Air Change Rate and SARS-CoV-2 Exposure in Hospitals and Residences: A Meta-Analysis.

As SARS-CoV-2 swept across the globe, increased ventilation and implementation of air cleaning were emphasized by the US CDC and WHO as important strategies to reduce the risk of inhalation exposure to the virus. To assess whether higher ventilation and air cleaning rates lead to lower exposure risk to SARS-CoV-2, 1274 manuscripts published between April 2020 and September 2022 were screened using key words "airborne SARS-CoV-2 or "SARS-CoV-2 aerosol". Ninety-three studies involved air sampling at locations with known sources (hospitals and residences) were selected and associated data were compiled. Two metrics were used to assess exposure risk: SARS-CoV-2 concentration and SARS-CoV-2 detection rate in air samples. Locations were categorized by type (hospital or residence) and proximity to the sampling location housing the isolated/quarantined patient (primary or secondary). The results showed that hospital wards had lower airborne virus concentrations than residential isolation rooms. A negative correlation was found between airborne virus concentrations in primary-occupancy areas and air changes per hour (ACH). In hospital settings, sample positivity rates were significantly reduced in secondary-occupancy areas compared to primary-occupancy areas, but they were similar across sampling locations in residential settings. ACH and sample positivity rates were negatively correlated, though the effect was diminished when ACH values exceeded 8. While limitations associated with diverse sampling protocols exist, data considered by this meta-analysis support the notion that higher ACH may reduce exposure risks to the virus in ambient air.

The BioCascade Impactor: A novel device for direct collection of size-fractionated bioaerosols into liquid medium.

The ability to collect size-fractionated airborne particles that contain viable bacteria and fungi directly into liquid medium while also maintaining their viability is critical for assessing exposure risks. In this study, we present the BioCascade impactor, a novel device designed to collect airborne particles into liquid based on their aerodynamic diameter in three sequential stages (>9.74 µm, 3.94-9.74 µm, and 1.38-3.94 µm when operated at 8.5 L/min). Aerosol samples containing microorganisms - either Saccharomyces kudriavzevii or Micrococcus luteus, were used to evaluate the performance of the BioCascade (BC) paired with either the VIable Virus Aerosol Sampler (VIVAS) or a gelatin filter (GF) as stage 4 to collect particles <1.38 µm. Stages 2 and 3 collected the largest fractions of viable S. kudriavzevii when paired with VIVAS (0.468) and GF (0.519), respectively. Stage 3 collected the largest fraction of viable M. luteus particles in both BC+VIVAS (0.791) and BC+GF (0.950) configurations. The distribution function of viable microorganisms was consistent with the size distributions measured by the Aerodynamic Particle Sizer. Testing with both bioaerosol species confirmed no internal loss and no re-aerosolization occurred within the BC. Irrespective of the bioaerosol tested, stages 1, 3 and 4 maintained ≥80% of viability, while stage 2 maintained only 37% and 73% of viable S. kudriavzevii and M. luteus, respectively. The low viability that occurred in stage 2 warrants further investigation. Our work shows that the BC can efficiently size-classify and collect bioaerosols without re-aerosolization and effectively maintain the viability of collected microorganisms.

The BioCascade-VIVAS system for collection and delivery of virus-laden size-fractionated airborne particles.

The size of virus-laden particles determines whether aerosol or droplet transmission is dominant in the airborne transmission of pathogens. Determining dominant transmission pathways is critical to implementing effective exposure risk mitigation strategies. The aerobiology discipline greatly needs an air sampling system that can collect virus-laden airborne particles, separate them by particle diameter, and deliver them directly onto host cells without inactivating virus or killing cells. We report the use of a testing system that combines a BioAerosol Nebulizing Generator (BANG) to aerosolize Human coronavirus (HCoV)-OC43 (OC43) and an integrated air sampling system comprised of a BioCascade impactor (BC) and Viable Virus Aerosol Sampler (VIVAS), together referred to as BC-VIVAS, to deliver the aerosolized virus directly onto Vero E6 cells. Particles were collected into four stages according to their aerodynamic diameter (Stage 1: >9.43 µm, Stage 2: 3.81-9.43 µm, Stage 3: 1.41-3.81 µm and Stage 4: <1.41 µm). OC43 was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses of samples from all BC-VIVAS stages. The calculated OC43 genome equivalent counts per cm3 of air ranged from 0.34±0.09 to 70.28±12.56, with the highest concentrations in stage 3 (1.41-3.81 µm) and stage 4 (<1.41 µm). Virus-induced cytopathic effects appeared only in cells exposed to particles collected in stages 3 and 4, demonstrating the presence of viable OC43 in particles <3.81 µm. This study demonstrates the dual utility of the BC-VIVAS as particle size-fractionating air sampler and a direct exposure system for aerosolized viruses. Such utility may help minimize conventional post-collection sample processing time required to assess the viability of airborne viruses and increase the understanding about transmission pathways for airborne pathogens.

Coding-complete genomic sequence of a rhinovirus C-32 in a human nasal swab sample that tested false positive in a SARS-CoV-2 antigen test.

Rhinovirus-A was previously shown to cause false-positive results in a Japanese SARS-CoV-2 antigen test. We report that a false-positive result was obtained in a specimen with rhinovirus C-32 that had been tested using an American SARS-CoV-2 antigen test.

Inactivation of SARS CoV-2 on porous and nonporous surfaces by compact portable plasma reactor.

We report the inactivation of SARS CoV-2 and its surrogate-Human coronavirus OC43 (HCoV-OC43), on representative porous (KN95 mask material) and nonporous materials (aluminum and polycarbonate) using a Compact Portable Plasma Reactor (CPPR). The CPPR is a compact (48 cm3), lightweight, portable and scalable device that forms Dielectric Barrier Discharge which generates ozone using surrounding atmosphere as input gas, eliminating the need of source gas tanks. Iterative CPPR exposure time experiments were performed on inoculated material samples in 3 operating volumes. Minimum CPPR exposure times of 5-15 min resulted in 4-5 log reduction of SARS CoV-2 and its surrogate on representative material samples. Ozone concentration and CPPR energy requirements for virus inactivation are documented. Difference in disinfection requirements in porous and non-porous material samples is discussed along with initial scaling studies using the CPPR in 3 operating volumes. The results of this feasibility study, along with existing literature on ozone and CPPR decontamination, show the potential of the CPPR as a powerful technology to reduce fomite transmission of enveloped respiratory virus-induced infectious diseases such as COVID-19. The CPPR can overcome limitations of high temperatures, long exposure times, bulky equipment, and toxic residuals related to conventional decontamination technologies.

A novel bluetongue virus serotype 2 strain isolated from a farmed Florida white-tailed deer (Odocoileus virginianus) arose from reassortment of gene segments derived from co-circulating serotypes in the Southeastern USA.

Bluetongue disease is a reportable animal disease that affects wild and farmed ruminants, including white-tailed deer (WTD). This report documents the clinical findings, ancillary diagnostics, and genomic characterization of a novel reassortant bluetongue virus serotype 2 (BTV-2) strain isolated from a dead Florida farmed WTD in 2022. Our analyses support that this BTV-2 strain likely stemmed from the acquisition of genome segments from co-circulating BTV strains in Florida and Louisiana. In addition, our analyses also indicate that genetically uncharacterized BTV strains may be circulating in the Southeastern USA; however, the identity and reassortant status of these BTV strains cannot be determined based on the VP2 and VP5 genome sequences. Hence, continued surveillance based on complete genome characterization is needed to understand the genetic diversity of BTV strains in this region and the potential threat they may pose to the health of deer and other ruminants.

Detection and isolation of infectious SARS-CoV-2 omicron subvariants collected from residential settings.

Airborne transmission of infectious (viable) SARS-CoV-2 is increasingly accepted as the primary manner by which the virus is spread from person to person. Risk of exposure to airborne virus is higher in enclosed and poorly ventilated spaces. We present a study focused on air sampling within residences occupied by individuals with COVID-19. Air samplers (BioSpot-VIVAS, VIVAS, and BC-251) were positioned in primary- and secondary-occupancy regions in seven homes. Swab samples were collected from high-touch surfaces. Isolation of SARS-CoV-2 was attempted for samples with virus detectable by RT-qPCR. Viable virus was quantified by plaque assay, and complete virus genome sequences were obtained for selected samples from each sampling day. SARS-CoV-2 was detected in 24 of 125 samples (19.2%) by RT-qPCR and isolated from 14 (11.2%) in cell cultures. It was detected in 80.9% (17/21) and cultured from 61.9% (13/21) of air samples collected using water condensation samplers, compared to swab samples which had a RT-qPCR detection rate of 10.5% (4/38) and virus isolation rate of 2.63% (1/38). No statistically significant differences existed in the likelihood of virus detection by RT-qPCR or amount of infectious virus in the air between areas of primary and secondary occupancy within residences. Our work provides information about the presence of SARS-CoV-2 in the air within homes of individuals with COVID-19. Information herein can help individuals make informed decisions about personal exposure risks when sharing indoor spaces with infected individuals isolating at home and further inform health departments and the public about SARS-CoV-2 exposure risks within residences.

Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism.

Introduction: SARS-CoV-2 subverts host cell processes to facilitate rapid replication and dissemination, and this leads to pathological inflammation. Methods: We used niclosamide (NIC), a poorly soluble anti-helminth drug identified initially for repurposed treatment of COVID-19, which activates the cells' autophagic and lipophagic processes as a chemical probe to determine if it can modulate the host cell's total lipid profile that would otherwise be either amplified or reduced during SARS-CoV-2 infection. Results: Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of SARS-CoV-2 infected Vero E6 cells, especially with triglycerides, which were elevated early during virus replication, but decreased thereafter, as well as plasmalogens, which were elevated at later timepoints during virus replication, but were also elevated under normal cell growth. These findings suggested a complex interplay of lipid profile reorganization involving plasmalogen metabolism. We also observed that NIC treatment of both low and high viral loads does not affect virus entry. Instead, NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which we found elevated during virus infection in the absence of NIC, resulting in a significant reduction in the production of infectious virions. Unexpectedly, at higher viral loads, NIC treatment also resulted in elevated triglyceride levels, and induced significant changes in phospholipid metabolism. Discussion: We posit that future screens of approved or new partner drugs should prioritize compounds that effectively counter SARS-CoV-2 subversion of lipid metabolism, thereby reducing virus replication, egress, and the subsequent regulation of key lipid mediators of pathological inflammation.

Toxicity assessment of CeO2 and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth.

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 µg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 µg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Retrospective Analysis of Wastewater-Based Epidemiology of SARS-CoV-2 in Residences on a Large College Campus: Relationships between Wastewater Outcomes and COVID-19 Cases across Two Semesters with Different COVID-19 Mitigation Policies.

Wastewater-based epidemiology (WBE) has been utilized for outbreak monitoring and response efforts in university settings during the coronavirus disease 2019 (COVID-19) pandemic. However, few studies examined the impact of university policies on the effectiveness of WBE to identify cases and mitigate transmission. The objective of this study was to retrospectively assess relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater outcomes and COVID-19 cases in residential buildings of a large university campus across two academic semesters (August 2020-May 2021) under different COVID-19 mitigation policies. Clinical case surveillance data of student residents were obtained from the university COVID-19 response program. We collected and processed building-level wastewater for detection and quantification of SARS-CoV-2 RNA by RT-qPCR. The odds of obtaining a positive wastewater sample increased with COVID-19 clinical cases in the fall semester (OR = 1.50, P value = 0.02), with higher odds in the spring semester (OR = 2.63, P value < 0.0001). We observed linear associations between SARS-CoV-2 wastewater concentrations and COVID-19 clinical cases (parameter estimate = 1.2, P value = 0.006). Our study demonstrated the effectiveness of WBE in the university setting, though it may be limited under different COVID-19 mitigation policies. As a complementary surveillance tool, WBE should be accompanied by robust administrative and clinical testing efforts for the COVID-19 pandemic response.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.

Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed a portable device that integrates sample preparation with real-time, reverse-transcription, loop-mediated isothermal amplification (rRT-LAMP). First, we designed a rRT-LAMP assay targeting MAYV's non-structural protein (NS1) gene and determined the limit of detection of at least 10 viral genome equivalents per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. The rRT-LAMP assay was integrated with a sample preparation device (SPD) wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting, while subsequent real-time amplification device (RAD) enables virus detection at the point of care (POC). The functions of our platform were demonstrated using purified MAYV RNA or blood samples containing viable viruses. We have used the devices for detection of MAYV in as short as 13 min, with limit of detection to as low as 10 GEs/reaction.

Lack of SARS-CoV-2 in environmental samples collected from September 2020-February 2021 in a university that followed CDC reopening guidance.

This study aimed to provide environmental surveillance data for evaluating the risk of acquiring SARS-CoV-2 in public areas with high foot traffic in a university. Air and surface samples were collected at a university that had the second highest number of COVID-19 cases among public higher education institutions in the U.S. during Fall 2020. A total of 60 samples were collected in 16 sampling events performed during Fall 2020 and Spring 2021. Nearly 9800 students traversed the sites during the study period. SARS-CoV-2 was not detected in any air or surface samples. The university followed CDC guidance, including COVID-19 testing, case investigations, and contact tracing. Students, faculty, and staff were asked to maintain physical distancing and wear face coverings. Although COVID-19 cases were relatively high at the university, the possibility of acquiring SARS-CoV-2 infections at the sites tested was low.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected > 6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant in Children and Utility of Rapid Antigen Testing as an Indicator of Culturable Virus.

We screened 65 longitudinally collected nasal swab samples from 31 children aged 0-16 years who were positive for severe acute respiratory syndrome coronavirus 2 Omicron BA.1. By day 7 after onset of symptoms, 48% of children remained positive by rapid antigen test. In a sample subset, we found 100% correlation between antigen test results and virus culture.

Assessment of a mass balance equation for estimating community-level prevalence of COVID-19 using wastewater-based epidemiology in a mid-sized city.

Wastewater-based epidemiology (WBE) has emerged as a valuable epidemiologic tool to detect the presence of pathogens and track disease trends within a community. WBE overcomes some limitations of traditional clinical disease surveillance as it uses pooled samples from the entire community, irrespective of health-seeking behaviors and symptomatic status of infected individuals. WBE has the potential to estimate the number of infections within a community by using a mass balance equation, however, it has yet to be assessed for accuracy. We hypothesized that the mass balance equation-based approach using measured SARS-CoV-2 wastewater concentrations can generate accurate prevalence estimates of COVID-19 within a community. This study encompassed wastewater sampling over a 53-week period during the COVID-19 pandemic in Gainesville, Florida, to assess the ability of the mass balance equation to generate accurate COVID-19 prevalence estimates. The SARS-CoV-2 wastewater concentration showed a significant linear association (Parameter estimate = 39.43, P value < 0.0001) with clinically reported COVID-19 cases. Overall, the mass balance equation produced accurate COVID-19 prevalence estimates with a median absolute error of 1.28%, as compared to the clinical reference group. Therefore, the mass balance equation applied to WBE is an effective tool for generating accurate community-level prevalence estimates of COVID-19 to improve community surveillance.

Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza.

Analysis of Zika Virus Sequence Data Associated with a School Cohort in Haiti.

Zika virus (ZIKV) infections occurred in epidemic form in the Americas in 2014-2016, with some of the earliest isolates in the region coming from Haiti. We isolated ZIKV from 20 children with acute undifferentiated febrile illness who were part of a cohort of children seen at a school clinic in the Gressier region of Haiti. The virus was also isolated from three pools of Aedes aegypti mosquitoes collected at the same location. On phylogenetic analysis, three distinct ZIKV clades were identified. Strains from all three clades were present in Haiti in 2014, making them among the earliest isolates identified in the Western Hemisphere. Strains from all three clades were also isolated in 2016, indicative of their persistence across the time period of the epidemic. Mosquito isolates were collected in 2016 and included representatives from two of the three clades; in one instance, ZIKV was isolated from a pool of male mosquitoes, suggestive of vertical transmission of the virus. The identification of multiple ZIKV clades in Haiti at the beginning of the epidemic suggests that Haiti served as a nidus for transmission within the Caribbean.