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.

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.retrovirus | RNA genome | Gag-RNA complex | virus assembly | RNA-binding protein

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.

Background SARS-CoV-2 epidemiology implicates airborne transmission; aerosol infectiousness and impacts of masks and variants on aerosol shedding are not well understood. Methods We recruited COVID-19 cases to give blood, saliva, mid-turbinate and fomite (phone) swabs, and 30-minute breath samples while vocalizing into a Gesundheit-II, with and without masks at up to two visits two days apart. We quantified and sequenced viral RNA, cultured virus, and assayed sera for anti-spike and anti-receptor binding domain antibodies. Results We enrolled 49 seronegative cases (mean days post onset 3.8 ±2.1), May 2020 through April 2021. We detected SARS-CoV-2 RNA in 45% of fine (≤5 µm), 31% of coarse (>5 µm) aerosols, and 65% of fomite samples overall and in all samples from four alpha-variant cases. Masks reduced viral RNA by 48% (95% confidence interval [CI], 3 to 72%) in fine and by 77% (95% CI, 51 to 89%) in coarse aerosols; cloth and surgical masks were not significantly different. The alpha variant was associated with a 43-fold (95% CI, 6.6 to 280-fold) increase in fine aerosol viral RNA, compared with earlier viruses, that remained a significant 18-fold (95% CI, 3.4 to 92-fold) increase adjusting for viral RNA in saliva, swabs, and other potential confounders. Two fine aerosol samples, collected while participants wore masks, were culture-positive. Conclusion SARS-CoV-2 is evolving toward more efficient aerosol generation and loose-fitting masks provide significant but only modest source control. Therefore, until vaccination rates are very high, continued layered controls and tight-fitting masks and respirators will be necessary.

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 complete discrimination of these two groups. 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.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.

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A B S T R A C TStrategies to protect building occupants from the risk of acute respiratory infection (ARI) need to consider ventilation for its ability to dilute and remove indoor bioaerosols. Prior studies have described an association of increased self-reported colds and influenza-like symptoms with low ventilation but have not combined rigorous characterization of ventilation with assessment of laboratory confirmed infections. We report a study designed to fill this gap. We followed laboratory confirmed ARI rates and measured CO 2 concentrations for four months during the winter-spring of 2018 in two campus residence halls: (1) a high ventilation building (HVB) with a dedicated outdoor air system that supplies 100% of outside air to each dormitory room, and (2) a low ventilation building (LVB) that relies on infiltration as ventilation. We enrolled 11 volunteers for a total of 522 person-days in the HVB and 109 volunteers for 6069 person-days in the LVB, and tested upper-respiratory swabs from symptomatic cases and their close contacts for the presence of 44 pathogens using a molecular assay. We observed one ARI case in the HVB (0.70/person-year) and 47 in the LVB (2.83/person-year). Simultaneously, 154 CO 2 sensors distributed primarily in the dormitory rooms collected 668,390 useful data points from over 1 million recorded data points. Average and standard deviation of CO 2 concentrations were 1230 ppm and 408 ppm in the HVB, and 1492 ppm and 837 ppm in the LVB, respectively. Importantly, this study developed and calibrated multi-zone models for the HVB with 229 zones and 983 airflow paths, and for the LVB with 529 zones and 1836 airflow paths by using a subset of CO 2 data for model calibration. The models were used to calculate ventilation rates in the two buildings and potential for viral aerosol migration between rooms in the LVB. With doors and windows closed, the average ventilation rate was 12 L/s in the HVB dormitory rooms and 4 L/s in the LVB dormitory rooms. As a result, residents had on average 6.6 L/(s person) of outside air in the HVB and 2.3 L/(s person) in the LVB. LVB rooms located at the leeward side of the building had smaller average ventilation rates, as well as a somewhat higher ARI incidence rate and average CO 2 concentrations when compared to those values in the rooms located at the windward side of the building. Average ventilation rates in twenty LVB dormitory rooms increased from 2.3 L/s to 7.5 L/s by opening windows, 3.6 L/s by opening doors, and 8.8 L/s by opening both windows and doors. Therefore, opening both windows and doors in the LVB dormitory rooms can increase ventilation rates to the levels comparable to those in the HVB. But it can also have a negative effect on thermal comfort due to low outdoor temperatures. Simulation results identified an aerobiologic pathway from a room occupied by an index case of influenza A to a room occupied by a possible secondary case.