Quantitative risk assessment of COVID-19 aerosol transmission indoors: a mechanistic stochastic web application.

An increasing body of literature suggests that aerosol inhalation plays a primary role in COVID-19 transmission, particularly in indoor settings. Mechanistic stochastic models can help public health professionals, engineers, and space planners understand the risk of aerosol transmission of COVID-19 to mitigate it. We developed such model and a user-friendly web application to meet the need of accessible risk assessment tools during the COVID-19 pandemic. We built our model based on the Wells-Riley model of respiratory disease transmission, using quanta emission rates obtained from COVID-19 outbreak investigations. In this report, three modelled scenarios were evaluated and compared to epidemiological studies looking at similar settings: classrooms, weddings, and heavy exercise sessions. We found that the risk of long-range aerosol transmission increased 309-332% when people were not wearing masks, and 424-488% when the room was poorly ventilated in addition to no masks being worn across the scenarios. Also, the risk of transmission could be reduced by ∼40-60% with ventilation rates of 5 ACH for 1-4 h exposure events, and ∼70% with ventilation rates of 10 ACH for 4 h exposure events. Relative humidity reduced the risk of infection (inducing viral inactivation) by a maximum of ∼40% in a 4 h exposure event at 70% RH compared to a dryer indoor environment with 25% RH. Our web application has been used by more than 1000 people in 52 countries as of September 1st, 2021. Future work is needed to obtain SARS-CoV-2 dose-response functions for more accurate risk estimates.

Metagenomic analysis of endemic viruses in oral secretions from Chinese pigs.

BACKGROUND: Pigs are unique reservoirs for virus ecology. Despite the increased use of improved biosecurity measures, pig viruses readily circulate in Chinese swine farms. OBJECTIVES: The main objective of this study was to examine archived swine oral secretion samples with a panel of pan-species viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms. METHODOLOGY: Two hundred (n = 200) swine oral secretion samples, collected during 2015 and 2016 from healthy pigs on six swine farms in two provinces in China, were screened with molecular pan-species assays for coronaviruses (CoVs), adenoviruses (AdVs), enteroviruses (EVs), and paramyxoviruses (PMV). Samples were also screened for porcine circovirus (PCV) 3, porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). RESULTS: Among 200 swine oral secretion samples, 152 (76.0%) were found to have at least one viral detection. Thirty-four samples (17%) were positive for more than one virus, including 24 (70.5%) with dual detection and 10 (29.5%) with triple detection. Seventy-eight (39.0%) samples were positive for porcine AdVs, 22 (11.0%) were positive for porcine CoVs, 21 (10.5%) were positive for IAVs, 13 (6.5%) were positive for PCV, 7 (3.5%) were positive for PMV, six (3.0%) were positive for PRRSV and five (2.5%) were positive for porcine EV. CONCLUSION: Our findings underscore the high prevalence of numerous viruses among production pigs in China and highlight the need for routine, periodic surveillance for novel virus emergence with the goal of protecting pigs.

EpidemiologiCal POpulatioN STudy of SARS-CoV-2 in Lake CounTy, Illinois (CONTACT): Methodology and Baseline Characteristics of a Community-Based Surveillance Study.

INTRODUCTION: EpidemiologiCal POpulatioN STudy of SARS-CoV-2 in Lake CounTy, Illinois (CONTACT) is an observational, epidemiological study with a 9-month longitudinal follow-up of nonhospitalized persons aged 18 years or older currently living or employed in Lake County, IL. We describe the study design and report baseline characteristics of the study participants, including the proportion of participants with acute or previous SARS-CoV-2 infection at enrollment. METHODS: At enrollment and subsequent timepoints, participants recruited through digital and paper-based advertising campaigns reported their occupational and school-based exposure, risk factors, and behaviors, and provided nasal and serum specimens. Stratified enrichment was used to enhance enrollment into medium- and higher-risk groups within four occupational risk groups for SARS-CoV-2 infection. RT-PCR and serologic (IgG) testing were conducted to detect acute or previous SARS-CoV-2 infection in participants, respectively. RESULTS: Between November 2020 and January 2021, 1008 participants (female 70.7%, mean age ± SD 51 ± 13.8 years) completed the questionnaire and diagnostic testing. Among participants, 41.8% (n = 421) were considered low risk, 24.6% (n = 248) were medium-to-low risk, 22.3% (n = 225) were medium-to-high risk, and 11.3% (n = 114) were high risk. Of 56 (5.6%) participants with evidence of acute or previous SARS-CoV-2 infection at baseline, 11 (19.6%) were RT-PCR-positive, 36 (64.3%) were IgG-seropositive, and 9 (16.1%) were positive by both assays. Participants who were adherent vs nonadherent to social distancing measures (odds ratio [95% CI] 0.8 [0.4-1.8]) were less likely, while those in higher vs lower occupational risk groups (2.0 [1.0-4.4]) were more likely to have evidence for acute or previous SARS-CoV-2 infection. CONCLUSION: In fall/winter 2020/21, 5.6% of adults in a Lake County convenience sample had evidence for acute or previous SARS-CoV-2 infection at baseline. Nonadherence to social distancing measures and high-risk professions were associated with SARS-CoV-2 infection. The study is ongoing and future analyses will assess infection status over time. CLINICAL TRIAL REGISTRATION: NCT04611230.

Development of flow cytometry-based assays to assess the ability of antibodies to bind to SARS-CoV-2-infected and spike-transfected cells and mediate NK cell degranulation.

Since the beginning of the SARS-CoV-2 pandemic, antibody responses and antibody effector functions targeting SARS-CoV-2-infected cells have been understudied. Consequently, the role of these types of antibodies in SARS-CoV-2 disease (COVID-19) and immunity is still undetermined. To provide tools to study these responses, we used plasma from SARS-CoV-2-infected individuals (n = 50) and SARS-CoV-2 naive healthy controls (n = 20) to develop four specific and reproducible flow cytometry-based assays: (i) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2-infected cells and (ii) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2 Spike-transfected cells. All four assays demonstrated the ability to detect the presence of these functional antibody responses in a specific and reproducible manner. Interestingly, we found weak to moderate correlations between the four assays (Spearman rho ranged from 0.50 to 0.74), suggesting limited overlap in the responses captured by the individual assays. Lastly, while we initially developed each assay with multiple dilutions in an effort to capture the full relationship between antibody titers and assay outcome, we explored the relationship between fewer antibody dilutions and the full dilution series for each assay to reduce assay costs and improve assay efficiency. We found high correlations between the full dilution series and fewer or single dilutions of plasma. Use of single or fewer sample dilutions to accurately determine the response rates and magnitudes of the responses allows for high-throughput use of these assays platforms to facilitate assessment of antibody responses elicited by SARS-CoV-2 infection and vaccination in large clinical studies.

Bioaerosol Sampling at a Live Animal Market in Kunshan, China: A Noninvasive Approach for Detecting Emergent Viruses.

From October to December 2018, periodic bioaerosol sampling was conducted at a live bird market in Kunshan, China. Sixty-six (55%) of 120 samples had molecular evidence of avian influenza viruses. Four yielded live H9N2 virus after egg culture.

A pan-coronavirus RT-PCR assay for rapid viral screening of animal, human, and environmental specimens.

We examined a collection of 386 animal, 451 human, and 109 archived bioaerosol samples with a new pan-species coronavirus molecular assay. Thirty-eight (4.02%) of 946 specimens yielded evidence of human or animal coronaviruses. Our findings demonstrate the utility of employing the pan-CoV RT-PCR assay in detecting varied coronavirus among human, animal, and environmental specimens. This RT-PCR assay might be employed as a screening diagnostic for early detection of coronaviruses incursions or prepandemic coronavirus emergence in animal or human populations.

Novel Canine Coronavirus Isolated from a Hospitalized Patient With Pneumonia in East Malaysia.

BACKGROUND: During the validation of a highly sensitive panspecies coronavirus (CoV) seminested reverse-transcription polymerase chain reaction (RT-PCR) assay, we found canine CoV (CCoV) RNA in nasopharyngeal swab samples from 8 of 301 patients (2.5%) hospitalized with pneumonia during 2017-2018 in Sarawak, Malaysia. Most patients were children living in rural areas with frequent exposure to domesticated animals and wildlife. METHODS: Specimens were further studied with universal and species-specific CoV and CCoV 1-step RT-PCR assays, and viral isolation was performed in A72 canine cells. Complete genome sequencing was conducted using the Sanger method. RESULTS: Two of 8 specimens contained sufficient amounts of CCoVs as confirmed by less-sensitive single-step RT-PCR assays, and 1 specimen demonstrated cytopathic effects in A72 cells. Complete genome sequencing of the virus causing cytopathic effects identified it as a novel canine-feline recombinant alphacoronavirus (genotype II) that we named CCoV-human pneumonia (HuPn)-2018. Most of the CCoV-HuPn-2018 genome is more closely related to a CCoV TN-449, while its S gene shared significantly higher sequence identity with CCoV-UCD-1 (S1 domain) and a feline CoV WSU 79-1683 (S2 domain). CCoV-HuPn-2018 is unique for a 36-nucleotide (12-amino acid) deletion in the N protein and the presence of full-length and truncated 7b nonstructural protein, which may have clinical relevance. CONCLUSIONS: This is the first report of a novel canine-feline recombinant alphacoronavirus isolated from a human patient with pneumonia. If confirmed as a pathogen, it may represent the eighth unique coronavirus known to cause disease in humans. Our findings underscore the public health threat of animal CoVs and a need to conduct better surveillance for them.

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.

Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider.

Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human-animal interfaces in geographical hotspots for emerging infectious diseases. Where human-animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics.

Panspecies molecular assays detect viral pathogens missed by real-time PCR/reverse-transcriptase PCR among pneumonia patients, Sarawak, Malaysia.

BACKGROUND: In a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients' nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed. METHODS: Specimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software. RESULTS: There was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected. CONCLUSIONS: The observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

While We Endure This Pandemic, What New Respiratory Virus Threats Are We Missing?

In this paper, we review recent human respiratory virus epidemics, their zoonotic nature, and our current inability to identify future prepandemic threats. We propose a cost-efficient, One Health surveillance strategy that will be more efficient and more sustainable than previous efforts.

Environmental and Aerosolized Severe Acute Respiratory Syndrome Coronavirus 2 Among Hospitalized Coronavirus Disease 2019 Patients.

During April and May 2020, we studied 20 patients hospitalized with coronavirus disease 2019 (COVID-19), their hospital rooms (fomites and aerosols), and their close contacts for molecular and culture evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among >400 samples, we found molecular evidence of virus in most sample types, especially the nasopharyngeal (NP), saliva, and fecal samples, but the prevalence of molecular positivity among fomites and aerosols was low. The agreement between NP swab and saliva positivity was high (89.5%; κ = 0.79). Two NP swabs collected from patients on days 1 and 7 post-symptom onset had evidence of infectious virus (2 passages over 14 days in Vero E6 cells). In summary, the low molecular prevalence and lack of viable SARS-CoV-2 virus in fomites and air samples implied low nosocomial risk of SARS-CoV-2 transmission through inanimate objects or aerosols.

Outbreak of severe acute respiratory infection in Southern Province, Sri Lanka in 2018: a cross-sectional study.

OBJECTIVES: To determine aetiology of illness among children and adults presenting during outbreak of severe respiratory illness in Southern Province, Sri Lanka, in 2018. DESIGN: Prospective, cross-sectional study. SETTING: 1600-bed, public, tertiary care hospital in Southern Province, Sri Lanka. PARTICIPANTS: 410 consecutive patients, including 371 children and 39 adults, who were admitted with suspected viral pneumonia (passive surveillance) or who met case definition for acute respiratory illness (active surveillance) in May to June 2018. RESULTS: We found that cocirculation of influenza A (22.6% of cases), respiratory syncytial virus (27.8%) and adenovirus (AdV) (30.7%; type B3) was responsible for the outbreak. Mortality was noted in 4.5% of paediatric cases identified during active surveillance. Virus type and viral coinfection were not significantly associated with mortality. CONCLUSIONS: This is the first report of intense cocirculation of multiple respiratory viruses as a cause of an outbreak of severe acute respiratory illness in Sri Lanka, and the first time that AdV has been documented as a cause of a respiratory outbreak in the country. Our results emphasise the need for continued vigilance in surveying for known and emerging respiratory viruses in the tropics.

Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients.

Understanding the particle size distribution in the air and patterns of environmental contamination of SARS-CoV-2 is essential for infection prevention policies. Here we screen surface and air samples from hospital rooms of COVID-19 patients for SARS-CoV-2 RNA. Environmental sampling is conducted in three airborne infection isolation rooms (AIIRs) in the ICU and 27 AIIRs in the general ward. 245 surface samples are collected. 56.7% of rooms have at least one environmental surface contaminated. High touch surface contamination is shown in ten (66.7%) out of 15 patients in the first week of illness, and three (20%) beyond the first week of illness (p = 0.01, χ2 test). Air sampling is performed in three of the 27 AIIRs in the general ward, and detects SARS-CoV-2 PCR-positive particles of sizes >4 µm and 1-4 µm in two rooms, despite these rooms having 12 air changes per hour. This warrants further study of the airborne transmission potential of SARS-CoV-2.

A RT-PCR assay for the detection of coronaviruses from four genera.

BACKGROUND: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats. OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily. STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types. CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Virus detections among patients with severe acute respiratory illness, Northern Vietnam.

Severe acute respiratory illness (SARI) is a major cause of death and morbidity in low- and middle-income countries, however, the etiologic agents are often undetermined due to the lack of molecular diagnostics in hospitals and clinics. To examine evidence for select viral infections among patients with SARI in northern Vietnam, we studied 348 nasopharyngeal samples from military and civilian patients admitted to 4 hospitals in the greater Hanoi area from 2017-2019. Initial screening for human respiratory viral pathogens was performed in Hanoi, Vietnam at the National Institute of Hygiene and Epidemiology (NIHE) or the Military Institute of Preventative Medicine (MIPM), and an aliquot was shipped to Duke-NUS Medical School in Singapore for validation. Patient demographics were recorded and used to epidemiologically describe the infections. Among military and civilian cases of SARI, 184 (52.9%) tested positive for one or more respiratory viruses. Influenza A virus was the most prevalent virus detected (64.7%), followed by influenza B virus (29.3%), enterovirus (3.8%), adenovirus (1.1%), and coronavirus (1.1%). Risk factor analyses demonstrated an increased risk of influenza A virus detection among military hospital patients (adjusted OR, 2.0; 95% CI, 1.2-3.2), and an increased risk of influenza B virus detection among patients enrolled in year 2017 (adjusted OR, 7.9; 95% CI, 2.7-22.9). As influenza A and B viruses were commonly associated with SARI and are treatable, SARI patients entering these hospitals would benefit if the hospitals were able to adapt onsite molecular diagnostics.