repair, microtubule organization, and RNA transport. Expression of Neg- and Pos-MLS genes is modulated by interventions, including mTOR and PI3K inhibition. Regulatory networks analysis showed that Neg-MLS genes are under circadian regulation possibly to avoid persistent high expression, whereas Pos-MLS genes are targets of master pluripotency regulators OCT4 and NANOG and are upregulated during somatic cell reprogramming. Pos-MLS genes are highly expressed during embryogenesis but significantly downregulated after birth. This work provides targets for anti-aging interventions by defining pathways correlating with longevity across mammals and uncovering circadian and pluripotency networks as central regulators of longevity.

Nonadditive gene expression is correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow (Salix spp.).

Many studies have highlighted the complex and diverse basis for heterosis in inbred crops. Despite the lack of a consensus model, it is vital that we turn our attention to understanding heterosis in undomesticated, heterozygous, and polyploid species, such as willow (Salix spp.). Shrub willow is a dedicated energy crop bred to be fast-growing and high yielding on marginal land without competing with food crops. A trend in willow breeding is the consistent pattern of heterosis in triploids produced from crosses between diploid and tetraploid species. Here, we test whether differentially expressed genes are associated with heterosis in triploid families derived from diploid Salix purpurea, diploid Salix viminalis, and tetraploid Salix miyabeana parents. Three biological replicates of shoot tips from all family progeny and parents were collected after 12 weeks in the greenhouse and RNA extracted for RNA-Seq analysis. This study provides evidence that nonadditive patterns of gene expression are correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow. Expression-level dominance was most correlated with heterosis for biomass yield traits and was highly enriched for processes involved in starch and sucrose metabolism. In addition, there was a global dosage effect of parent alleles in triploid hybrids, with expression proportional to copy number variation. Importantly, differentially expressed genes between family parents were most predictive of heterosis for both field and greenhouse collected traits. Altogether, these data will be used to progress models of heterosis to complement the growing genomic resources available for the improvement of heterozygous perennial bioenergy crops.

Predicting antimicrobial mechanism-of-action from transcriptomes: A generalizable explainable artificial intelligence approach.

To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action [MOA], represent a major research priority in biomedical science. However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.

Hepatocyte Growth Factor-Dependent Antiviral Activity of Activated cdc42-Associated Kinase 1 Against Hepatitis B Virus.

Activated cdc42-associated kinase 1 (ACK1) is a well-known non-receptor tyrosine kinase that regulates cell proliferation and growth through activation of cellular signaling pathways, including mitogen-activated protein kinase (MAPK). However, the anti-HBV activity of ACK1 has not been elucidated. This study aimed to investigate the role of ACK1 in the HBV life cycle and the mechanism underlying the anti-HBV activity of ACK1. To examine the antiviral activity of ACK1, we established HepG2-ACK1 cells stably overexpressing ACK1. The HBV life cycle, including HBeAg/HBsAg secretion, HBV DNA/transcription, and enhancer activity, was analyzed in HepG2 and HepG2-ACK1 cells with HBV replication-competent HBV 1.2mer (HBV 1.2). Finally, the anti-HBV activity of ACK1 was examined in an HBV infection system. ACK1 suppressed HBV gene expression and transcription in HepG2 and HepG2-ACK1 cells. Furthermore, ACK1 inhibited HBV replication by decreasing viral enhancer activity. ACK1 exhibited its anti-HBV activity via activation of Erk1/2, which consequently downregulated the expression of HNF4α binding to HBV enhancers. Furthermore, hepatocyte growth factor (HGF) induced ACK1 expression at an early stage. Finally, ACK1 mediated the antiviral effect of HGF in the HBV infection system. These results indicated that ACK1 induced by HGF inhibited HBV replication at the transcriptional level by activating the MAPK-HNF signaling pathway. Our findings suggest that ACK1 is a potentially novel upstream molecule of MAPK-mediated anti-HBV activity.

Conserved Genomic Terminals of SARS-CoV-2 as Coevolving Functional Elements and Potential Therapeutic Targets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 40 million people worldwide, with over 1 million deaths as of October 2020 and with multiple efforts in the development and testing of antiviral drugs and vaccines under way. In order to gain insights into SARS-CoV-2 evolution and drug targets, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from those of other well-characterized human and animal coronavirus genomes, as well as how polymorphic SARS-CoV-2 genomes are generally. We ultimately sought to identify features in the SARS-CoV-2 genome that may contribute to its viral replication, host pathogenicity, and vulnerabilities. Our analyses suggest the presence of unique sequence signatures in the 3' untranslated region (3'-UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2 and SARS-CoV as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host-microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on the results of multiple computational target prediction analyses and an assessment of similar interactions involving the influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.IMPORTANCE The coronavirus disease 2019 (COVID-19) outbreak is having a dramatic global effect on public health and the economy. As of October 2020, SARS-CoV-2 has been detected in over 189 countries, has infected over 40 million people, and is responsible for more than 1 million deaths. The genome of SARS-CoV-2 is small but complex, and its functions and interactions with human host factors are being studied extensively. The significance of our study is that, using extensive SARS-CoV-2 genome analysis techniques, we identified potential interacting human host microRNA targets that share similarity with those of influenza A virus H1N1. Our study results will allow the development of virus-host interaction models that will enhance our understanding of SARS-CoV-2 pathogenesis and motivate the exploitation of both the interacting viral and host factors as therapeutic targets.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

OBJECTIVES: To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. METHODS: Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. RESULTS: Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/ß-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. CONCLUSIONS: A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets.

To identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3'-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3'-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3'-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3'-UTR as therapeutic targets warrant further investigation.

Mechanism-of-Action Classification of Antibiotics by Global Transcriptome Profiling.

Antimicrobial resistance (AMR) is an ever-growing public health problem worldwide. The low rate of antibiotic discovery coupled with the rapid spread of drug-resistant bacterial pathogens is causing a global health crisis. To facilitate the drug discovery processes, we present a large-scale study of reference antibiotic challenge bacterial transcriptome profiles, which included 37 antibiotics across 6 mechanisms of actions (MOAs) and provide an economical approach to aid in antimicrobial dereplication in the discovery process. We demonstrate that classical MOAs can be sorted based upon the magnitude of gene expression profiles despite some overlap in the secondary effects of antibiotic exposures across MOAs. Additionally, using gene subsets, we were able to subdivide broad MOA classes into subMOAs. Furthermore, we provide a biomarker gene set that can be used to classify most antimicrobial challenges according to their canonical MOA. We also demonstrate the ability of this rapid MOA diagnostic tool to predict and classify the expression profiles of pure compounds and crude extracts to their expression profile-associated MOA class.

Enhanced Stability and CO/Formate Selectivity of Plasma-Treated SnO x/AgO x Catalysts during CO2 Electroreduction.

CO2 electroreduction into useful chemicals and fuels is a promising technology that might be used to minimize the impact that the increasing industrial CO2 emissions are having on the environment. Although plasma-oxidized silver surfaces were found to display a considerably decreased overpotential for the production of CO, the hydrogen evolution reaction (HER), a competing reaction against CO2 reduction, was found to increase over time. More stable and C1-product-selective SnO x/AgO x catalysts were obtained by electrodepositing Sn on O2-plasma-pretreated Ag surfaces. In particular, a strong suppression of HER (below 5% Faradaic efficiency (FE) at -0.8 V vs the reversible hydrogen electrode, RHE) during 20 h was observed. Ex situ scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDS), quasi in situ X-ray photoelectron spectroscopy (XPS), and operando X-ray absorption near-edge structure spectroscopy (XANES) measurements showed that our synthesis led to a highly roughened surface containing stable Snδ+/Sn species that were found to be key in the enhanced activity and stable CO/formate (HCOO-) selectivity. Our study highlights the importance of roughness, composition, and chemical state effects in CO2 electrocatalysis.

Co-occurrence of Anaerobes in Human Chronic Wounds.

Chronic wounds are wounds that have failed to heal after 3 months of appropriate wound care. Previous reports have identified a diverse collection of bacteria in chronic wounds, and it has been postulated that bacterial profile may contribute to delayed healing. The purpose of this study was to perform a microbiome assessment of the Wound Healing and Etiology (WE-HEAL) Study cohort, including underlying comorbidities less commonly studied in the context of chronic wounds, such as autoimmune diseases, and investigate possible relationships of the wound microbiota with clinical healing trends. We examined chronic wound specimens from 60 patients collected through the WE-HEAL Study using 16S ribosomal RNA gene sequencing. A group of co-occurring obligate anaerobes was identified from taxonomic analysis guided by Dirichlet multinomial mixtures (DMM) modeling. The group includes members of the Gram-positive anaerobic cocci (GPAC) of the Clostridia class (i.e., Anaerococcus, Finegoldia, and Peptoniphilus) and additional strict anaerobes (i.e., Porphyromonas and Prevotella). We showed that the co-occurring group of obligate anaerobes not only co-exists with commonly identified wound species (such as Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas, Corynebacterium, and Streptococcus), but importantly, they could also predominate the wound microbiota. Furthermore, examination of clinical comorbidities of the WE-HEAL specimens showed that specific obligate and facultative anaerobes were significantly reduced in wounds presented with autoimmune disease. With respect to future healing trends, no association with the wound microbiome community or the abundance of individual wound species could be established. In conclusion, we identified a co-occurring obligate anaerobic community type that predominated some human chronic wounds and underrepresentation of anaerobes in wounds associated with autoimmune diseases. Possible elucidation of host environments or key factors that influence anaerobe colonization warrants further investigation in a larger cohort.

Highly active single-layer MoS2 catalysts synthesized by swift heavy ion irradiation.

Two-dimensional molybdenum-disulfide (MoS2) catalysts can achieve high catalytic activity for the hydrogen evolution reaction upon appropriate modification of their surface. The intrinsic inertness of the compound's basal planes can be overcome by either increasing the number of catalytically active edge sites or by enhancing the activity of the basal planes via a controlled creation of sulfur vacancies. Here, we report a novel method of activating the MoS2 surface using swift heavy ion irradiation. The creation of nanometer-scale structures by an ion beam, in combination with the partial sulfur depletion of the basal planes, leads to a large increase of the number of low-coordinated Mo atoms, which can form bonds with adsorbing species. This results in a decreased onset potential for hydrogen evolution, as well as in a significant enhancement of the electrochemical current density by over 160% as compared to an identical but non-irradiated MoS2 surface.

Multidrug resistant pathogens respond differently to the presence of co-pathogen, commensal, probiotic and host cells.

In light of the ongoing antimicrobial resistance crisis, there is a need to understand the role of co-pathogens, commensals, and the local microbiome in modulating virulence and antibiotic resistance. To identify possible interactions that influence the expression of virulence or survival mechanisms in both the multidrug-resistant organisms (MDROs) and human host cells, unique cohorts of clinical isolates were selected for whole genome sequencing with enhanced assembly and full annotation, pairwise co-culturing, and transcriptome profiling. The MDROs were co-cultured in pairwise combinations either with: (1) another MDRO, (2) skin commensals (Staphylococcus epidermidis and Corynebacterium jeikeium), (3) the common probiotic Lactobacillus reuteri, and (4) human fibroblasts. RNA-Seq analysis showed distinct regulation of virulence and antimicrobial resistance gene responses across different combinations of MDROs, commensals, and human cells. Co-culture assays demonstrated that microbial interactions can modulate gene responses of both the target and pathogen/commensal species, and that the responses are specific to the identity of the pathogen/commensal species. In summary, bacteria have mechanisms to distinguish between friends, foe and host cells. These results provide foundational data and insight into the possibility of manipulating the local microbiome when treating complicated polymicrobial wound, intra-abdominal, or respiratory infections.

Comparison of phasing strategies for whole human genomes.

Humans are a diploid species that inherit one set of chromosomes paternally and one homologous set of chromosomes maternally. Unfortunately, most human sequencing initiatives ignore this fact in that they do not directly delineate the nucleotide content of the maternal and paternal copies of the 23 chromosomes individuals possess (i.e., they do not 'phase' the genome) often because of the costs and complexities of doing so. We compared 11 different widely-used approaches to phasing human genomes using the publicly available 'Genome-In-A-Bottle' (GIAB) phased version of the NA12878 genome as a gold standard. The phasing strategies we compared included laboratory-based assays that prepare DNA in unique ways to facilitate phasing as well as purely computational approaches that seek to reconstruct phase information from general sequencing reads and constructs or population-level haplotype frequency information obtained through a reference panel of haplotypes. To assess the performance of the 11 approaches, we used metrics that included, among others, switch error rates, haplotype block lengths, the proportion of fully phase-resolved genes, phasing accuracy and yield between pairs of SNVs. Our comparisons suggest that a hybrid or combined approach that leverages: 1. population-based phasing using the SHAPEIT software suite, 2. either genome-wide sequencing read data or parental genotypes, and 3. a large reference panel of variant and haplotype frequencies, provides a fast and efficient way to produce highly accurate phase-resolved individual human genomes. We found that for population-based approaches, phasing performance is enhanced with the addition of genome-wide read data; e.g., whole genome shotgun and/or RNA sequencing reads. Further, we found that the inclusion of parental genotype data within a population-based phasing strategy can provide as much as a ten-fold reduction in phasing errors. We also considered a majority voting scheme for the construction of a consensus haplotype combining multiple predictions for enhanced performance and site coverage. Finally, we also identified DNA sequence signatures associated with the genomic regions harboring phasing switch errors, which included regions of low polymorphism or SNV density.

Dominance and Sexual Dimorphism Pervade the Salix purpurea L. Transcriptome.

The heritability of gene expression is critical in understanding heterosis and is dependent on allele-specific regulation by local and remote factors in the genome. We used RNA-Seq to test whether variation in gene expression among F1 and F2 intraspecific Salix purpurea progeny is attributable to cis- and trans-regulatory divergence. We assessed the mode of inheritance based on gene expression levels and allele-specific expression for F1 and F2 intraspecific progeny in two distinct tissue types: shoot tip and stem internode. In addition, we explored sexually dimorphic patterns of inheritance and regulatory divergence among F1 progeny individuals. We show that in S. purpurea intraspecific crosses, gene expression inheritance largely exhibits a maternal dominant pattern, regardless of tissue type or pedigree. A significantly greater number of cis- and trans-regulated genes coincided with upregulation of the maternal parent allele in the progeny, irrespective of the magnitude, whereas the paternal allele was higher expressed for genes showing cis × trans or compensatory regulation. Importantly, consistent with previous genetic mapping results for sex in shrub willow, we have delimited sex-biased gene expression to a 2 Mb pericentromeric region on S. purpurea chr15 and further refined the sex determination region. Altogether, our results offer insight into the inheritance of gene expression in S. purpurea as well as evidence of sexually dimorphic expression which may have contributed to the evolution of dioecy in Salix.

Enhanced Carbon Dioxide Electroreduction to Carbon Monoxide over Defect-Rich Plasma-Activated Silver Catalysts.

Efficient, stable catalysts with high selectivity for a single product are essential if electroreduction of CO2 is to become a viable route to the synthesis of industrial feedstocks and fuels. A plasma oxidation pre-treatment of silver foil enhances the number of low-coordinated catalytically active sites, which dramatically lowers the overpotential and increases the activity of CO2 electroreduction to CO. At -0.6 V versus RHE more than 90 % Faradaic efficiency towards CO was achieved on a pre-oxidized silver foil. While transmission electron microscopy (TEM) and operando X-ray absorption spectroscopy showed that oxygen species can survive in the bulk of the catalyst during the reaction, quasi in situ X-ray photoelectron spectroscopy showed that the surface is metallic under reaction conditions. DFT calculations reveal that the defect-rich surface of the plasma-oxidized silver foils in the presence of local electric fields drastically decrease the overpotential of CO2 electroreduction.

HSV-1 clinical isolates with unique in vivo and in vitro phenotypes and insight into genomic differences.

Strain-specific factors contribute in significant but undefined ways to the variable incidence of herpes simplex virus (HSV) recrudescence. Studies that investigate these strain-specific factors are needed. Here, we used qPCR, in vitro assays, and genomic sequencing to identify important relationships between in vitro and clinical phenotypes of unique HSV-1 clinical isolates. Nine HSV-1 isolates from individuals displaying varying reactivation patterns were studied. Isolates associated with frequent recurrent herpes labialis (RHL) (1) displayed higher rates of viral shedding in the oral cavity than those associated with rare RHL and (2) tended to replicate more efficiently at 33 °C than 39 °C. HSV-1 isolates also displayed a more stable phenotype during propagation in U2OS cells than in Vero cells. Draft genome sequences of four isolates and one variant spanning 95.6 to 97.2 % of the genome were achieved, and whole-genome alignment demonstrated that the majority of these isolates clustered with known North American/European isolates. These findings revealed procedures that could help identify unique genotypes and phenotypes associated with HSV-1 isolates, which can be important for determining viral factors critical for regulating HSV-1 reactivation.