Biology subjects

Jerome, K. R.

Publications and source records attributed to Jerome, K. R..

27 records · Page 2Linked to original sources

bioRxiv2020

In vivo antiviral host response to SARS-CoV-2 by viral load, sex, and age

Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3, and Th1 chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-{kappa}B signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.

microbiology↗

bioRxiv2020

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗

bioRxiv2020

Herpes Simplex Virus Mistyping due to HSV-1 x HSV-2 Interspecies Recombination in Viral Gene Encoding Glycoprotein B

Human herpes simplex viruses (HSV) 1 and 2 are most often typed via molecular assays. Here we describe the first known case of HSV mistyping due to a previously undescribed HSV-1 x HSV-2 recombination event in UL27, the gene that encodes glycoprotein B. This is the first reported HSV interspecies recombination event impacting this gene, which is frequently used as a target for diagnostics and experimental therapeutics.

genomics↗

bioRxiv2018

The impact of interspecies recombination on human herpes simplex virus evolution and host immune recognition

Among the most ubiquitous of human pathogens, HSV-1 and HSV-2 are distinct viral species that diverged about six million years ago. At least four ancient HSV-1 x HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and non-recombinants at each locus circulating today. Though interspecies recombination has occurred in the past, its importance in HSV evolution remains incompletely defined. Using 255 newly-sequenced and 219 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV. The novel recombinants we identify demonstrate that the sizes and locations of interspecies recombination events in HSV-2 are more variable than previously appreciated. One novel recombinant arose in its current host, showing for the first time that interspecies recombination occurs in contemporary HSV populations. We also demonstrate that interspecies recombination affects T-cell recognition of HSV. Our findings indicate that interspecies recombination can significantly influence genetic variation in and host immunologic response to HSV-2.

genomics↗

bioRxiv2018

Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.\n\nImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

epidemiology↗

bioRxiv2018

Viral diversity is an obligate consideration in CRISPR/Cas9 designs for HIV cure

RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. To address this, we identified a set of gRNAs targeting HIV LTR, gag and pol using publicly available sequences for these genes. We ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally-available sequences. We then selected 59 gRNAs from our list of highly-conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon ART cessation even with many doses.\n\nAuthor summaryThe field of genome engineering has exploded over the last decade with the discovery of targeted endonucleases such as CRISPR/Cas9. Endonucleases are now being used to develop a wide range of therapeutics and their use has expanded into antiviral therapy against latent viral infections like HIV. The idea is to induce mutations in latent viral genomes that will render them replication-incompetent, thereby producing a functional cure. Although a great deal of progress has been made, most studies to date have relied on molecular clones that represent \"ideal\" targets. For clinical success and broad applicability, these therapies need to account for viral genetic diversity within and between individuals. Our paper examines the impact of HIV diversity on CRISPR-based cure strategies to determine the predictors of future clinical success. We performed an exhaustive and detailed computational analysis to identify optimal CRISPR target sites, taking into consideration both within-host and global viral diversity. We coupled this with laboratory testing of highly-conserved guides and compared measured activity to predicted results. Finally, we developed a mathematical model to predict the impact of enzyme activity and viral diversity on the number of doses of a CRISPR-based therapy needed to achieve a functional cure of HIV.

bioinformatics↗

bioRxiv2017

Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains

Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6 is important for detection of viral reactivation and inherited chromosomally integrated HHV-6 in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV6 candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Copy number measured between 4-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6 strain with a HHV-6A GS backbone and >5.5kb region from HHV-6B Z29 from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at UL1/UL2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6 strains derived from cord blood mononuclear cells from different labs on different continents revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.\n\nImportanceAnything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very usefulness through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6 biology and the standard selection process.

genomics↗

bioRxiv2017

Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of 4 respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.

genomics↗

bioRxiv2016

Standard Issue: Copy number heterogeneity of JC virus standards discovered through next-generation sequencing

Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to gain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and ExactTM v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T-antigen region. No such variation was seen for a clinical sample with high copy-number of JCV nor a plasmid control. Intriguingly, several of the JCV standards sequenced in this study with large T-antigen deletions were cultured in cell lines immortalized using SV40 T-antigen, suggesting the possibility of trans-complementation in cell culture. Using a cut-off of 2% variant allele fraction for junctional reads to define the presence of a strain, 11 different strains were present in the WHO standard. In summary, targeting of different regions of the same international standard could result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.

microbiology↗