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The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes. COVID-19 diagnosis is commonly performed by PCR testing, however, serologic methods are more accurate and versatile for monitoring disease burden and epidemiology. Here the authors report a protein microarray with antigens from SARS-CoV-2, SARS-CoV, MERS-CoV as well as common human respiratory viruses.

Evaluation of population‐based COVID‐19 control measures informs strategies to quell the current pandemic and reduce the impact of those yet to come. Effective COVID‐19 control measures may simultaneously reduce the incidence of other acute respiratory infections (ARIs) due to shared transmission modalities. To assess the impact of stay‐at‐home orders and other physical distancing measures on the prevalence of ARI‐related symptoms, we compared symptoms reported by prospective college cohorts enrolled during two consecutive academic years. ARI‐related symptoms declined following campus closure and implementation of stay‐at‐home orders, demonstrating the impact of population‐based physical distancing measures on control of a broad range of respiratory infections.

Objectives/Goals: Mathematical models of airborne virus transmission lack supporting field and clinical data such as viral aerosol emission rates and airborne infectious doses. Here, we aim to measure inhalation exposure to influenza aerosols in a room shared with persons with community-acquired influenza and estimate the infectious dose via inhalation. Methods/Study Population: We recruited healthy volunteer recipients and influenza donors with polymerase chain reaction (PCR)-confirmed community-acquired infection. On admission to a hotel quarantine, recipients provided sera to determine baseline immunity to influenza virus, and donor infections were confirmed by quantitative real-time polymerase chain reaction. Donors and recipients were housed in separate rooms and interacted in an “event room” with controlled ventilation (0.2 – 0.5 air changes/hour) and relative humidity (20–40%). We collected ambient bioaerosol exposure samples using NIOSH BC-251 samplers. Donors provided exhaled breath samples collected by a Gesundheit-II (G-II). We analyzed aerosol samples using dPCR and fluorescent focus assays for influenza A and sera by hemagglutinin inhibition assay (HAI) against donor viruses and vaccine strains. Results/Anticipated Results: Among two cohorts (24b and 24c), we exposed 11 recipients (mean age: 36; 55% female) to 5 donors (mean age: 21; 80% female) infected with influenza A H1N1 or H3N2. Eight G-II and two NIOSH bioaerosol samples (1–4 µm and ≥4 µm) were PCR positive. We cultured virus from one G-II sample. Based on previous literature, we hypothesized that ~50% of immunologically naïve people (HAI Discussion/Significance of Impact: We demonstrated that it is feasible to recruit donors with community-acquired influenza and expose recipients to measurable virus quantities under controlled conditions. However, baseline immunity was high among volunteers. Our work sets the stage for designing studies with increased sample sizes comprising immunologically naïve volunteers.

OBJECTIVES/GOALS: We designed the Biocascade Exhaled Breath Sampler (BEBS) to characterize viral aerosol shedding among individuals with influenza and other respiratory virus infections. We first aimed to test the BEBS on volunteer COVID-19 cases and report the aerodynamic size distribution of exhaled breath aerosol particles carrying SARS-CoV-2 RNA. METHODS/STUDY POPULATION: From June 15 through December 15, 2022, we recruited 27 PCR-confirmed COVID-19 cases from a college campus and the surrounding community to provide 30-minute breath samples into a well-validated Gesundheit-II (G-II) exhaled breath aerosol sampler. Among these individuals, 17 provided an additional exhaled breath sample into the newly designed BEBS. We quantified samples for viral RNA using reverse transcription digital polymerase chain reaction (RT-dPCR) and determined the viral RNA copies collected within two aerosol size fractions (≤5 µm and >5 µm in diameter) from the G-II, and four aerosol size fractions (<1.15 µm, 1.15–3.2 µm, 3.3–8.2 µm, and >8.2 µm) from the BEBS. RESULTS/ANTICIPATED RESULTS: Individuals with a SARS-CoV-2 Omicron BA.4 or BA.5 infection shed virus in aerosols at an average rate of 7.5x103 RNA copies per 30-minute G-II sample, with 78% of the total RNA in aerosols ≤5 µm in diameter. Among the BEBS samples, 10% of the total viral RNA was detected in aerosols <1.15 µm, 43% in 1.15–3.2 µm, 37% in 3.3–8.2 µm, and 10% in the >8.2 µm size fraction. Based on viral RNA loads, our results indicate that exhaled aerosols ≤3.2 µm contribute the majority of SARS-CoV-2 inhalation exposure. DISCUSSION/SIGNIFICANCE: Our data provide additional evidence that respirable aerosols contribute to the spread of SARS-CoV-2. Thus, our data suggest that mitigation measures designed to reduce infectious aerosol inhalation, such as ventilation and the use of air cleaners and respirators, are needed to control the spread.

A previous controlled human influenza transmission trial produced minimal transmission using nasal inoculation of an egg adapted virus. Therefore, we implemented a new trial with naturally infected Donors. We recruited healthy Recipients for four, two-week hotel quarantine cohorts and naturally infected, qRT-PCR confirmed Donors for two cohorts. Five Donors (mean age: 21; 80% female; two H1N1, three H3N2, one for cohort 24b and 4 for 24c, Jan-Feb 2024) exposed Recipients (mean age: 36; 54% female, eight in cohort 24b and 3 in 24c) in a hotel room with limited ventilation but a high air recirculation rate. We collected exhaled breath, ambient and personal bioaerosols, fomite swabs, and sera, and analyzed samples using dPCR and fluorescent focus assays, hemagglutination inhibition (HAI) assay, and enzyme-linked immunosorbent assay (ELISA). Compared with previously studied community-acquired influenza cases, we detected viral RNA (44%) and culturable virus (6%) less frequently and measured fewer viral RNA copies (79 – 8.9 × 10 3 copies/30-min) in Donors’ exhaled fine aerosols. One of 23 surface swab samples was culture positive. At admission, 8 of 11 Recipients had HAI titers ≤10 but 9 of 11 had stronger binding antibody responses than Donors against vaccine strains corresponding to Donor viruses. No Recipient developed influenza-like illness, PCR-positive respiratory samples, or serological evidence of infection. Potential explanations and insights regarding lack of transmission include importance of cough and seasonal variation in viral aerosol shedding by Donors, of potential cross-reactive immunity in middle-aged Recipients with decades of exposure, and of exposure to concentrated exhaled breath plumes limited by rapid air mixing from environmental controls that distributed aerosols evenly. Future trials over multiple seasons, Donors that cough, younger recipients, and environments that preserve normal exhaled breath plumes will be required to observe transmission from naturally infected Donors under controlled conditions and generate new insights into influenza transmission dynamics.

Background Nasally inoculated influenza cases reported milder symptoms and shed lower viral RNA load in exhaled breath aerosols (EBA) than people with classic influenza‐like illness in a previous study. Whether nasally inoculated influenza is representative of mild natural influenza infection is unknown. We extend previous analyses to include a broader range of community‐acquired cases. Methods We previously studied (A) volunteers intranasally inoculated with a dose of 5.5 log10TCID50 of influenza A/Wisconsin/67/2005 (H3N2) and (B) cases with classic influenza‐like illness including fever recruited in 2013. We now add (C) cases from a 2017–2019 surveillance cohort of college dormitory residents and their contacts and (D) cases from a university health center in 2019. All cases had an influenza A(H3) infection. We collected 30‐min EBA samples using a Gesundheit‐II sampler. Results Community‐acquired cases from the surveillance cohort (C) shed more EBA viral RNA and were more symptomatic than the inoculated cases (A) but shed less viral RNA than the symptom‐selected natural cases (B) from 2013, but not (D) from 2019. Despite similar symptoms to the 2013 selected cases (B), the 2019 community‐acquired cases (D) recruited post‐infection had lower fine aerosol viral RNA. Conclusions Nasal inoculation of influenza virus did not reproduce EBA viral RNA shedding or symptoms observed in mild natural infection. Circulating strains of influenza A(H3) may differ year‐to‐year in the extent to which symptomatic cases shed virus into fine aerosols. New models, including possibly aerosol inoculation, are needed to study viral aerosol shedding from the human respiratory tract.

Background The SARS‐CoV‐2 pandemic focused attention on airborne‐inhalation transmission and building ventilation. However, investment in solutions lags because few epidemiologic studies demonstrate a causal effect of ventilation on acute respiratory infection (ARI) transmission. This highlights a need for improved study designs to support causal inference. Methods To investigate the potential for causal inference, we analyzed prospective cohorts residing in a high‐ventilation (HVent, ≥ 5 L/s per person) or a neighboring low‐ventilation (LVent, < 5 L/s per person) college residence hall during two spring semesters (2018 and 2019). Swab samples, analyzed using a PCR panel for respiratory pathogens, were collected based on self‐reported symptoms and contacts. Our analysis focused on roommate pairs where both had been tested within a 2‐week period. Roommate pairs with concordant positive PCR results were categorized as possible transmission events. We used genetic sequencing and phylogenetic analysis to identify probable transmission clusters and events. Results We analyzed data from 368 cohort participants (82 HVent and 286 LVent), including 60 person‐infections, with a trend toward 54% lower ARI risk among students living in HVent versus LVent residence halls. We identified 97 roommate pairs, 64 two‐week intervals where both members were tested, 36 (2 HVent and 34 LVent) intervals with ≥ 1 infection, and four possible transmission events (all LVent). Sequence data available for two of the four events confirmed one probable transmission cluster and one probable transmission event. Conclusions Future college dorm transmission studies should prioritize enrolling roommates rather than individuals, measuring ventilation, and confirming transmission events through whole genome sequencing.

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.