A flow cytometry-based assay to measure neutralizing antibodies against SARS-CoV-2 virus.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.

Persistent pollution of genetic materials in a typical laboratory environment.

From the onset of coronavirus disease (COVID-19) pandemic, there are concerns regarding the disease spread and environmental pollution of biohazard since studies on genetic engineering flourish and numerous genetic materials were used such as the nucleic acid test of the severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this work, we studied genetic material pollution in an institute during a development cycle of plasmid, one of typical genetic materials, with typical laboratory settings. The pollution source, transmission routes, and pollution levels in laboratory environment were examined. The Real-Time quantitative- Polymerase Chain Reaction results of all environmental mediums (surface, aerosol, and liquid) showed that a targeted DNA segment occurred along with routine experimental operations. Among the 79 surface and air samples collected in the genetic material operation, half of the environment samples (38 of 79) are positive for nucleic acid pollution. Persistent nucleic acid contaminations were observed in all tested laboratories and spread in the public area (hallway). The highest concentration for liquid and surface samples were 1.92 × 108 copies/uL and 5.22 × 107 copies/cm2, respectively. Significant amounts of the targeted gene (with a mean value of 74 copies/L) were detected in the indoor air of laboratories utilizing centrifuge devices, shaking tables, and cell homogenizers. Spills and improper disposal of plasmid products were primary sources of pollution. The importance of establishing designated experimental zones, employing advanced biosafety cabinets, and implementing highly efficient cleaning systems in laboratories with lower biosafety levels is underscored. SYNOPSIS: STATEMENT. Persistent environmental pollutions of genetic materials are introduced by typical experiments in laboratories with low biosafety level.

Experiencias de las enfermeras en primera línea contagiadas con COVID-19 que recibieron atención domiciliaria / Experiências de enfermeiros da linha de frente infectados com COVID-19 que receberam atendimento domiciliar / Experiences of frontline nurses infected with COVID-19 who received home care

Introducción: Las enfermeras han brindado cuidados a lospacientes con COVID-19, y muchas tuvieron que lidiar coneste virus al ser infectadas.Objetivo: Describir las experiencias de las enfermeras enprimera línea contagiadas con COVID-19 que recibieronatención domiciliaria.Método: Investigación cualitativa descriptiva, el tamaño dela muestra fue con 12 enfermeras infectadas con COVID-19que recibieron tratamiento domiciliario en Chiclayo, Perú. Elmuestreo fue por la técnica de bola de nieve. Para recolectarlos datos se utilizó la entrevista semiestructurada a travésde los medios virtuales previo consentimiento informado.Resultados: Surgieron tres categorías: a) Incertidumbre antemodo de contagio, medios de diagnóstico, sintomatología yevolución por la COVID-19, b) Cuidados recuperativos enel hogar: tratamiento médico, uso de medicina tradicional ymedidas de bioseguridad, y c) Impacto en la vida familiar,laboral, social, emocional y espiritual al sufrir de la COVID-19.Conclusiones: Las enfermeras presentaron síntomas leves dela COVID-19 y se recuperaron en su domicilio, cumplieroncon el tratamiento médico, algunas usaron remedios caseros.Practicaron estrictamente las medidas de bioseguridad paraevitar que su familia se contagie. Utilizaron la tecnologíadigital y reforzaron su fe para afrontar el impacto familiar,emocional y social.(AU)

Introduction: Nurses have provided care to patients with COVID-19,and many have had to deal with this virus when infected.Objective: To describe the experiences of frontline nursesinfected with COVID-19 who received home care.Method: Descriptive qualitative research, the sample sizewas 12 nurses infected with COVID-19 who received hometreatment in Chiclayo, Peru. Sampling was by snowballtechnique. To collect the data, the semi-structured interviewwas used through virtual media with prior informed consent.Results: Three categories emerged: a) Uncertainty regardingthe mode of transmission, means of diagnosis, symptomatologyand evolution due to COVID-19, b) Recuperative care athome: medical treatment, use of traditional medicine andbiosafety measures, and c) Impact on the family, work, social,emotional and spiritual life when suffering from COVID-19.Conclusions: The nurses presented mild symptoms ofCOVID-19 and recovered at home, complied with medicaltreatment, some used home remedies. They strictly practicedbiosecurity measures to prevent their family from gettinginfected. They used digital technology and strengthened theirfaith to cope with the family, emotional and social impact.(AU)

Introdução: Enfermeiras têm prestado atendimento a pacientescom COVID-19, e muitos tiveram que lidar com esse vírusquando infectados.Objetivo: Descrever as experiências de enfermeiros dalinha de frente infectados com COVID-19 que receberamatendimento domiciliar.Método: Emergiram três categorias: a) Incerteza quanto aomodo de contágio, meios de diagnóstico, sintomatologiae evolução da COVID-19, b) Cuidados de recuperação nodomicílio: tratamento médico, uso da medicina tradicional emedidas de biossegurança, e c) Impacto na saúde vida familiar,laboral, social, emocional e espiritual ao sofrer de COVID-19. Resultados: a) Participação do pessoal de saúde na atenção domiciliar: Consulta médica,administração de medicamentos e educação em saúde, b) Participação da família na atençãodomiciliar: Satisfação das necessidades básicas, apoio emocional e espiritual, c) Anedotas familiaressobre oferta de oxigênio, cuidados com oxigenoterapia e pós -Terapia respiratória COVID-19, d)Controvérsias para aplicação de protocolos de biossegurança durante o atendimento domiciliar.Conclusões: As enfermeiras apresentaram sintomas leves de COVID-19 e se recuperaram em casa,cumpriram tratamento médico, alguns usaram remédios caseiros. Eles praticavam rigorosamenteas medidas de biossegurança para evitar que sua família fosse infectada. Eles usaram a tecnologiadigital e fortaleceram sua fé para lidar com o impacto familiar, emocional e social.(AU)

Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system.

Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-△env backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.

Acute murine-betacoronavirus infection impairs testicular steroidogenesis and the quality of sperm production.

Although several testicular alterations promoted by coronavirus infection have been demonstrated, the extent, causes, and players of testicular pathogenesis are not totally understood. The present study aimed to investigate the short-term effects on male fertility of intranasally administered murine hepatitis virus strain 3 (MHV-3), a member of the genus Betacoronavirus, which causes a severe systemic acute infection. This mouse model might be used as a in vivo prototype for investigating the impact of betacoronavirus on the endocrine and exocrine testicular functions with the advantage to be performed in a biosafety level 2 condition. Herein, we performed virological, histopathological, and molecular studies regarding the testicular spermatogenesis and the spermatic quality analyses in an MHV-3-infected C57BL/6 mice. The main outcomes showed that MHV-3 infects mouse testis and induces a testicular inflammatory state, impairing the steroidogenic pathway. The infection led to several alterations in the testicular parenchyma, such as: seminiferous epithelium sloughing, retention of residual bodies, germ cell apoptosis, alterations in intercellular junction proteins, and worse spermatogenic parameters. Moreover, the levels of plasmatic testosterone as well as the quality of sperm production reduced. Therefore, the present data suggest that the viral/inflammatory impairment of the steroidogenic pathway and the consequent imbalance of androgen levels is critical in testicular pathology, disturbing the SC barrier function and the germ cell differentiation. Our study is important for comprehending the effects of beta coronavirus infections on testis function in order to develop treatments that could prevent virus-mediated male infertility.

Effects of primary health care and socioeconomic aspects on the dispersion of COVID-19 in the Brazilian Northeast: Ecological study of the first pandemic wave.

BACKGROUND: The COVID-19 pandemic has had a negative impact on socioeconomic and public health conditions of the population. AIM: To measure the temporal evolution of COVID-19 cases in cities near the countryside outside metropolitan areas of northeastern Brazil and the impact of the primary care organization in its containment. METHODS: This is a time-series study, based on the first three months of COVID-19 incidence in northeastern Brazil. Secondary data were used, the outcome was number of COVID-19 cases. Independent variables were time, coverage and quality score of basic health services, and demographic, socioeconomic and social isolation variables. Generalizable Linear Models with first order autoregression were applied. RESULTS: COVID-19 spreads heterogeneously in cities near the countryside of Northeastern Brazilian cities, showing associations with the city size, socioeconomic and organizational indicators of services. The Family Health Strategy seems to mitigate the speed of progression and burden of the disease, in addition to measures such as social isolation and closure of commercial activities. CONCLUSION: The spread of COVID-19 reveals multiple related factors, which require coordinated intersectoral actions in order to mitigate its problems, especially in biologically and socially vulnerable populations.

Exploring COVID-related relationship extraction: Contrasting data sources and analyzing misinformation.

The COVID-19 pandemic presented an unparalleled challenge to global healthcare systems. A central issue revolves around the urgent need to swiftly amass critical biological and medical knowledge concerning the disease, its treatment, and containment. Remarkably, text data remains an underutilized resource in this context. In this paper, we delve into the extraction of COVID-related relations using transformer-based language models, including Bidirectional Encoder Representations from Transformers (BERT) and DistilBERT. Our analysis scrutinizes the performance of five language models, comparing information from both PubMed and Reddit, and assessing their ability to make novel predictions, including the detection of "misinformation." Key findings reveal that, despite inherent differences, both PubMed and Reddit data contain remarkably similar information, suggesting that Reddit can serve as a valuable resource for rapidly acquiring information during times of crisis. Furthermore, our results demonstrate that language models can unveil previously unseen entities and relations, a crucial aspect in identifying instances of misinformation.

Change in body weight of older adults before and during the COVID-19 pandemic: longitudinal results from the Berlin Aging Study II.

OBJECTIVES: Change in body weight during the COVID-19 pandemic as an unintended side effect of lockdown measures has been predominantly reported for younger and middle-aged adults. However, information on older adults for which weight loss is known to result in adverse outcomes, is scarce. In this study we describe the body weight change in older adults before, during, and after the COVID-19 lockdown measures and explore putative associated factors with a focus on the period that includes the first six months of the COVID-19 containment measures. DESIGN: Prospective cohort study with three follow-up examinations over the course of 10 years. SETTING AND PARTICIPANTS: In this study, we analyzed the longitudinal weight change of 472 participants of the Berlin Aging Study II (mean age of 67.5 years at baseline). MEASUREMENTS: Body weight was assessed at four time points. Additionally, differences between subgroups characterized by socio-economic, cognitive, and psychosocial variables as well as morbidity burden, biological age markers (epigenetic clocks, telomere length), and frailty were compared. RESULTS: On average, women and men lost 0.87% (n = 227) and 0.5% (n = 245) of their body weight per year in the study period covering the first six months of the COVID-19 pandemic. Weight loss among men was particularly pronounced among groups characterized by change in physical activity due to COVID-19 lockdown, low positive affect, premature epigenetic age (7-CpG clock), diagnosed metabolic syndrome, and a more masculine gender score (all variables: p < 0.05, n = 245). CONCLUSION: During the COVID-19 pandemic, older participants lost weight with a 2.5-times (women) and 2-times (men) higher rate than what is expected in this age.

Experimental and analytical pipeline for sub-genomic RNA landscape of coronavirus by Nanopore sequencer.

Coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2, can infect a variety of mammalian and avian hosts with significant medical and economic consequences. During the life cycle of CoV, a coordinated series of subgenomic RNAs, including canonical subgenomic messenger RNA and non-canonical defective viral genomes (DVGs), are generated with different biological implications. Studies that adopted the Nanopore sequencer (ONT) to investigate the landscape and dynamics of viral RNA subgenomic transcriptomes applied arbitrary bioinformatics parameters without justification or experimental validation. The current study used bovine coronavirus (BCoV), which can be performed under biosafety level 2 for library construction and experimental validation using traditional colony polymerase chain reaction and Sanger sequencing. Four different ONT protocols, including RNA direct and cDNA direct sequencing with or without exonuclease treatment, were used to generate RNA transcriptomic libraries from BCoV-infected cell lysates. Through rigorously examining the k-mer, gap size, segment size, and bin size, the optimal cutoffs for the bioinformatic pipeline were determined to remove the sequence noise while keeping the informative DVG reads. The sensitivity and specificity of identifying DVG reads using the proposed pipeline can reach 82.6% and 99.6% under the k-mer size cutoff of 15. Exonuclease treatment reduced the abundance of RNA transcripts; however, it was not necessary for future library preparation. Additional recovery of clipped BCoV nucleotide sequences with experimental validation expands the landscape of the CoV discontinuous RNA transcriptome, whose biological function requires future investigation. The results of this study provide the benchmarks for library construction and bioinformatic parameters for studying the discontinuous CoV RNA transcriptome.IMPORTANCEFunctional defective viral genomic RNA, containing all the cis-acting elements required for translation or replication, may play different roles in triggering cell innate immune signaling, interfering with the canonical subgenomic messenger RNA transcription/translation or assisting in establishing persistence infection. This study does not only provide benchmarks for library construction and bioinformatic parameters for studying the discontinuous coronavirus RNA transcriptome but also reveals the complexity of the bovine coronavirus transcriptome, whose functional assays will be critical in future studies.

A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2-Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation.

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2-based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19-convalescent or NVX-CoV2373-vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3-based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva.

Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 µl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.

An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements.

Percepção de enfermeiros acerca dos processos de cuidado durante a pandemia da covid-19 / Nurses' perception about care processes during the covid-19 pandemic / Percepción de las enfermeras sobre los procesos de atención durante la pandemia de covid-19

Objetivo: analisar a percepção do enfermeiro acerca dos processos de cuidado durante a pandemia da COVID-19. Métodos: estudo de campo, descritivo e abordagem qualitativa, realizado entre dezembro de 2020 a abril de 2021. Participaram 45 enfermeiros atuantes há três anos ou mais na Atenção Primária de municípios paraibanos classificados em urbano, intermediário adjacente e remoto, rural adjacente e remoto. Realizou-se a coleta através de entrevistas semiestruturadas pela plataforma Google Meet, essas eram gravadas pela própria plataforma e transcritas na íntegra para análise. A análise de dados deu-se por meio da Análise de Conteúdo considerando as etapas de pré-análise, codificação do texto e a inferência e interpretação dos resultados. O estudo atendeu ao que preconiza os padrões éticos. Resultados: no contexto pandêmico, os enfermeiros identificaram déficits existentes em suas práticas, elencando desafios e limitações nas condutas, como as de biossegurança e prevenção de doenças, fazendo-os repensar sobre futuras mudanças a serem realizadas após o período de pandemia. Conclusão: infere-se que os enfermeiros atuantes da Atenção Primária obtiveram novas percepções relacionadas ao cuidado, a preocupação com a saúde física e mental no planejamento desse cuidado, não apenas do usuário, mas de si mesmo e às pessoas do convívio. (AU)

Objective: to analyze nurses' perceptions of care processes during the COVID-19 pandemic. Methods: field study, descriptive and qualitative approach, carried out between December 2020 and April 2021. Participated 45 nurses working for three years or more in Primary Care in municipalities in Paraíba classified as urban, intermediate adjacent and remote, rural adjacent and remote. The collection was carried out through semi-structured interviews through the Google Meet platform, these were recorded by the platform itself and transcribed in full for analysis. Data analysis was carried out through Content Analysis considering the pre-analysis steps, text coding and the inference and interpretation of results. The study complied with ethical standards. Results: in the pandemic context, nurses identified existing deficits in their practices, listing challenges and limitations in conduct, such as biosecurity and disease prevention, making them rethink about future changes to be made after the period of pandemic. Conclusion: it is inferred that nurses working in Primary Care obtained new perceptions related to care, the concern with physical and mental health in the planning of this care, not only of the user, but of himself and the people around him. (AU)

Objetivo: analizar las percepciones de los enfermeros sobre los procesos de atención durante la pandemia de COVID-19. Métodos: estudio de campo, de abordaje descriptivo y cualitativo, realizado entre diciembre de 2020 y abril de 2021. Participaron 45 enfermeros que actúan desde hace tres años o más en Atención Básica en municipios de Paraíba clasificados como urbano, intermedio adyacente y remoto, rural adyacente y remoto. La recolección se realizó a través de entrevistas semiestructuradas a través de la plataforma Google Meet, estas fueron grabadas por la propia plataforma y transcritas en su totalidad para su análisis. El análisis de los datos se realizó a través del Análisis de Contenido considerando las etapas del preanálisis, la codificación del texto y la inferencia e interpretación de los resultados. El estudio cumplió con las normas éticas. Resultados: en el contexto de la pandemia, los enfermeros identificaron déficits existentes en sus prácticas, enumerando desafíos y limitaciones en la conducta, como la bioseguridad y la prevención de enfermedades, lo que les hizo repensar sobre los cambios futuros que se deben realizar después del período de pandemia. Conclusión: se infiere que los enfermeros que actúan en la Atención Primaria obtuvieron nuevas percepciones relacionadas con el cuidado, la preocupación por la salud física y mental en la planificación de este cuidado, no sólo del usuario, sino de sí mismo y de las personas que lo rodean. (AU)

Frequency, kinetics and determinants of viable SARS-CoV-2 in bioaerosols from ambulatory COVID-19 patients infected with the Beta, Delta or Omicron variants.

Airborne transmission of SARS-CoV-2 aerosol remains contentious. Importantly, whether cough or breath-generated bioaerosols can harbor viable and replicating virus remains largely unclarified. We performed size-fractionated aerosol sampling (Andersen cascade impactor) and evaluated viral culturability in human cell lines (infectiousness), viral genetics, and host immunity in ambulatory participants with COVID-19. Sixty-one percent (27/44) and 50% (22/44) of participants emitted variant-specific culture-positive aerosols <10µm and <5µm, respectively, for up to 9 days after symptom onset. Aerosol culturability is significantly associated with lower neutralizing antibody titers, and suppression of transcriptomic pathways related to innate immunity and the humoral response. A nasopharyngeal Ct <17 rules-in ~40% of aerosol culture-positives and identifies those who are probably highly infectious. A parsimonious three transcript blood-based biosignature is highly predictive of infectious aerosol generation (PPV > 95%). There is considerable heterogeneity in potential infectiousness i.e., only 29% of participants were probably highly infectious (produced culture-positive aerosols <5µm at ~6 days after symptom onset). These data, which comprehensively confirm variant-specific culturable SARS-CoV-2 in aerosol, inform the targeting of transmission-related interventions and public health containment strategies emphasizing improved ventilation.

Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Ongoing Cooperative Engagement Facilitates Agile Pandemic and Outbreak Response: Lessons Learned Through Cooperative Engagement Between Uganda and the United States.

Pathogens threaten human lives and disrupt economies around the world. This has been clearly illustrated by the current COVID-19 pandemic and outbreaks in livestock and food crops. To manage pathogen emergence and spread, cooperative engagement programs develop and strengthen biosafety, biosecurity, and biosurveillance capabilities among local researchers to detect pathogens. In this case study, we describe the efforts of a collaboration between the Los Alamos National Laboratory and the Uganda Virus Research Institute, the primary viral diagnostic laboratory in Uganda, to implement and ensure the sustainability of sequencing for biosurveillance. We describe the process of establishing this capability along with the lessons learned from both sides of the partnership to inform future cooperative engagement efforts in low- and middle-income countries. We found that by strengthening sequencing capabilities at the Uganda Virus Research Institute before the COVID-19 pandemic, the institute was able to successfully sequence SARS-CoV-2 samples and provide data to the scientific community. We highlight the need to strengthen and sustain capabilities through in-country training, collaborative research projects, and trust.

The SARS-CoV-2 Mpro Dimer-Based Screening System: A Synthetic Biology Tool for Identifying Compounds with Dimerization Inhibitory Potential.

The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (Mpro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 Mpro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-Mpro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 µg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 µg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 Mpro and also minimize the use of a high biosafety level laboratory.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Effect of chemical and physical agents on monkeypox virus infectivity and downstream research applications.

The 2022 global spread of Monkeypox Virus (MPXV) underlined the need to investigate safe-handling procedures of clinical and research samples. Here we evaluated the efficiency in reducing MPXV infectious titer of Triton X-100 (0.1 and 0.2%), UV-C irradiation (15 or 30 min), and heat (56 °C 30 min or 70 °C 5 min). The treatment of MPXV at 70 °C resulted in the strongest decrease of MPXV infectious titer (5.4 Log TCID50/mL), 56 °C and UV-C had a lighter impact (3.9 and 4.3Log), Triton X-100 was less efficient (1.8-2.5Log). Notably, SARS-CoV-2 was much more susceptible to Triton X-100 (4.0 Log decrease). UV-C had the highest impact on MPXV DNA detection by PCR (2.2-4.3 Ct value increase); protein detection by ELISA was dramatically impaired by heating. Overall, UV-C and heating were more effective in lowering MPXV infectious titer but their impact on nucleic acids or protein detection assays must be considered.

Inactivation mechanism of cold plasma combined with 222 nm ultraviolet for spike protein and its application in disinfecting of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus that has precipitated a worldwide pandemic of coronavirus disease since 2019. Developing an effective disinfection strategy is crucial to prevent the risk of surface cross-contamination by SARS-CoV-2. This study employed pseudovirus and the receptor-binding domain (RBD) protein of SARS-CoV-2 as models to investigate the spike protein inactivation process and its underlying mechanisms using a novel nonthermal technology. Cold plasma combined with 222 nm ultraviolet (CP+UV) treatment was applied to accelerate the generation of reactive species and enhance sterilization efficiency. The results indicated that the binding activity of RBD protein was completely inhibited at specific concentrations (0.01-0.05 mg/cm2) with corresponding treatment times of 15-30 s. The mechanism potentially involves the reactive species generated by CP+UV, which react with the spike protein RBD of SARS-CoV-2, leading to the loss of SARS-CoV-2 infectivity by causing damage to the ß-sheet structure and chemical bonds in the RBD protein. Validated by a biosafety level 3 (BSL3) laboratory, the CP+UV treatment for 30 s could completely inactivate SARS-CoV-2 with a concentration of 19054 ± 1112 TCID50/cm2. Therefore, this study potentially provides a novel disinfection strategy for the inactivation of SARS-CoV-2 on surface cross-contamination.