Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments.

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

Staufen1 Regulates Multiple Alternative Splicing Events either Positively or Negatively in DM1 Indicating Its Role as a Disease Modifier.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.

Transcriptomic evidence of erythropoietic adaptation from the International Space Station and from an Earth-based space analog.

Space anemia affects astronauts and the underlying molecular alterations remain unknown. We evaluated the response of erythropoiesis-modulating genes to spaceflight through the analysis of leukocyte transcriptomes from astronauts during long-duration spaceflight and from an Earth model of microgravity. Differential expression analysis identified 50 genes encoding ribosomal proteins with reduced expression at the transition to bed rest and increased during the bed rest phase; a similar trend was observed in astronauts. Additional genes associated with anemia (15 genes), erythrocyte maturation (3 genes), and hemoglobin (6 genes) were down-regulated during bed rest and increased during reambulation. Transcript levels of the erythropoiesis transcription factor GATA1 and nine of most enriched erythrocyte proteins increased at reambulation after bed rest and at return to Earth from space. Dynamic changes of the leukocyte transcriptome composition while in microgravity and during reambulation supported an erythropoietic modulation accompanying the hemolysis of space anemia and of immobility-induced anemia.

Clinical and serological predictors of post COVID-19 condition-findings from a Canadian prospective cohort study.

Introduction: More than 3 years into the pandemic, there is persisting uncertainty as to the etiology, biomarkers, and risk factors of Post COVID-19 Condition (PCC). Serological research data remain a largely untapped resource. Few studies have investigated the potential relationships between post-acute serology and PCC, while accounting for clinical covariates. Methods: We compared clinical and serological predictors among COVID-19 survivors with (n = 102 cases) and without (n = 122 controls) persistent symptoms ≥12 weeks post-infection. We selected four primary serological predictors (anti-nucleocapsid (N), anti-Spike, and anti-receptor binding domain (RBD) IgG titres, and neutralization efficiency), and specified clinical covariates a priori. Results: Similar proportions of PCC-cases (66.7%, n = 68) and infected-controls (71.3%, n = 87) tested positive for anti-N IgG. More cases tested positive for anti-Spike (94.1%, n = 96) and anti-RBD (95.1%, n = 97) IgG, as compared with controls (anti-Spike: 89.3%, n = 109; anti-RBD: 84.4%, n = 103). Similar trends were observed among unvaccinated participants. Effects of IgG titres on PCC status were non-significant in univariate and multivariate analyses. Adjusting for age and sex, PCC-cases were more likely to be efficient neutralizers (OR 2.2, 95% CI 1.11-4.49), and odds was further increased among cases to report deterioration in quality of life (OR 3.4, 95% CI 1.64-7.31). Clinical covariates found to be significantly related to PCC included obesity (OR 2.3, p = 0.02), number of months post COVID-19 (OR 1.1, p < 0.01), allergies (OR 1.8, p = 0.04), and need for medical support (OR 4.1, p < 0.01). Conclusion: Despite past COVID-19 infection, approximately one third of PCC-cases and infected-controls were seronegative for anti-N IgG. Findings suggest higher neutralization efficiency among cases as compared with controls, and that this relationship is stronger among cases with more severe PCC. Cases also required more medical support for COVID-19 symptoms, and described complex, ongoing health sequelae. More data from larger cohorts are needed to substantiate results, permit subgroup analyses of IgG titres, and explore for differences between clusters of PCC symptoms. Future assessment of IgG subtypes may also elucidate new findings.

The effect of dose-interval on antibody response to mRNA COVID-19 vaccines: a prospective cohort study.

Background: Vaccination against COVID-19 is highly effective in preventing severe disease and hospitalization, but primary COVID mRNA vaccination schedules often differed from those recommended by the manufacturers due to supply chain issues. We investigated the impact of delaying the second dose on antibody responses to COVID mRNA-vaccines in a prospective cohort of health-care workers in Quebec. Methods: We recruited participants from the McGill University Health Centre who provided serum or participant-collected dried blood samples (DBS) at 28-days, 3 months, and 6 months post-second dose and at 28-days after a third dose. IgG antibodies to SARS-CoV2 spike (S), the receptor-binding domain (RBD), nucleocapsid (N) and neutralizing antibodies to the ancestral strain were assessed by enzyme-linked immunosorbent assay (ELISA). We examined associations between long (≤89 days) versus short (<89 days) between-dose intervals and antibody response through multivariable mixed-effects models adjusted for age, sex, prior covid infection status, time since vaccine dose, and assay batch. Findings: The cohort included 328 participants who received up to three vaccine doses (>80% Pfizer-BioNTech). Weighted averages of the serum (n=744) and DBS (n=216) cohort results from the multivariable models showed that IgG anti-S was 31% higher (95% CI: 12% to 53%) and IgG anti-RBD was 37% higher (95% CI: 14% to 65%) in the long vs. short interval participants, across all time points. Interpretation: Our study indicates that extending the covid primary series between-dose interval beyond 89 days (approximately 3 months) provides stronger antibody responses than intervals less than 89 days. Our demonstration of a more robust antibody response with a longer between dose interval is reassuring as logistical and supply challenges are navigated in low-resource settings.

Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30.

BACKGROUND: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. METHODS: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. RESULTS: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-ß promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively. CONCLUSIONS: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

The Characteristic Response of the Human Leukocyte Transcriptome to 60 Days of Bed Rest and to Reambulation.

INTRODUCTION: We sought to isolate the microgravity effect of spaceflight from other space stressors by characterizing the leukocytes' transcriptome of participants to a 60-d bed rest study; an Earth model of microgravity. METHODS: Twenty healthy men received a nutritional supplement or not and 10 blood samples were collected throughout three study phases: baseline data collection (BDC) (BDC-12, BDC-11), head-down tilt (HDT) bed rest (HDT1, HDT2, HDT30, HDT60), and reambulation (R1, R2, R12, R30). We measured gene expression through RNA sequencing of leukocytes, applied generalized linear models to assess differential expression followed by enrichment analysis to identify temporal changes (model 1) and to measure the impact of a nutritional supplement (model 2). RESULTS: Baseline transcriptomes included 14,624 protein-coding transcripts and showed both high intraindividual correlations (mean Kendall coefficient, 0.91 ± 0.04) and interindividual homogeneity (0.89 ± 0.03). We identified 2415 differentially expressed protein-coding transcripts grouping into six clusters (C1-C6). At phase transitions, clusters showed either a decrease-then-increase (C3 and C5) or an increase-then-decrease (C1, C2, C6) pattern. All six clusters converged toward average expression at HDT30 and HDT60. Gene ontology terms at baseline related to immune functions while in bed rest and reambulation related to sequestration of ions, immune response, cellular stress, and mineralization. The nutritional intervention had no effect. CONCLUSIONS: The temporal profiles of leukocytes' transcriptomes emphasized the dynamic nature of gene expression occurring during and after bed rest. Enriched biological processes among the differentially expressed genes included immune related and unrelated responses. The convergence toward no differential expression at days 30 and 60 of bed rest suggests a hypometabolic state. Current findings can guide future work on the complex responses and adaptation mechanisms to microgravity.

The transcriptome response of astronaut leukocytes to long missions aboard the International Space Station reveals immune modulation.

Introduction: Spaceflight leads to the deconditioning of multiple body systems including the immune system. We sought to characterize the molecular response involved by capturing changes in leukocyte transcriptomes from astronauts transitioning to and from long-duration spaceflight. Methods: Fourteen male and female astronauts with ~6-month- long missions aboard the International Space Station (ISS) had 10 blood samples collected throughout the three phases of the study: one pre-flight (PF), four in-flight (IF) while onboard the ISS, and five upon return to Earth (R). We measured gene expression through RNA sequencing of leukocytes and applied generalized linear modeling to assess differential expression across all 10 time points followed by the analysis of selected time points and functional enrichment of changing genes to identify shifts in biological processes. Results: Our temporal analysis identified 276 differentially expressed transcripts grouped into two clusters (C) showing opposite profiles of expression with transitions to and from spaceflight: (C1) decrease-then-increase and (C2) increase-then-decrease. Both clusters converged toward average expression between ~2 and ~6 months in space. Further analysis of spaceflight transitions identified the decrease-then-increase pattern with most changes: 112 downregulated genes between PF and early spaceflight and 135 upregulated genes between late IF and R. Interestingly, 100 genes were both downregulated when reaching space and upregulated when landing on Earth. Functional enrichment at the transition to space related to immune suppression increased cell housekeeping functions and reduced cell proliferation. In contrast, egress to Earth is related to immune reactivation. Conclusion: The leukocytes' transcriptome changes describe rapid adaptations in response to entering space followed by opposite changes upon returning to Earth. These results shed light on immune modulation in space and highlight the major adaptive changes in cellular activity engaged to adapt to extreme environments.

Cross-sectional Characterization of SARS-CoV-2 Antibody Levels and Decay Rates Following Infection of Unvaccinated Elderly Individuals.

Background: SARS-CoV-2 infections have disproportionally burdened elderly populations with excessive mortality. While several contributing factors exists, questions remain about the quality and duration of humoral antibody-mediated responses resulting from infections in unvaccinated elderly individuals. Methods: Residual serum/plasma samples were collected from individuals undergoing routine SARS-CoV-2 polymerase chain reaction testing in a community laboratory in Canada. The samples were collected in 2020, before vaccines became available. IgG, IgA, and IgM antibodies against SARS-CoV-2 nucleocapsid, trimeric spike, and its receptor-binding domain were quantified via a high-throughput chemiluminescent enzyme-linked immunosorbent assay. Neutralization efficiency was also quantified through a surrogate high-throughput protein-based neutralization assay. Results: This study analyzed SARS-CoV-2 antibody levels in a large cross-sectional cohort (N = 739), enriched for elderly individuals (median age, 82 years; 75% >65 years old), where 72% of samples tested positive for SARS-CoV-2 by polymerase chain reaction. The age group ≥90 years had higher levels of antibodies than that <65 years. Neutralization efficiency showed an age-dependent trend, where older persons had higher levels of neutralizing antibodies. Antibodies targeting the nucleocapsid had the fastest decline. IgG antibodies targeting the receptor-binding domain remained stable over time, potentially explaining the lack of neutralization decay observed in this cohort. Conclusions: Despite older individuals having the highest levels of antibodies postinfection, they are the cohort in which antibody decay was the fastest. Until a better understanding of correlates of protection is acquired, along with the protective role of nonneutralizing antibodies, booster vaccinations remain important in this demographic.

Cohort profile: investigating SARS-CoV-2 infection and the health and psychosocial impact of the COVID-19 pandemic in the Canadian CHILD Cohort.

The coronavirus disease 2019 (COVID-19) pandemic has affected all Canadian families, with some impacted differently than others. Our study aims to: (1) determine the prevalence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among Canadian families, (2) identify predictors of infection susceptibility and severity of SARS-CoV-2, and (3) identify health and psychosocial impacts of the COVID-19 pandemic. This study builds upon the CHILD Cohort Study, an ongoing multi-ethnic general population prospective cohort consisting of 3,454 Canadian families with children born in Vancouver, Edmonton, Manitoba, and Toronto between 2009 and 2012. During the pandemic, CHILD households were invited to participate in the CHILD COVID-19 Add-On Study involving: (1) brief biweekly surveys about COVID-19 symptoms and testing; (2) quarterly questionnaires assessing COVID-19 exposure and testing, vaccination status, physical and mental health, and pandemic-driven life changes; and (3) in-home biological sampling kits to collect blood and stool. In total, 1,462 households (5,378 participants) consented to the CHILD COVID-19 Add-On Study: 2,803 children (mean±standard deviation [SD], 9.0±2.7 years; range, 0-17 years) and 2,576 adults (mean±SD, 43.0±6.5 years; range, 18-85 years). We will leverage the wealth of pre-pandemic CHILD data to identify risk and resilience factors for susceptibility and severity to the direct and indirect pandemic effects. Our short-term findings will inform key stakeholders and knowledge users to shape current and future pandemic responses. Additionally, this study provides a unique resource to study the long-term impacts of the pandemic as the CHILD Cohort Study continues.

Tomato RNA polymerase II interacts with the rod-like conformation of the left terminal domain of the potato spindle tuber viroid positive RNA genome.

Potato spindle tuber viroid (PSTVd) is a small, single-stranded, circular, non-coding RNA pathogen. Host DNA-dependent RNA polymerase II (RNAP II) was proposed to be critical for its replication, but no interaction site for RNAP II on the PSTVd RNA genome was identified. Using a co-immunoprecipitation strategy involving a mAb specific for the conserved heptapeptide (i.e. YSPTSPS) located at the carboxy-terminal domain of the largest subunit of RNAP II, we established the interaction of tomato RNAP II with PSTVd RNA and showed that RNAP II associates with the left terminal domain of PSTVd (+) RNA. RNAP II did not interact with any of several PSTVd (-) RNAs tested. Deletion and site-directed mutagenesis of a shortened model PSTVd (+) RNA fragment were used to identify the role of specific nucleotides and structural motifs in this interaction. Our results provide evidence for the interaction of a RNAP II complex from a natural host with the rod-like conformation of the left terminal domain of PSTVd (+) RNA.

Hepatitis C virus: the role of molecular mimicry in response to interferon treatment.

Chronic hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In order for HCV to persist, the virus must escape immune recognition or inhibit the host immune response. The NS5A protein contains the interferon sensitivity-determining region (ISDR) and is able to repress dsRNA-dependent protein kinase (PKR) thus influencing the response to interferon (IFN) therapy. Patients who respond to IFN therapy have stronger antibody reactivity against the NS5A compared to IFN non-responders. Therefore, given the possible role for the ISDR in IFN resistance and differential antibody reactivity, it is possible that variation in ISDR may be involved in viral immune escape and development of persistent HCV infection employing aspects of host mimicry. In this study, pre-treatment samples obtained from HCV infected patients were used to investigate the effect of different NS5A ISDR variants on the IFN antiviral response and their involvement in immune evasion. The NS5A was identified as a homologue of the variable region of immunoglobulins (Ig). The IFN resistant genotypes had higher levels of similarity to Ig compared to IFN sensitive genotypes. Expression of NS5A-6003 (HCV genotype 1b) and NS5A-6074 (HCV genotype 2a) was able to rescue vesicular stomatitis virus (VSV) from IFN inhibition and restore luciferase activity. A correlation between Ig-like NS5A structure and also antibody response with the outcome of IFN treatment was observed.

Role of RNA Polymerase II Promoter-Proximal Pausing in Viral Transcription.

The promoter-proximal pause induced by the binding of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) to RNAP II is a key step in the regulation of metazoan gene expression. It helps maintain a permissive chromatin landscape and ensures a quick transcriptional response from stimulus-responsive pathways such as the innate immune response. It is also involved in the biology of several RNA viruses such as the human immunodeficiency virus (HIV), the influenza A virus (IAV) and the hepatitis delta virus (HDV). HIV uses the pause as one of its mechanisms to enter and maintain latency, leading to the creation of viral reservoirs resistant to antiretrovirals. IAV, on the other hand, uses the pause to acquire the capped primers necessary to initiate viral transcription through cap-snatching. Finally, the HDV RNA genome is transcribed directly by RNAP II and requires the small hepatitis delta antigen to displace NELF from the polymerase and overcome the transcriptional block caused by RNAP II promoter-proximal pausing. In this review, we will discuss the RNAP II promoter-proximal pause and the roles it plays in the life cycle of RNA viruses such as HIV, IAV and HDV.

Effect of rehabilitation on biologic and transcriptomic responses after hospital-acquired deconditioning: a prospective longitudinal feasibility study.

PURPOSE: The objective of this study is to explore the transcriptomic and biologic variables characterizing the longitudinal rehabilitation intervention of patients with hospital-acquired deconditioning (HAD). METHODS: This prospective clinical trial recruited HAD patients (n = 10) who spent ≥3 weeks hospitalized and then received inpatient rehabilitation. Functional improvement was measured using the Functional Independence Measure (FIM). Transcriptomic and biological variables were recorded at rehabilitation admission and 1, 2, 4, and 6 weeks post-admission. RNA sequencing studied the temporal changes of gene expression in leukocytes. Between-subject transcriptome comparisons were performed using principle component analysis. Within-subject changes in gene expression were analyzed using a gene ontology hierarchical clustering to identify common biological terms. Heart rate, weight, albumin, creatinine, and complete blood counts were analyzed. RESULTS: Patients average age was 50.6 ± 7.2, FIM increased during inpatient rehabilitation (p = 0.01), weight increased (p = 0.01), lymphocytes decreased (p = 0.05), neutrophil increased (0.03) and red cell distribution width decreased (p = 0.05). The temporal profiles of gene expression revealed within-patient homogeneity and between-patients heterogeneity. The biological terms "bone morphogenesis" and "muscle cell development" were the most significantly enriched differentially expressed genes. CONCLUSION: Transcriptomic and biologic markers paralleled the functional improvements of HAD patients during inpatient rehabilitation. Transcriptomic analyses were consistent with the cohort heterogeneity. Enrichment of the biological pathways bone morphogenesis and muscle cell development constituted evidence at the gene expression level of the effect of rehabilitation. Larger studies of various rehabilitation patient groups may increase gene expression profile homogeneity. Objective transcriptomic and biologic markers have the potential to improve the rehabilitation of HAD patients.IMPLICATIONS FOR REHABILITATIONNovel gene expression methods are increasingly being integrated into clinical practice and may apply to rehabilitation.Patients with hospital-acquired deconditioning (HAD) enriched gene expression of pathways targeted by inpatient rehabilitation such as bone morphogenesis and muscle cell development.The gene expression paralleled functional improvement of HAD patients.These data demonstrated the feasibility of molecular methods to identify markers of rehabilitation success in HAD patients.

Antibody Seronegativity in COVID-19 RT-PCR-Positive Children.

This substudy of a prospective case-ascertained household transmission study investigated severe acute respiratory syndrome coronavirus 2 reverse transcription polymerase chain reaction-positive individuals without antibody development and factors associated with nonseroconversion. Approximately 1 of 8 individuals with coronavirus disease 2019 did not seroconvert. Children, particularly the youngest, were approximately half as likely to seroconvert compared with adults. Apart from the absence of fever/chills, individual symptoms did not strongly predict nonseroconversion.

Household transmission of SARS-CoV-2 from unvaccinated asymptomatic and symptomatic household members with confirmed SARS-CoV-2 infection: an antibody-surveillance study.

BACKGROUND: Household transmission contributes to SARS-CoV-2 spread, but the role of children in transmission is unclear. We conducted a study that included symptomatic and asymptomatic children and adults exposed to SARS-CoV-2 in their households with the objective of determining how SARS-CoV-2 is transmitted within households. METHODS: In this case-ascertained antibody-surveillance study, we enrolled households in Ottawa, Ontario, in which at least 1 household member had tested positive for SARS-CoV-2 on reverse transcription polymerase chain reaction testing. The enrolment period was September 2020 to March 2021. Potentially eligible participants were identified if they had tested positive for SARS-CoV-2 at an academic emergency department or affiliated testing centre; people who learned about the study through the media could also self-identify for participation. At least 2 participants were required for a household to be eligible for study participation, and at least 1 enrolled participant per household had to be a child (age < 18 yr). Enzyme-linked immunosorbent assays were used to evaluate SARS-CoV-2-specific IgA, IgM and IgG against the spike-trimer and nucleocapsid protein. The primary outcome was household secondary attack rate, defined as the proportion of household contacts positive for SARS-CoV-2 antibody among the total number of household contacts participating in the study. We performed descriptive statistics at both the individual and household levels. To estimate and compare outcomes between patient subgroups, and to examine predictors of household transmission, we fitted a series of multivariable logistic regression with robust standard errors to account for clustering of individuals within households. RESULTS: We enrolled 695 participants from 180 households: 180 index participants (74 children, 106 adults) and 515 of their household contacts (266 children, 249 adults). A total of 487 household contacts (94.6%) (246 children, 241 adults) had SARS-CoV-2 antibody testing, of whom 239 had a positive result (secondary attack rate 49.1%, 95% confidence interval [CI] 42.9%-55.3%). Eighty-eight (36.8%, 95% CI 29.3%-43.2%) of the 239 were asymptomatic; asymptomatic rates were similar for children (51/130 [39.2%, 95% CI 30.7%-48.5%]) and adults (37/115 [32.2%, 95% CI 24.2%-41.4%]) (odds ratio [OR] 1.3, 95% CI 0.8-2.1). Adults were more likely than children to transmit SARS-CoV-2 (OR 2.2, 95% CI 1.3-3.6). The odds of transmission from asymptomatic (OR 0.6, 95% CI 0.2-1.4) versus symptomatic (OR 0.9, 95% CI 0.6-1.4) index participants to household contacts was uncertain. Predictors of household transmission included household density (number of people per bedroom), relationship to index participant and number of cases in the household. INTERPRETATION: The rate of SARS-CoV-2 transmission within households was nearly 50% during the study period, and children were an important source of spread. The findings suggest that children are an important driver of the COVID-19 pandemic; this should inform public health policy.

Seropositivity and risk factors for SARS-CoV-2 infection in a South Asian community in Ontario: a cross-sectional analysis of a prospective cohort study.

BACKGROUND: Early in the COVID-19 pandemic, the South Asian community in the Greater Toronto Area (GTA) was identified as having risk factors for exposure and specific barriers to accessing testing and reliable health information, rendering them particularly vulnerable to SARS-CoV-2 infection. We sought to investigate the burden of SARS-CoV-2 infection among South Asian people in the GTA, and to characterize the demographic characteristics, risk perceptions and trusted sources of health information in this group. METHODS: We conducted a cross-sectional analysis from the baseline assessment of participants in a prospective cohort study. Participants from the GTA were enrolled from Apr. 14 to July 28, 2021. Seropositivity for antispike and antinucleocapsid antibodies was determined from dried blood spots, and estimates of seropositivity were age and sex standardized to the South Asian population in Ontario. Demographic characteristics, risk perceptions and sources of COVID-19 information were collected via questionnaire and reported descriptively. RESULTS: Among the 916 South Asian participants enrolled (mean age 41 yr), the age- and sex-standardized seropositivity was 23.6% (95% confidence interval 20.8%-26.4%). Of the 693 respondents to the questionnaire, 228 (32.9%) identified as essential workers, and 125 (19.1%) reported living in a multigenerational household. A total of 288 (49.4%) perceived that they were at high COVID-19 risk owing to their geographic location, and 149 (34.3%) owing to their type of employment. The top 3 most trusted sources of information related to COVID-19 included health care providers and public health, traditional media sources and social media. INTERPRETATION: By the third wave of the COVID-19 pandemic, about one-quarter of a sample of South Asian individuals in Ontario had serologic evidence of prior SARS-CoV-2 infection. Insight into factors that put certain populations at risk can help future pandemic planning and disease control efforts.

Real-world serological responses to extended-interval and heterologous COVID-19 mRNA vaccination in frail, older people (UNCoVER): an interim report from a prospective observational cohort study.

BACKGROUND: The use of COVID-19 vaccines has been prioritised to protect the most vulnerable-notably, older people. Because of fluctuations in vaccine availability, strategies such as delayed second dose and heterologous prime-boost have been used. However, the effectiveness of these strategies in frail, older people are unknown. We aimed to assess the antigenicity of mRNA-based COVID-19 vaccines in frail, older people in a real-world setting, with a rationed interval dosing of 16 weeks between the prime and boost doses. METHODS: This prospective observational cohort study was done across 12 long-term care facilities of the Montréal Centre-Sud - Integrated University Health and Social Services Centre in Montréal, Québec, Canada. Under a rationing strategy mandated by the provincial government, adults aged 65 years and older residing in long-term care facilities in Québec, Canada, with or without previously documented SARS-CoV-2 infection, were administered homologous or heterologous mRNA vaccines, with an extended 16-week interval between doses. All older residents in participating long-term care facilities who received two vaccine doses were eligible for inclusion in this study. Participants were enrolled from Dec 31, 2020, to Feb 16, 2021, and data were collected up to June 9, 2021. Clinical data and blood samples were serially collected from participants at the following timepoints: at baseline, before the first dose; 4 weeks after the first dose; 6-10 weeks after the first dose; 16 weeks after the first dose, up to 2 days before administration of the second dose; and 4 weeks after the second dose. Sera were tested for SARS-CoV-2-specific IgG antibodies (to the trimeric spike protein, the receptor-binding domain [RBD] of the spike protein, and the nucleocapsid protein) by automated chemiluminescent ELISA. Two cohorts were used in this study: a discovery cohort, for which blood samples were collected before administration of the first vaccine dose and longitudinally thereafter; and a confirmatory cohort, for which blood samples were only collected from 4 weeks after the prime dose. Analyses were done in the discovery cohort, with validation in the confirmatory cohort, when applicable. FINDINGS: The total study sample consisted of 185 participants. 65 participants received two doses of mRNA-1273 (Spikevax; Moderna), 36 received two doses of BNT162b2 (Comirnaty; Pfizer-BioNTech), and 84 received mRNA-1273 followed by BNT162b2. In the discovery cohort, after a significant increase in anti-RBD and anti-spike IgG concentrations 4 weeks after the prime dose (from 4·86 log binding antibody units [BAU]/mL to 8·53 log BAU/mL for anti-RBD IgG and from 5·21 log BAU/mL to 8·05 log BAU/mL for anti-spike IgG), there was a significant decline in anti-RBD and anti-spike IgG concentrations until the boost dose (7·10 log BAU/mL for anti-RBD IgG and 7·60 log BAU/mL for anti-spike IgG), followed by an increase 4 weeks later for both vaccines (9·58 log BAU/mL for anti-RBD IgG and 9·23 log BAU/mL for anti-spike IgG). SARS-CoV-2-naive individuals showed lower antibody responses than previously infected individuals at all timepoints tested up to 16 weeks after the prime dose, but achieved similar antibody responses to previously infected participants by 4 weeks after the second dose. Individuals primed with the BNT162b2 vaccine showed a larger decrease in mean anti-RBD and anti-spike IgG concentrations with a 16-week interval between doses (from 8·12 log BAU/mL to 4·25 log BAU/mL for anti-RBD IgG responses and from 8·18 log BAU/mL to 6·66 log BAU/mL for anti-spike IgG responses) than did those who received the mRNA-1273 vaccine (two doses of mRNA-1273: from 8·06 log BAU/mL to 7·49 log BAU/mL for anti-RBD IgG responses and from 6·82 log BAU/mL to 7·56 log BAU/mL for anti-spike IgG responses; mRNA-1273 followed by BNT162b2: from 8·83 log BAU/mL to 7·95 log BAU/mL for anti-RBD IgG responses and from 8·50 log BAU/mL to 7·97 log BAU/mL for anti-spike IgG responses). No differences in antibody responses 4 weeks after the second dose were noted between the two vaccines, in either homologous or heterologous combinations. INTERPRETATION: Interim results of this ongoing longitudinal study show that among frail, older people, previous SARS-CoV-2 infection and the type of mRNA vaccine influenced antibody responses when used with a 16-week interval between doses. In these cohorts of frail, older individuals with a similar age and comorbidity distribution, we found that serological responses were similar and clinically equivalent between the discovery and confirmatory cohorts. Homologous and heterologous use of mRNA vaccines was not associated with significant differences in antibody responses 4 weeks following the second dose, supporting their interchangeability. FUNDING: Public Health Agency of Canada, Vaccine Surveillance Reference Group; and the COVID-19 Immunity Task Force. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

A scalable serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination.

Objectives: Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. Methods: We developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results: Our single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions: Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter-laboratory data comparison and aggregation.

Identification of a High-Frequency Intrahost SARS-CoV-2 Spike Variant with Enhanced Cytopathic and Fusogenic Effects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intrahost viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and will not make significant contributions to variations within and between infected hosts. Herein, we profiled the intrasample genetic diversity of SARS-CoV-2 variants, also known as quasispecies, using high-throughput sequencing data sets from 15,289 infected individuals and infected cell lines. Despite high mutational background, we identified recurrent intragenetic variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral spike (S) protein. Strikingly, we observed a high frequency of C→A missense mutations resulting in the S protein lacking the last 20 amino acids (SΔ20). We found that this truncated S protein undergoes increased processing and increased syncytium formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intrahost disease cytopathic effects. IMPORTANCE The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection. Many SARS-CoV-2 variants have been recently described and show phenotypic selection toward more infectious viruses. Our study describes another type of variant that does not contribute to interhost heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the spike protein removes an important endoplasmic reticulum (ER) retention signal, which consequently results in a spike variant that easily travels through the Golgi complex toward the plasma membrane in a preactivated conformation, leading to increased syncytium formation.