Publications by authors named "Alexander L Greninger"

168 Publications

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

Sci Rep 2021 02 22;11(1):4290. Epub 2021 Feb 22.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA, USA.

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-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).
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http://dx.doi.org/10.1038/s41598-021-83730-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900118PMC
February 2021

Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

J Infect Dis 2021 02;223(2):197-205

Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.
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http://dx.doi.org/10.1093/infdis/jiaa618DOI Listing
February 2021

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Clin Infect Dis 2021 01;72(2):323-326

Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine, Seattle, Washington, USA.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.
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http://dx.doi.org/10.1093/cid/ciaa722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314163PMC
January 2021

Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus.

PLoS Pathog 2021 Jan 26;17(1):e1009088. Epub 2021 Jan 26.

Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles.
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http://dx.doi.org/10.1371/journal.ppat.1009088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864422PMC
January 2021

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.

Mol Ther Methods Clin Dev 2021 Mar 26;20:258-275. Epub 2020 Nov 26.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR- ()Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-Cas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of Cas9 delivery to HBV human hepatocytes than those without gene editing. HBV-specific AAV-Cas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.
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http://dx.doi.org/10.1016/j.omtm.2020.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803634PMC
March 2021

CrAssphage and its bacterial host in cat feces.

Sci Rep 2021 Jan 12;11(1):815. Epub 2021 Jan 12.

Vitalant Research Institute, 270 Masonic Avenue, San Francisco, CA, 94118, USA.

CrAssphages are a diverse group of related phages detected in human feces where they are the most prevalent and abundant prokaryotic virus. CrAssphages' cellular host has been identified as the anaerobic Bacteroides intestinalis. CrAssphage has also been reported in non-human primates and environmental samples and has been proposed as a marker of human fecal contamination. Here we describe crAssphage DNA in a feline fecal sample. 95% of the ~ 100 Kb genome could be assembled and classified in genus 1 of the recently proposed Alphacrassvirinae subfamily. The cat origin of the fecal sample was confirmed by partial mitochondrial DNA sequencing. High levels of Bacteroides intestinalis DNA could also be detected in this cat's feces. Fecal samples longitudinally collected over a 4-week period showed the continuous shedding of crAssphage DNA. We therefore report the first genome sequence-confirmed detection of crAssphage in fecal samples of a non-primate mammal.
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http://dx.doi.org/10.1038/s41598-020-80076-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804022PMC
January 2021

SARS-CoV-2 Viral Load on Admission Is Associated With 30-Day Mortality.

Open Forum Infect Dis 2020 Dec 3;7(12):ofaa535. Epub 2020 Nov 3.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load on admission was associated with a significantly increased 30-day mortality (odds ratio [OR], 4.20; 95% CI, 1.62-10.86), and anti-SARS-CoV-2 nucleocapisid IgG seropositivity on admission trended toward a reduced 30-day mortality (OR, 0.43; 95% CI, 0.15-1.26). Reporting of quantitative SARS-CoV-2 viral load and serologic assays may offer prognostic clinical information.
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http://dx.doi.org/10.1093/ofid/ofaa535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665729PMC
December 2020

Analytical Sensitivity of the Abbott BinaxNOW COVID-19 Ag CARD.

J Clin Microbiol 2020 Dec 11. Epub 2020 Dec 11.

Department of Laboratory Medicine & Pathology, Virology Division, University of Washington, Seattle, WA

Multiple rapid antigen tests for SARS-CoV-2 have recently received Emergency Use Authorization (EUA) from the United States Food and Drug Administration (FDA). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for cheap, quick, decentralized testing. Robust analytical sensitivity data in comparison to qRT-PCR is currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of Abbott BinaxNOW COVID-19 Ag CARD using SARS-CoV-2 positive clinical specimens quantified by RT-ddPCR and multiple FDA EUA qRT-PCR platforms using RNA standards. Initial and confirmatory limits of detection for the BinaxNOW COVID-19 Ag CARD were determined to be equivalent to 4.04 - 8.06x10 copies/swab. We further confirmed this limit of detection with 72 additional clinical samples positive for SARS-CoV-2 in either phosphate-buffered saline or viral transport media.100% of samples with viral loads >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag CARD has the approximate analytical sensitivity equivalent to a generic qRT-PCR C of 29-30.
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http://dx.doi.org/10.1128/JCM.02880-20DOI Listing
December 2020

Hydroxychloroquine as Postexposure Prophylaxis to Prevent Severe Acute Respiratory Syndrome Coronavirus 2 Infection : A Randomized Trial.

Ann Intern Med 2020 Dec 8. Epub 2020 Dec 8.

University of Washington, Seattle, Washington (H.C.S., T.T.S., M.L.K., K.K.T., S.M., H.S.H., L.K., M.W., C.C., H.Y.C., J.M.B.).

Background: Effective prevention against coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently limited to nonpharmaceutical strategies. Laboratory and observational data suggested that hydroxychloroquine had biological activity against SARS-CoV-2, potentially permitting its use for prevention.

Objective: To test hydroxychloroquine as postexposure prophylaxis for SARS-CoV-2 infection.

Design: Household-randomized, double-blind, controlled trial of hydroxychloroquine postexposure prophylaxis. (ClinicalTrials.gov: NCT04328961).

Setting: National U.S. multicenter study.

Participants: Close contacts recently exposed (<96 hours) to persons with diagnosed SARS-CoV-2 infection.

Intervention: Hydroxychloroquine (400 mg/d for 3 days followed by 200 mg/d for 11 days) or ascorbic acid (500 mg/d followed by 250 mg/d) as a placebo-equivalent control.

Measurements: Participants self-collected mid-turbinate swabs daily (days 1 to 14) for SARS-CoV-2 polymerase chain reaction (PCR) testing. The primary outcome was PCR-confirmed incident SARS-CoV-2 infection among persons who were SARS-CoV-2 negative at enrollment.

Results: Between March and August 2020, 671 households were randomly assigned: 337 (407 participants) to the hydroxychloroquine group and 334 (422 participants) to the control group. Retention at day 14 was 91%, and 10 724 of 11 606 (92%) expected swabs were tested. Among the 689 (89%) participants who were SARS-CoV-2 negative at baseline, there was no difference between the hydroxychloroquine and control groups in SARS-CoV-2 acquisition by day 14 (53 versus 45 events; adjusted hazard ratio, 1.10 [95% CI, 0.73 to 1.66];  > 0.20). The frequency of participants experiencing adverse events was higher in the hydroxychloroquine group than the control group (66 [16.2%] versus 46 [10.9%], respectively;  = 0.026).

Limitation: The delay between exposure, and then baseline testing and the first dose of hydroxychloroquine or ascorbic acid, was a median of 2 days.

Conclusion: This rigorous randomized controlled trial among persons with recent exposure excluded a clinically meaningful effect of hydroxychloroquine as postexposure prophylaxis to prevent SARS-CoV-2 infection.

Primary Funding Source: Bill & Melinda Gates Foundation.
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http://dx.doi.org/10.7326/M20-6519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732017PMC
December 2020

Estimation of Full-Length TprK Diversity in Treponema pallidum subsp. .

mBio 2020 10 27;11(5). Epub 2020 Oct 27.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA

Immune evasion and disease progression of subsp. are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of during infection in the human host is still lacking. Furthermore, prior studies examining how subsp. uses its repertoire of genomic donor sites to generate diversity within the variable regions of the have yielded a partial understanding of this process due to the limited number of alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length alleles from subsp. collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese subsp. specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows subsp. to establish lifelong infection. Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. subsp. , the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical subsp. strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that subsp. can potentially generate. Our results support how the subsp. TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.
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http://dx.doi.org/10.1128/mBio.02726-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593977PMC
October 2020

Inhibition of Coronavirus Entry and by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain.

mBio 2020 10 20;11(5). Epub 2020 Oct 20.

Department of Pediatrics, Columbia University Medical Center, New York, New York, USA

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses. SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers () and human airway tissues (). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.
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http://dx.doi.org/10.1128/mBio.01935-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587434PMC
October 2020

Sensitive Recovery of Complete SARS-CoV-2 Genomes from Clinical Samples by Use of Swift Biosciences' SARS-CoV-2 Multiplex Amplicon Sequencing Panel.

J Clin Microbiol 2020 12 17;59(1). Epub 2020 Dec 17.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

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http://dx.doi.org/10.1128/JCM.02226-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771467PMC
December 2020

Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment.

J Clin Microbiol 2020 Nov 18;58(12). Epub 2020 Nov 18.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.
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http://dx.doi.org/10.1128/JCM.01605-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685877PMC
November 2020

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

PLoS Biol 2020 10 2;18(10):e3000896. Epub 2020 Oct 2.

Division of Immunobiology, Department of Medicine, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.
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http://dx.doi.org/10.1371/journal.pbio.3000896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556528PMC
October 2020

Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection.

J Infect Dis 2020 Sep 30. Epub 2020 Sep 30.

Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Most individuals infected with SARS-CoV-2 develop neutralizing antibodies that target the viral spike protein. Here we quantify how levels of these antibodies change in the months following SARS-CoV-2 infection by examining longitudinal samples collected between ~30 and 152 days post symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about four-fold from one to four months post symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B-cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.
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http://dx.doi.org/10.1093/infdis/jiaa618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543487PMC
September 2020

Reinfection with SARS-CoV-2 and Failure of Humoral Immunity: a case report.

medRxiv 2020 Sep 25. Epub 2020 Sep 25.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a new strain harboring the spike variant D614G. With antibody and B cell analytics, we show correlates of adaptive immunity, including a differential response to D614G. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.09.22.20192443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523175PMC
September 2020

Retrospective clinical evaluation of 4 lateral flow assays for the detection of SARS-CoV-2 IgG.

Diagn Microbiol Infect Dis 2020 Nov 2;98(3):115161. Epub 2020 Aug 2.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Phoenix, AZ, USA. Electronic address:

In a Clinical Laboratory Improvement Amendments laboratory setting, we evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG detection with 4 lateral flow immunoassays [LFIAs; 2 iterations from BTNX Inc. (n = 457) and 1 each from ACON Laboratories (n = 200) and SD BIOSENSOR (n = 155)]. In a cohort of primarily hospitalized, reverse-transcription polymerase chain reaction-confirmed coronavirus disease 2019 cases, sensitivity at ≥14 days from symptom onset was: BTNX kit 1, 95%; BTNX kit 2, 91%; ACON, 95%; and SD, 92%. All assays showed good concordance with the Abbott SARS-CoV-2 IgG assay at ≥14 days from symptom onset: BTNX kit 1, 99%; BTNX kit 2, 94%; ACON, 99%; and SD, 100%. Specificity, measured using specimens collected prior to SARS-CoV-2 circulation in the United States and "cross-reactivity challenge" specimens, was 98% for BTNX kit 1 and ACON and 100% for BTNX kit 2 and SD. These results suggest that LFIAs may provide adequate results for rapid detection of SARS-CoV-2.
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http://dx.doi.org/10.1016/j.diagmicrobio.2020.115161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395943PMC
November 2020

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2.

J Virol Methods 2020 12 15;286:113972. Epub 2020 Sep 15.

Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, AB, Canada; Clinical Section of Microbiology, Alberta Precision Laboratories, Calgary, AB, Canada; Department Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada; Department of Medicine, University of Calgary, Calgary, AB, Canada. Electronic address:

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.
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http://dx.doi.org/10.1016/j.jviromet.2020.113972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490281PMC
December 2020

Cryptic transmission of SARS-CoV-2 in Washington state.

Science 2020 10 10;370(6516):571-575. Epub 2020 Sep 10.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

After its emergence in Wuhan, China, in late November or early December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus rapidly spread globally. Genome sequencing of SARS-CoV-2 allows the reconstruction of its transmission history, although this is contingent on sampling. We analyzed 453 SARS-CoV-2 genomes collected between 20 February and 15 March 2020 from infected patients in Washington state in the United States. We find that most SARS-CoV-2 infections sampled during this time derive from a single introduction in late January or early February 2020, which subsequently spread locally before active community surveillance was implemented.
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http://dx.doi.org/10.1126/science.abc0523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810035PMC
October 2020

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

PLoS Biol 2020 09 8;18(9):e3000849. Epub 2020 Sep 8.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America.

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (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 (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. 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 the 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 cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) 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.
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http://dx.doi.org/10.1371/journal.pbio.3000849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478592PMC
September 2020

Prolonged persistence of PCR-detectable virus during an outbreak of SARS-CoV-2 in an inpatient geriatric psychiatry unit in King County, Washington.

Am J Infect Control 2021 03 20;49(3):293-298. Epub 2020 Aug 20.

Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA.

Background: We describe key characteristics, interventions, and outcomes of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak within an inpatient geriatric psychiatry unit at the University of Washington Medical Center - Northwest.

Methods: After identifying 2 patients with SARS-CoV-2 infection on March 11, 2020, we conducted an outbreak investigation and employed targeted interventions including: screening of patients and staff; isolation and cohorting of confirmed cases; serial testing; and enhanced infection prevention measures.

Results: We identified 10 patients and 7 staff members with SARS-CoV-2 infection. Thirty percent of patients (n = 3) remained asymptomatic over the course of infection. Among SARS-CoV-2 positive patients, fever (n = 5, 50%) and cough (n = 4, 40%) were the most common symptoms. Median duration of reverse transcription polymerase chain reaction (RT-PCR) positivity was 25.5 days (interquartile range [IQR] 22.8-41.8) among symptomatic patients and 22.0 days (IQR 19.5-25.5) among asymptomatic patients. Median initial (19.0, IQR 18.7-25.7 vs 21.7, IQR 20.7-25.6) and nadir (18.9, IQR 18.2-20.3 vs 19.8, IQR 17.0-20.7) cycle threshold values were similar across symptomatic and asymptomatic patients, respectively.

Conclusions: Asymptomatic infection was common in this cohort of hospitalized, elderly individuals despite similar duration of SARS-CoV-2 RT-PCR positivity and cycle threshold values among symptomatic and asymptomatic patients.
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http://dx.doi.org/10.1016/j.ajic.2020.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438365PMC
March 2021

Neutralizing Antibodies Correlate with Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High Attack Rate.

J Clin Microbiol 2020 10 21;58(11). Epub 2020 Oct 21.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have been performed only in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral reverse transcription-PCR (RT-PCR) testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range, 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR-positive viral test with a cycle threshold ( ) of <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested that the outbreak originated largely from a single viral clade. Only three crew members tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of the crew members with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against reinfection (Fisher's exact test,  = 0.002).
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http://dx.doi.org/10.1128/JCM.02107-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587101PMC
October 2020

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate.

medRxiv 2020 Aug 14. Epub 2020 Aug 14.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA.

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fisher's exact test, p=0.002).
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http://dx.doi.org/10.1101/2020.08.13.20173161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430625PMC
August 2020

Gene editing and elimination of latent herpes simplex virus in vivo.

Nat Commun 2020 08 18;11(1):4148. Epub 2020 Aug 18.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.
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http://dx.doi.org/10.1038/s41467-020-17936-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435201PMC
August 2020

Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

J Clin Virol 2020 10 2;131:104570. Epub 2020 Aug 2.

Department of Laboratory Medicine and Pathology, Virology Division, University of Washington, Seattle, WA, United States; Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States. Electronic address:

Background: SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing.

Objective: To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR.

Study Design: Individual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based RT-PCR laboratory developed test. Positive samples were serially diluted and theoretical and empirical PCR cycle thresholds were evaluated. Thirty-two distinct positive samples were pooled into negative specimens and individual CTs were compared to pooled CTs. Low positive samples were repeated for reproducibility and 32 four-way pools of negative specimens were assayed to determine specificity.

Results: Four-way pooling was associated with a loss of sensitivity of 1.7 and 2.0 CTs for our N1 and N2 targets, respectively. Pooling correctly identified SARS-CoV-2 in 94 % (n = 30/32) of samples tested. The two low positive specimens (neat CT > 35) not detected by pooling were individually repeated and detected 75 % (n=6/8) and 37.5 % (n = 3/8) of the time, respectively. All specimens individually determined negative were also negative by pooling.

Conclusion: We report that 1:4 pooling of samples is specific and associated with an expected 2 CT loss in analytical sensitivity. Instead of running each sample individually, pooling of four samples will allow for a greater throughput and conserve scarce reagents.
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http://dx.doi.org/10.1016/j.jcv.2020.104570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396208PMC
October 2020

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

Viruses 2020 08 6;12(8). Epub 2020 Aug 6.

Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.

Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-based "typing" assays. Here we describe a case of HSV mistyping caused by a previously undescribed HSV-1 × HSV-2 recombination event in UL27, the HSV gene that encodes glycoprotein B. This is the first documented case of HSV mistyping caused by an HSV-1 × HSV-2 recombination event and the first description of an HSV interspecies recombination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and probe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that about 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.
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http://dx.doi.org/10.3390/v12080860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472045PMC
August 2020

Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration out of Africa.

Mol Biol Evol 2021 01;38(1):96-107

Institut für Virologie, Freie Universität Berlin, Berlin, Germany.

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or "reactivation" of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.
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http://dx.doi.org/10.1093/molbev/msaa190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782865PMC
January 2021

Reliability of self-sampling for accurate assessment of respiratory virus viral and immunologic kinetics.

J Infect Dis 2020 Jul 25. Epub 2020 Jul 25.

Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center.

The SARS-CoV-2 pandemic demonstrates the need for accurate and convenient approaches to diagnose and therapeutically monitor respiratory viral infections. We demonstrated that self-sampling with foam swabs is well-tolerated and provides quantitative viral output concordant with flocked swabs. Using longitudinal home-based self-sampling, we demonstrate nasal cytokine levels correlate and cluster according to immune cell of origin. Periods of stable viral loads are followed by rapid elimination, which could be coupled with cytokine expansion and contraction. Nasal foam swab self-sampling at home provides a precise, mechanistic readout of respiratory virus shedding and local immune responses.
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http://dx.doi.org/10.1093/infdis/jiaa451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454707PMC
July 2020

The First Quarter of SARS-CoV-2 Testing: the University of Washington Medicine Experience.

J Clin Microbiol 2020 Jul 23;58(8). Epub 2020 Jul 23.

Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, USA.

In early March 2020, the University of Washington Medical Center clinical virology laboratory became one of the first clinical laboratories to offer testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). When we first began test development in mid-January, neither of us believed there would be more than 2 million confirmed SARS-CoV-2 infections nationwide or that we would have performed more than 150,000 real-time PCR (RT-PCR) tests, with many more to come. This article will be a chronological summary of how we rapidly validated tests for SARS-CoV-2, increased our testing capacity, and addressed the many problems that came up along the way.
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http://dx.doi.org/10.1128/JCM.01416-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383557PMC
July 2020