Publications by authors named "Meei-Li Huang"

193 Publications

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays.

J Clin Microbiol 2021 Jul 21:JCM0052721. Epub 2021 Jul 21.

Vaccine and Infectious Diseases Division, Fred Hutch Cancer Research Center, Seattle WA.

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson = 0.81-0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD ( = 0.63-0.89), but moderately correlated with nucleoprotein IgG ( = 0.46-0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 249.
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http://dx.doi.org/10.1128/JCM.00527-21DOI Listing
July 2021

Cytomegalovirus viremia and clinical outcomes in Kenyan children diagnosed with HIV in hospital.

Clin Infect Dis 2021 Jul 2. Epub 2021 Jul 2.

Kenyatta National Hospital, Nairobi, Kenya.

Background: Cytomegalovirus (CMV) viremia is common in HIV infection, and is associated with worse long-term outcomes. To date, no studies have assessed CMV viremia in children diagnosed with HIV in hospital.

Methods: We studied CMV viremia and clinical outcomes in 163 Kenyan children aged 2 months-12 years, diagnosed with HIV in hospital. CMV DNA levels in plasma were measured using quantitative PCR. Regression models were used to assess associations between CMV viremia >1000 IU/mL and the risk of continued hospitalization or death at 15 days, duration of hospitalization, and 6-month mortality.

Results: At enrollment, 62/114 (54%) children had CMV viremia, and 20 (32%) were >1000 IU/mL. Eleven CMV reactivations were observed after admission. The prevalence and level of CMV viremia were highest in children <2 years and lowest in children >5 years old. CMV viremia >1000 IU/mL was independently associated with age < 2 years (p=0.03), higher log10 HIV RNA level (p=0.01), and height-for-age z score <-2 (p=0.02). Adjusting for age and log10 HIV RNA, the relative risk of death or continued hospitalization at 15 days was 1.74 (95%CI=1.04, 2.90), and the hazard ratio of 6-month mortality was 1.97 (95%CI=0.57, 5.07) for children with CMV DNA ≥1000 IU/ml compared to lower-level or undetectable CMV DNA. Children with CMV DNA ≥1000 IU/ml were hospitalized a median ~5 days longer than children with lower-level or undetectable CMV DNA (p=0.002).

Conclusions: In this nested observational study, CMV viremia was common in hospitalized children with HIV, and levels ≥1000 IU/mL were associated with increased risk of mortality and longer hospitalization.
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http://dx.doi.org/10.1093/cid/ciab604DOI Listing
July 2021

Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State.

J Med Virol 2021 Jun 25. Epub 2021 Jun 25.

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

Real-time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real-time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT-ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS-CoV-2 detected in Washington State by using a combination of reverse-transcription polymerase chain reaction (RT-PCR), RT-ddPCR, and next-generation sequencing. We initially screened 1035 samples positive for SARS-CoV-2 by our CDC-based laboratory-developed assay using ThermoFisher's multiplex RT-PCR COVID-19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT-ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69-70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow-up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT-ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT-ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS-CoV-2.
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http://dx.doi.org/10.1002/jmv.27155DOI Listing
June 2021

Variants of concern are overrepresented among post-vaccination breakthrough infections of SARS-CoV-2 in Washington State.

Clin Infect Dis 2021 Jun 24. Epub 2021 Jun 24.

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

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOC) and had a median Ct of 20.2 (IQR=17.1-23.3). When compared to 5174 contemporaneous samples sequenced in our laboratory, VOC were significantly enriched among breakthrough infections (p < .05).
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http://dx.doi.org/10.1093/cid/ciab581DOI Listing
June 2021

Anti-SARS-CoV-2 antibody levels measured by the Advise Dx SARS-CoV-2 assay are concordant with previously available serologic assays but are not fully predictive of sterilizing immunity.

J Clin Microbiol 2021 Jun 24:JCM0098921. Epub 2021 Jun 24.

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

With the availability of widespread SARS-CoV-2 vaccination, high-throughput quantitative anti-spike serological testing will likely become increasingly important. Here, we investigated the performance characteristics of the recently FDA authorized semi-quantitative anti-spike AdviseDx SARS-CoV-2 IgG II assay compared to the FDA authorized anti-nucleocapsid Abbott Architect SARS-CoV-2 IgG, Roche elecsys Anti-SARS-CoV-2-S, EuroImmun Anti-SARS-CoV-2 ELISA, and GenScript surrogate virus neutralization assays and examined the humoral response associated with vaccination, natural protection, and breakthrough infection. The AdviseDx assay had a clinical sensitivity at 14 days post-symptom onset or 10 days post PCR detection of 95.6% (65/68, 95% CI: 87.8-98.8%) with two discrepant individuals seroconverting shortly thereafter. The AdviseDx assay demonstrated 100% positive percent agreement with the four other assays examined using the same symptom onset or PCR detection cutoffs. Using a recently available WHO International Standard for anti-SARS-CoV-2 antibody, we provide assay unit conversion factors to international units for each of the assays examined. We performed a longitudinal survey of healthy vaccinated individuals, finding median AdviseDx immunoglobulin levels peaked seven weeks post-first vaccine dose at approximately 4,000 IU/mL. Intriguingly, among the five assays examined, there was no significant difference in antigen binding level or neutralizing activity between two seropositive patients protected against SARS-CoV-2 infection in a previously described fishing vessel outbreak and five healthcare workers who experienced vaccine breakthrough of SARS-CoV-2 infection - all with variants of concern. These findings suggest that protection against SARS-CoV-2 infection cannot currently be predicted exclusively using antibody assays against wildtype SARS-CoV-2 spike. Further work is required to establish protective correlates for SARS-CoV-2 infection.
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http://dx.doi.org/10.1128/JCM.00989-21DOI Listing
June 2021

Examining the dynamics of Epstein-Barr virus shedding in the tonsils and the impact of HIV-1 coinfection on daily saliva viral loads.

PLoS Comput Biol 2021 Jun 21;17(6):e1009072. Epub 2021 Jun 21.

Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montréal, Québec, Canada.

Epstein-Barr virus (EBV) is transmitted by saliva and is a major cause of cancer, particularly in people living with HIV/AIDS. Here, we describe the frequency and quantity of EBV detection in the saliva of Ugandan adults with and without HIV-1 infection and use these data to develop a novel mathematical model of EBV infection in the tonsils. Eligible cohort participants were not taking antiviral medications, and those with HIV-1 infection had a CD4 count >200 cells/mm3. Over a 4-week period, participants provided daily oral swabs that we analysed for the presence and quantity of EBV. Compared with HIV-1 uninfected participants, HIV-1 coinfected participants had an increased risk of EBV detection in their saliva (IRR = 1.27, 95% CI = 1.10-1.47) and higher viral loads in positive samples. We used these data to develop a stochastic, mechanistic mathematical model that describes the dynamics of EBV, infected cells, and immune response within the tonsillar epithelium to analyse potential factors that may cause EBV infection to be more severe in HIV-1 coinfected participants. The model, fit using Approximate Bayesian Computation, showed high fidelity to daily oral shedding data and matched key summary statistics. When evaluating how model parameters differed among participants with and without HIV-1 coinfection, results suggest HIV-1 coinfected individuals have higher rates of B cell reactivation, which can seed new infection in the tonsils and lower rates of an EBV-specific immune response. Subsequently, both these traits may explain higher and more frequent EBV detection in the saliva of HIV-1 coinfected individuals.
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http://dx.doi.org/10.1371/journal.pcbi.1009072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248743PMC
June 2021

Association of Inherited Chromosomally Integrated Human Herpesvirus 6 with Neurologic Symptoms and Management after Allogeneic Hematopoietic Cell Transplantation.

Transplant Cell Ther 2021 Jun 7. Epub 2021 Jun 7.

Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, Washington; Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington. Electronic address:

Reactivation of human herpesvirus 6 (HHV-6) after allogeneic hematopoietic cell transplantation (HCT) is associated with neurologic complications, but the impact of donor and/or recipient inherited chromosomally integrated HHV-6 (iciHHV-6) on post-HCT central nervous system (CNS) symptoms and diagnostic and therapeutic interventions is not well understood. The aims of the present study were (1) to compare the cumulative incidence of CNS symptoms in the first 100 days following allogeneic HCT among patients with donor and/or recipient iciHHV-6 (iciHHV-6)with that of patients with neither donor nor recipient iciHHV-6 (iciHHV-6) and (2) to assess the role of HHV-6 detection in driving potentially unnecessary interventions in iciHHV-6 patients. We performed a retrospective matched cohort study of 87 iciHHV-6 and 174 iciHHV-6 allogeneic HCT recipients. HHV-6 testing was performed at the discretion of healthcare providers, who were unaware of iciHHV-6 status. The cumulative incidence of CNS symptoms was similar in iciHHV-6 (n = 37; 43%) and iciHHV-6 HCT recipients (n = 81; 47%; P = .63). HHV-6 plasma testing was performed in similar proportions of iciHHV-6 (n = 6; 7%) and iciHHV-6 (9%) patients and was detected in all tested iciHHV-6 HCTs and 2 (13%) iciHHV-6 HCTs. This resulted in more frequent HHV-6-targeted antiviral therapy after iciHHV-6 HCT (odds ratio, 12.8; 95% confidence interval, 1.5 to 108.2) with associated side effects. HHV-6 plasma detection in 2 iciHHV-6 patients without active CNS symptoms prompted unnecessary lumbar punctures. The cumulative incidence of CNS symptoms was similar after allogeneic HCT involving recipients or donors with and without iciHHV-6. Misattribution of HHV-6 detection as infection after iciHHV-6 HCT may lead to unnecessary interventions. Testing for iciHHV-6 may improve patient management.
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http://dx.doi.org/10.1016/j.jtct.2021.05.029DOI Listing
June 2021

Performance characteristics of the Abbott Alinity m SARS-CoV-2 assay.

J Clin Virol 2021 07 14;140:104869. Epub 2021 May 14.

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

Mass molecular diagnostic testing for the SARS-CoV-2 pandemic has drawn on laboratory developed tests, commercial assays, and fully-automated platforms to accommodate widespread demand. The Alinity m instrument by Abbott is capable of detecting several clinically relevant pathogens and has recently received FDA emergency use authorization for SARS-CoV-2 molecular testing. The Alinity m performs automatic sample preparation, RT-PCR assembly, amplification, detection, and result calculation in under two hours. Here, we validate the performance characteristics of the Alinity m SARS-CoV-2 assay in comparison with the Roche cobas 6800 and Hologic Panther Fusion platforms. Across 178 positive and 195 negative nasopharyngeal swab specimens (C range 14.30-38.84), the Alinity m detected one additional positive specimen that was found to be negative on the Roche cobas 6800 (PPA 100%, NPA 99.5%). Across a separate set of 30 positive and 174 negative nasopharyngeal swab specimens (C range 14.1-38.5), the Alinity m had 100% positive and negative agreement with the Hologic Panther Fusion. Using SeraCare SARS-CoV-2 RNA standards, the assay limit of detection was verified to be two-fold more sensitive than the parameters stated by the SARS-CoV-2 AMP kit package insert, at 50 virus copies/mL. Assay specificity was 100% over 20 specimens positive for other respiratory viruses and intraday precision was 100% concordant with <2% CV. These data illst u illustrate the Abbott Alinity m system's high concordance with reference assays and analyti high analytical for SARS-CoV-2 molecular detection.
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http://dx.doi.org/10.1016/j.jcv.2021.104869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118701PMC
July 2021

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Cell Rep Med 2021 Apr 12;2(4):100243. Epub 2021 Apr 12.

Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4 T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.
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http://dx.doi.org/10.1016/j.xcrm.2021.100243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080125PMC
April 2021

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State.

Sci Transl Med 2021 05 3;13(595). Epub 2021 May 3.

Seattle Children's Research Institute, Seattle, WA 98101, USA.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has gravely affected societies around the world. Outbreaks in different parts of the globe have been shaped by repeated introductions of new viral lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State (USA) to characterize how the spread of SARS-CoV-2 in Washington State in early 2020 was shaped by differences in timing of mitigation strategies across counties and by repeated introductions of viral lineages into the state. In addition, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G but not the other variant (614D) into the state. At an individual level, we observed evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we did not find any evidence that the 614G variant affects clinical severity or patient outcomes. Overall, this suggests that with regard to D614G, the behavior of individuals has been more important in shaping the course of the pandemic in Washington State than this variant of the virus.
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http://dx.doi.org/10.1126/scitranslmed.abf0202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158963PMC
May 2021

Retrospective Detection of SARS-CoV-2 in Symptomatic Patients prior to Widespread Diagnostic Testing in Southern California.

Clin Infect Dis 2021 May 3. Epub 2021 May 3.

Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles (UCLA), California, USA.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited.

Methods: We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples.

Results: We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January.

Conclusions: We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.
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http://dx.doi.org/10.1093/cid/ciab360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135745PMC
May 2021

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

medRxiv 2021 Apr 16. Epub 2021 Apr 16.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.
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http://dx.doi.org/10.1101/2021.04.10.21254091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057246PMC
April 2021

Phylogenetic estimates of SARS-CoV-2 introductions into Washington State.

medRxiv 2021 Apr 7. Epub 2021 Apr 7.

Background: The first confirmed case of SARS-CoV-2 in North America was identified in Washington state on January 21, 2020. We aimed to quantify the number and temporal trends of out-of-state introductions of SARS-CoV-2 into Washington.

Methods: We conducted a phylogenetic analysis of 11,422 publicly available whole genome SARS-CoV-2 sequences from GISAID sampled between December 2019 and September 2020. We used maximum parsimony ancestral state reconstruction methods on time-calibrated phylogenies to enumerate introductions/exports, their likely geographic source (e.g. US, non-US, and between eastern and western Washington), and estimated date of introduction. To incorporate phylogenetic uncertainty into our estimates, we conducted 5,000 replicate analyses by generating 25 random time-stratified samples of non-Washington reference sequences, 20 random polytomy resolutions, and 10 random resolutions of the reconstructed ancestral state.

Results: We estimated a minimum 287 separate introductions (median, range 244-320) into Washington and 204 exported lineages (range 188-227) of SARS-CoV-2 out of Washington. Introductions began in mid-January and peaked on March 29, 2020. Lineages with the Spike D614G variant accounted for the majority (88%) of introductions. Overall, 61% (range 55-65%) of introductions into Washington likely originated from a source elsewhere within the US, while the remaining 39% (range 35-45%) likely originated from outside of the US. Intra-state transmission accounted for 65% and 28% of introductions into eastern and western Washington, respectively.

Conclusions: There is phylogenetic evidence that the SARS-CoV-2 epidemic in Washington is continually seeded by a large number of introductions, and that there was significant inter- and intra-state transmission. Due to incomplete sampling our data underestimate the true number of introductions.
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http://dx.doi.org/10.1101/2021.04.05.21254924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045029PMC
April 2021

SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.

mBio 2021 04 13;12(2). Epub 2021 Apr 13.

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

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth , they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.
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http://dx.doi.org/10.1128/mBio.00065-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092196PMC
April 2021

Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

J Virol 2021 05 24;95(12). Epub 2021 May 24.

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

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology. Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.
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http://dx.doi.org/10.1128/JVI.00250-21DOI Listing
May 2021

Hydroxychloroquine with or without azithromycin for treatment of early SARS-CoV-2 infection among high-risk outpatient adults: A randomized clinical trial.

EClinicalMedicine 2021 Mar 27;33:100773. Epub 2021 Feb 27.

Division of Allergy and Infectious Diseases, University of Washington, United States.

Background: Treatment options for outpatients with COVID-19 could reduce morbidity and prevent SARS-CoV-2 transmission.

Methods: In this randomized, double-blind, three-arm (1:1:1) placebo-equivalent controlled trial conducted remotely throughout the United States, adult outpatients with laboratory-confirmed SARS-CoV-2 infection were recruited. Participants were randomly assigned to receive hydroxychloroquine (HCQ) (400 mg BID x1day, followed by 200 mg BID x9days) with or without azithromycin (AZ) (500 mg, then 250 mg daily x4days) or placebo-equivalent (ascorbic acid (HCQ) and folic acid (AZ)), stratified by risk for progression to severe COVID-19 (high-risk vs. low-risk). Self-collected nasal swabs for SARS-CoV-2 PCR, FLUPro symptom surveys, EKGs and vital signs were collected daily. Primary endpoints were: (a) 14-day progression to lower respiratory tract infection (LRTI), 28-day COVID-19 related hospitalization, or death; (b) 14-day time to viral clearance; secondary endpoints included time to symptom resolution (ClinicalTrials.gov: NCT04354428). Due to the low rate of clinical outcomes, the study was terminated for operational futility.

Findings: Between 15th April and 27th July 2020, 231 participants were enrolled and 219 initiated medication a median of 5.9 days after symptom onset. Among 129 high-risk participants, incident LRTI occurred in six (4.7%) participants (two control, four HCQ/AZ) and COVID-19 related hospitalization in seven (5.4%) (four control, one HCQ, two HCQ/AZ); no LRTI and two (2%) hospitalizations occurred in the 102 low-risk participants (one HCQ, one HCQ/AZ). There were no deaths. Among 152 participants with viral shedding at enrollment, median time to clearance was 5 days (95% CI=4-6) in HCQ, 6 days (95% CI=4-8) in HCQ/AZ, and 8 days (95% CI=6-10) in control. Viral clearance was faster in HCQ (HR=1.62, 95% CI=1.01-2.60,  = 0.047) but not HCQ/AZ (HR=1.25,  = 0.39) compared to control. Among 197 participants who met the COVID-19 definition at enrollment, time to symptom resolution did not differ by group (HCQ: HR=1.02, 95% CI-0.63-1.64,  = 0.95, HCQ/AZ: HR=0.91, 95% CI=0.57-1.45,  = 0.70).

Interpretation: Neither HCQ nor HCQ/AZ shortened the clinical course of outpatients with COVID-19, and HCQ, but not HCQ/AZ, had only a modest effect on SARS-CoV-2 viral shedding. HCQ and HCQ/AZ are not effective therapies for outpatient treatment of SARV-CoV-2 infection.

Funding: The COVID-19 Early Treatment Study was funded by the Bill & Melinda Gates Foundation (INV-017062) through the COVID-19 Therapeutics Accelerator. University of Washington Institute of Translational Health Science (ITHS) grant support (UL1 TR002319), KL2 TR002317, and TL1 TR002318 from NCATS/NIH funded REDCap. The content is solely the responsibility of the authors and does not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. PAN and MJA were supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. ClinicalTrials.gov number NCT04354428.
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http://dx.doi.org/10.1016/j.eclinm.2021.100773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912360PMC
March 2021

Association of HHV-6 With Outcomes in CMV-Seronegative Liver Transplant Recipients With CMV-Seropositive Donors Receiving Preemptive Antiviral Therapy.

Transplantation 2020 Dec 29. Epub 2020 Dec 29.

University of Pittsburgh, and VA Pittsburgh Healthcare System, Pittsburgh, PA University of California Los Angeles Medical Center, Los Angeles, CA Mayo Clinic, Rochester, MN Emory University, Atlanta, GA University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh PA Fred Hutchinson Cancer Research Center, Seattle, WA University of Washington, Seattle, WA.

Background: Risk factors, virologic parameters and outcomes associated with HHV-6 viremia in high-risk donor CMV-seropositive and recipient CMV-seronegative (D+R-) liver transplant recipients in the current era are incompletely defined.

Methods: The study population consisted of patients in the preemptive therapy (PET) arm of a randomized, controlled trial of PET versus valganciclovir prophylaxis for CMV prevention in D+R- liver transplant recipients. Weekly blood samples through 100 days in the PET group were tested for HHV-6 viremia using a real-time quantitative PCR. Assessments included virologic characteristics and relationship with CMV, risk factors and impact of HHV-6 viremia with outcomes through 12 months post-transplant.

Results: HHV-6 viremia at any level developed in 42% (40/96). Older patient age (p=0.03), longer hospitalization (p=0.015), and ICU stay at transplantation (p=0.029) were significantly associated with high-grade viremia. Concurrent HHV-6 and CMV viremia was associated with earlier onset of HHV-6 viremia (p=0.004), higher HHV-6 AUC (p=0.043), and higher peak HHV-6 viral load (p=0.006) vs HHV-6 viremia alone. High-grade viremia was independently associated with biopsy-proven rejection within 12 months (p=0.045) post-transplant.

Conclusions: Among D+R- liver transplant recipients receiving valganciclovir as PET, high-grade HHV-6 viremia was associated with increased age and critical-illness in ICU at time of transplant and was independently associated with allograft rejection.
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http://dx.doi.org/10.1097/TP.0000000000003604DOI Listing
December 2020

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

Detection of parvovirus B19 and human herpesvirus 6 in pediatric dilated cardiomyopathy: Impact after heart transplantation.

Ann Pediatr Cardiol 2020 Oct-Dec;13(4):301-308. Epub 2020 Sep 1.

Department of Laboratory Medicine, Division of Virology, University of Washington, Seattle, WA, USA.

Objectives: The aim of this study is to evaluate HHV-6 and PVB19 infection using polymerase chain reaction (PCR) and immunofluorescent assay (IFA) in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the impact of viral persistence in the cardiac allograft after heart transplantation (HT).

Methods: Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial samples from 48 pediatric DCM patients and 10 control subjects. Of the 48 DCM patients, 44 underwent HT. After HT, consecutive endomyocardial biopsy (EMB) samples were analyzed for the presence of PVB19 and HHV-6 antigens using IFA and the patients were evaluated for rejections, coronary vasculopathy, and graft loss.

Results: Of the 48 DCM patients, 14 had positive viral PCR results in explanted/autopsy hearts. Among them, PVB19 was found in 8/48, HHV6 in 4/48, both PVB19 and HHV6 in 1/48, and enterovirus in one, but no adenovirus was found. The EMB samples obtained after HT were positive for PVB19 and HHV-6 in 7/44 and 3/44 cases, respectively. Viral presence in both the explanted heart and the cardiac allograft was demonstrated in 4 patients, 3 of whom were positive for PVB19, and one of whom was positive for HHV-6 pretransplant. Coronary vasculopathy and graft loss were more common in patients with PVB19-positive myocardial tissues versus those who were PVB19-negative.

Conclusions: There is an association between PVB19 and HHV-6 infection and DCM in children. The study suggests the persistence of PVB19 and HHV-6 in the host can lead to subsequent viral reactivation in the transplanted heart, even in those recipients who do not have active myocarditis. PVB19 in the cardiac allograft tended toward higher adverse post-HT events.
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http://dx.doi.org/10.4103/apc.APC_124_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727911PMC
September 2020

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

J Clin Microbiol 2021 02 18;59(3). Epub 2021 Feb 18.

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

Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for inexpensive, quick, decentralized testing. Robust analytical sensitivity data in comparison to reverse transcription-quantitative PCR (qRT-PCR) are currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of the Abbott BinaxNOW COVID-19 Ag card using SARS-CoV-2-positive clinical specimens quantified by reverse transcription-droplet digital PCR (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 × 10 to 8.06 × 10 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 medium. One hundred percent of samples with viral loads of >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag card has an analytical sensitivity approximately equivalent to a generic qRT-PCR cycle threshold ( ) value of 29 to 30.
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http://dx.doi.org/10.1128/JCM.02880-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106729PMC
February 2021

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

Ann Intern Med 2021 03 8;174(3):344-352. 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
March 2021

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

Maternal Epstein-Barr Virus-Specific Antibodies and Risk of Infection in Ugandan Infants.

J Infect Dis 2021 Jun;223(11):1897-1904

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

Background: Epstein-Barr virus (EBV) infection is a major cause of malignancy worldwide. Maternal antibody is thought to prevent EBV infection because it is uncommon in early infancy. Maternal HIV infection is associated with an increased incidence of EBV infection in exposed infants, which we hypothesized results from impaired transfer of EBV-neutralizing maternal antibodies.

Methods: Among Ugandan infants followed for EBV acquisition from birth, we measured antibody binding to EBV glycoproteins (gp350, gH/gL) involved in B-cell and epithelial-cell entry, as well as viral neutralization and antibody-dependent cellular cytotoxicity (ADCC) activity in plasma samples prior to infection. These serologic data were analyzed for differences between HIV-exposed uninfected (HEU) and HIV-unexposed (HUU) infants, and for associations with incident infant EBV infection.

Results: HEU infants had significantly higher titers than HUU infants for all EBV-binding and neutralizing antibodies measured (P < .01) but not ADCC activity, which was similar between groups. No antibody measure was associated with a decreased risk of EBV acquisition in the cohort.

Conclusions: Our findings indicate that in this cohort maternal antibody did not protect infants against EBV infection through viral neutralization. The identification of protective nonneutralizing antibody functions would be invaluable for the development of an EBV vaccine.
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http://dx.doi.org/10.1093/infdis/jiaa654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176630PMC
June 2021

Association Between Cytomegalovirus and Epstein-Barr Virus Viremia And Human Immunodeficiency Virus DNA Levels in the Reservoir of Kenyan Infants Receiving Antiretroviral Therapy.

J Infect Dis 2021 Jun;223(11):1923-1927

Department of Global Health, University of Washington, Seattle, Washington, USA.

Identifying determinants of human immunodeficiency virus (HIV) reservoir levels may inform novel viral eradication strategies. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) coinfections were assessed as predictors of HIV proviral DNA level in 26 HIV RNA-suppressed Kenyan children starting antiretroviral therapy before 7 months of age. Earlier acquisition of CMV and EBV and higher cumulative burden of systemic EBV DNA viremia were each associated with higher HIV DNA level in the reservoir after 24 months of antiretroviral therapy, independent of HIV RNA levels over time. These data suggest that delaying or containing CMV and EBV viremia may be novel strategies to limit HIV reservoir formation.
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http://dx.doi.org/10.1093/infdis/jiaa640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176631PMC
June 2021

Subclinical Genital Herpes Shedding in HIV/Herpes Simplex Virus 2-Coinfected Women during Antiretroviral Therapy Is Associated with an Increase in HIV Tissue Reservoirs and Potentially Promotes HIV Evolution.

J Virol 2020 12 9;95(1). Epub 2020 Dec 9.

Seattle Children's Research Institute, Seattle, Washington, USA

Antigen (Ag)-specific immune responses to chronic infections, such as herpes simplex virus type 2 (HSV-2) in HIV/HSV-coinfected persons, may sustain HIV tissue reservoirs by promoting T-cell proliferation but are poorly studied in women on antiretroviral therapy (ART). Mixed anogenital swabs and cervical secretions were self-collected by nine HIV/HSV-2-coinfected women during ART for 28 days to establish subclinical HSV DNA shedding rates and detection of HIV RNA by real-time PCR. Typical herpes lesion site biopsy (TLSB) and cervical biopsy specimens were collected at the end of the daily sampling period. Nucleic acids (NA) isolated from biopsy specimens had HIV quantified and HIV C2-V5 single-genome amplification (SGA) and T-cell receptor (TCR) repertoires assessed. Women had a median CD4 count of 537 cells/μl (IQR: 483 to 741) at enrollment and HIV plasma viral loads of <40 copies/ml. HSV DNA was detected on 12% of days (IQR: 2 to 25%) from anogenital specimens. Frequent subclinical HSV DNA shedding was associated with increased HIV DNA tissue concentrations and increased divergence from the most recent common ancestor (MRCA), an indicator of HIV replication. Distinct predominant TCR clones were detected in cervical and TLSB specimens in a woman with frequent HSV DNA shedding, with mixing of minor variants between her tissues. In contrast, more limited TCR repertoire mixing was observed in two women with less frequent subclinical HSV DNA shedding. Subclinical HSV shedding in HIV/HSV-coinfected women during ART may sustain HIV tissue reservoirs via Ag exposure or HIV replication. This study provides evidence supporting further study of interventions targeting suppression of Ag-specific immune responses as a component of HIV cure strategies. Persons with HIV infection are frequently coinfected with chronic herpesviruses, which periodically replicate and produce viable herpes virions, particularly in anogenital and cervical tissues. Persistent protein expression results in proliferation of CD8 and CD4 T cells, and the latter could potentially expand and sustain HIV tissue reservoirs. We found HSV genital shedding rates were positively correlated with HIV DNA concentrations and HIV divergence from ancestral sequences in tissues. Our work suggests that immune responses to common coinfections, such as herpesviruses, may sustain HIV tissue reservoirs during suppressive ART, suggesting future cure strategies should study interventions to suppress replication or reactivation of chronic herpes infections.
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http://dx.doi.org/10.1128/JVI.01606-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737750PMC
December 2020

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State.

medRxiv 2020 Sep 30. Epub 2020 Sep 30.

University of Washington, Seattle, WA, USA.

The rapid spread of SARS-CoV-2 has gravely impacted societies around the world. Outbreaks in different parts of the globe are shaped by repeated introductions of new lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State to characterize how the spread of SARS-CoV-2 in Washington State (USA) was shaped by differences in timing of mitigation strategies across counties, as well as by repeated introductions of viral lineages into the state. Additionally, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G, but not the other variant (614D) into the state. At an individual level, we see evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we do not find any evidence that the 614G variant impacts clinical severity or patient outcomes. Overall, this suggests that at least to date, the behavior of individuals has been more important in shaping the course of the pandemic than changes in the virus.
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http://dx.doi.org/10.1101/2020.09.30.20204230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536883PMC
September 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

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Hum Gene Ther 2021 Jan 2;32(1-2):96-112. Epub 2020 Nov 2.

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

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4 T cells , are being explored for delivery of anti-HIV therapeutic modalities . However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.
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http://dx.doi.org/10.1089/hum.2020.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020555PMC
January 2021

CMV viral load kinetics as surrogate endpoints after allogeneic transplantation.

J Clin Invest 2021 Jan;131(1)

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

BACKGROUNDViral load (VL) surrogate endpoints transformed development of HIV and hepatitis C therapeutics. Surrogate endpoints for CMV-related morbidity and mortality could advance development of antiviral treatments. Although observational data support using CMV VL as a trial endpoint, randomized controlled trials (RCTs) demonstrating direct associations between virological markers and clinical endpoints are lacking.METHODSWe performed CMV DNA PCR on frozen serum samples from the only placebo-controlled RCT of ganciclovir for early treatment of CMV after hematopoietic cell transplantation (HCT). We used established criteria to assess VL kinetics as surrogates for CMV disease or death by weeks 8, 24, and 48 after randomization and quantified antiviral effects captured by each marker. We used ensemble-based machine learning to assess the predictive ability of VL kinetics and performed this analysis on a ganciclovir prophylaxis RCT for validation.RESULTSVL suppression with ganciclovir reduced cumulative incidence of CMV disease and death for 20 years after HCT. Mean VL, peak VL, and change in VL during the first 5 weeks of treatment fulfilled the Prentice definition for surrogacy, capturing more than 95% of ganciclovir's effect, and yielded highly sensitive and specific predictions by week 48. In the prophylaxis trial, the viral shedding rate satisfied the Prentice definition for CMV disease by week 24.CONCLUSIONSOur results support using CMV VL kinetics as surrogates for CMV disease, provide a framework for developing CMV preventative and therapeutic agents, and support reductions in VL as the mechanism through which antivirals reduce CMV disease.FUNDINGMerck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.
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http://dx.doi.org/10.1172/JCI133960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773411PMC
January 2021
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