Comparison of upper respiratory viral load distributions in asymptomatic and symptomatic children diagnosed with SARS-CoV-2 infection in pediatric hospital testing programs

The distribution of upper respiratory viral loads (VL) in asymptomatic children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We assessed PCR cycle threshold (Ct) values and estimated VL in infected asymptomatic children diagnosed in nine pediatric hospital testing programs. Records for asymptomatic and symptomatic patients with positive clinical SARS-CoV-2 tests were reviewed. Ct values were (i) adjusted by centering each value around the institutional median Ct value from symptomatic children tested with that assay and (ii) converted to estimated VL (numbers of copies per milliliter) using internal or manufacturer data. Adjusted Ct values and estimated VL for asymptomatic versus symptomatic children (118 asymptomatic versus 197 symptomatic children aged 0 to 4 years, 79 asymptomatic versus 97 symptomatic children aged 5 to 9 years, 69 asymptomatic versus 75 symptomatic children aged 10 to 13 years, 73 asymptomatic versus 109 symptomatic children aged 14 to 17 years) were compared. The median adjusted Ct value for asymptomatic children was 10.3 cycles higher than for symptomatic children

SARS-CoV-2 E gene variant alters analytical sensitivity characteristics of viral detection using a commercial reverse transcription-PCR assay

Diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for patient management, infection prevention, and the public health response for coronavirus disease 2019 (COVID-19). The efficacy and reliability of these assays are of paramount importance in both tracking and controlling the spread of the virus. Real-time reverse transcription-PCR (RT-PCR) assays rely on a fixed genetic sequence for primer and probe binding. Mutations can potentially alter the accuracy of these assays and lead to unpredictable analytical performance characteristics and false-negative results. Here, we identify a G-to-U transversion (nucleotide 26372) in the SARS-CoV-2 E gene in three specimens with reduced viral detection efficiency using a widely available commercial assay. Further analysis of the public GISAID repository led to the identification of 18 additional genomes with this mutation, which reflect five independent mutational events. This work supports the use of dual-target assays to reduce the number of false-negative PCR results

Tracking the prevalence and emergence of SARS-CoV-2 variants of concern using a regional genomic surveillance program

UNLABELLED: SARS-CoV-2 molecular testing coupled with whole-genome sequencing is instrumental for real-time genomic surveillance. Genomic surveillance is critical for monitoring the spread of variants of concern (VOCs) as well as discovery of novel variants. Since the beginning of the pandemic, millions of SARS-CoV-2 genomes have been deposited into public sequence databases. This is the result of efforts of both national and regional diagnostic laboratories. In this study, we describe the results of SARS-CoV-2 genomic surveillance from February 2021 to June 2022 at a metropolitan hospital in the United States. We demonstrate that consistent daily sampling is sufficient to track the regional prevalence and emergence of VOCs and recapitulate national trends. Similar sampling efforts should be considered a viable option for local SARS-CoV-2 genomic surveillance at other regional laboratories. IMPORTANCE: In our manuscript, we describe the results of SARS-CoV-2 genomic surveillance from February 2021 to June 2022 at a metropolitan hospital in the United States. We demonstrate that consistent daily sampling is sufficient to track the regional prevalence and emergence of variants of concern (VOCs). Similar sampling efforts should be considered a viable option for local SARS-CoV-2 genomic surveillance at other regional laboratories. While the SARS-CoV-2 pandemic has evolved into a more endemic form, we still believe that additional real-world information about sampling, procedures, and data interpretation is valuable for ongoing as well as future genomic surveillance efforts. Our study should be of substantial interest to clinical virologists

Virus infection is controlled by hematopoietic and stromal cell sensing of murine cytomegalovirus through STING

Recognition of DNA viruses, such as cytomegaloviruses (CMVs), through pattern-recognition receptor (PRR) pathways involving MyD88 or STING constitute a first-line defense against infections mainly through production of type I interferon (IFN-I). However, the role of these pathways in different tissues is incompletely understood, an issue particularly relevant to the CMVs which have broad tissue tropisms. Herein, we contrasted anti-viral effects of MyD88 versus STING in distinct cell types that are infected with murine CMV (MCMV). Bone marrow chimeras revealed STING-mediated MCMV control in hematological cells, similar to MyD88. However, unlike MyD88, STING also contributed to viral control in non-hematological, stromal cells. Infected splenic stromal cells produced IFN-I in a cGAS-STING-dependent and MyD88-independent manner, while we confirmed plasmacytoid dendritic cell IFN-I had inverse requirements. MCMV-induced natural killer cytotoxicity was dependent on MyD88 and STING. Thus, MyD88 and STING contribute to MCMV control in distinct cell types that initiate downstream immune responses

Cis-regulatory evolution of the recently expanded Ly49 gene family

Comparative genomics has revealed the rapid expansion of multiple gene families involved in immunity. Members within each gene family often evolved distinct roles in immunity. However, less is known about the evolution of their epigenome and cis-regulation. Here we systematically profile the epigenome of the recently expanded murine Ly49 gene family that mainly encode either inhibitory or activating surface receptors on natural killer cells. We identify a set of cis-regulatory elements (CREs) for activating Ly49 genes. In addition, we show that in mice, inhibitory and activating Ly49 genes are regulated by two separate sets of proximal CREs, likely resulting from lineage-specific losses of CRE activity. Furthermore, we find that some Ly49 genes are cross-regulated by the CREs of other Ly49 genes, suggesting that the Ly49 family has begun to evolve a concerted cis-regulatory mechanism. Collectively, we demonstrate the different modes of cis-regulatory evolution for a rapidly expanding gene family

ZBTB32 restrains antibody responses to murine cytomegalovirus infections, but not other repetitive challenges

ZBTB32 is a transcription factor that is highly expressed by a subset of memory B cells and restrains the magnitude and duration of recall responses against hapten-protein conjugates. To define physiological contexts in which ZBTB32 acts, we assessed responses by Zbtb32-/- mice or bone marrow chimeras against a panel of chronic and acute challenges. Mixed bone marrow chimeras were established in which all B cells were derived from either Zbtb32-/- mice or control littermates. Chronic infection of Zbtb32-/- chimeras with murine cytomegalovirus led to nearly 20-fold higher antigen-specific IgG2b levels relative to controls by week 9 post-infection, despite similar viral loads. In contrast, IgA responses and specificities in the intestine, where memory B cells are repeatedly stimulated by commensal bacteria, were similar between Zbtb32-/- mice and control littermates. Finally, an infection and heterologous booster vaccination model revealed no role for ZBTB32 in restraining primary or recall antibody responses against influenza viruses. Thus, ZBTB32 does not limit recall responses to a number of physiological acute challenges, but does restrict antibody levels during chronic viral infections that periodically engage memory B cells. This restriction might selectively prevent recall responses against chronic infections from progressively overwhelming other antibody specificities.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01AI99109, R01AI131680, U01AI131349, K08AI04991]; New York Stem Cell FoundationOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Distinct clonal identities of B-ALLs arising after lenolidomide therapy for multiple myeloma

Patients with multiple myeloma (MM) who are treated with lenalidomide rarely develop a secondary B-cell acute lymphoblastic leukemia (B-ALL). The clonal and biological relationship between these sequential malignancies is not yet clear. We identified 17 patients with MM treated with lenalidomide, who subsequently developed B-ALL. Patient samples were evaluated through sequencing, cytogenetics/fluorescence in situ hybridization (FISH), immunohistochemical (IHC) staining, and immunoglobulin heavy chain (IgH) clonality assessment. Samples were assessed for shared mutations and recurrently mutated genes. Through whole exome sequencing and cytogenetics/FISH analysis of 7 paired samples (MM vs matched B-ALL), no mutational overlap between samples was observed. Unique dominant IgH clonotypes between the tumors were observed in 5 paired MM/B-ALL samples. Across all 17 B-ALL samples, 14 (83%) had a TP53 variant detected. Three MM samples with sufficient sequencing depth (\u3e500×) revealed rare cells (average of 0.6% variant allele frequency, or 1.2% of cells) with the same TP53 variant identified in the subsequent B-ALL sample. A lack of mutational overlap between MM and B-ALL samples shows that B-ALL developed as a second malignancy arising from a founding population of cells that likely represented unrelated clonal hematopoiesis caused by a TP53 mutation. The recurrent variants in TP53 in the B-ALL samples suggest a common path for malignant transformation that may be similar to that of TP53-mutant, treatment-related acute myeloid leukemia. The presence of rare cells containing TP53 variants in bone marrow at the initiation of lenalidomide treatment suggests that cellular populations containing TP53 variants expand in the presence of lenalidomide to increase the likelihood of B-ALL development

Dual requirement of cytokine and activation receptor triggering for cytotoxic control of murine cytomegalovirus by NK cells

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570