Modifications in SARS-CoV-2 N Linker Region Regulate RNA Interactions and Phase Separation

The N protein of the SARS-CoV-2 virion is critical for viral genome packaging via RNA binding and regulation of viral transcription at the replication-transcription complex (RTC). The N protein can be divided into five main domains, and the central region is the linker, which is predicted to be primarily disordered and has not been heavily studied. The linker is Serine-Arginine Rich, which is phosphorylated at multiple sites by host kinases during infection, thereby promoting the N protein's role in viral transcription. Phosphorylation is a critical process for the regulation of many cellular processes and can provide recognition sites for binding complexes. In a study that examined the recognition of the SARS-CoV-2 N protein by the human 14-3-3 protein, the linker was found to contain critical phosphosites for 14-3-3 binding. The goals of this project are to determine the structure, dynamics, and RNA interactions of the Serine-Arginine Rich linker region. To accomplish this, we performed Nuclear Magnetic Resonance spectroscopy (NMR) experiments to analyze the structure of the linker region of the N protein and its ability to bind viral RNA. NMR confirms predictions that the linker is not entirely unstructured and it is able to bind RNA. The linker region of the N protein with phosphoserine incorporated at S188 was also examined via an NMR titration experiment with 1-1000 RNA. Compared to wild type, the incorporation of phosphorylation decreases binding. Other biophysical techniques such as Analytical Ultracentrifugation (AUC) and Multi-Angle Light Scattering (MALS) are used to identify the association state of the linker and the size of the resulting protein-RNA complex. We are currently working to biophysically characterize the structure, dynamics, and viral RNA binding ability of a mutation found in the Delta and Omicron variants: the R203M linker, which have been shown to enhance viral infectivity. This work was supported by the NSF EAGER grant NSF/ MCB 2034446 and URSA Engage. Support to facilities includes the Oregon State University NMR Facility funded in part by NIH, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

PLASMA LIPIDS AND AMINO ACIDS LINKED TO LOW SARS-CoV-2 SUSCEPTIBILITY

Background: The mechanisms driving SARS-CoV-2 susceptibility remain poorly understood, especially the factors determining why a subset of unvaccinated individuals remain uninfected despite high-risk exposures. Method(s): We studied an exceptional group of unvaccinated healthcare workers heavily exposed to SARS-CoV-2 ('nonsusceptible') from April to June 2020, who were compared against 'susceptible' individuals to SARS-CoV-2, including uninfected subjects who became infected during the follow-up, and hospitalized patients with different disease severity providing samples at early disease stages. We analyzed plasma samples using different mass spectrometry technique and obtained metabolites and lipids profiles. Result(s): We found that the metabolite profiles were predictive of the selected study groups and identified lipids profiles and metabolites linked to SARS-CoV-2 susceptibility and COVID-19 severity. More importantly, we showed that non-susceptible individuals exhibited unique metabolomics and lipidomic patterns characterized by upregulation of most lipids -especially ceramides and sphingomyelin-and amino acids related to tricarboxylic acid cycle and mitochondrial metabolism, which could be interpreted as markers of low susceptibility to SARS-CoV-2 infection. Lipids and metabolites pathways analysis revealed that metabolites related to energy production, mitochondrial and tissue dysfunction, and lipids involved in membrane structure and virus infectivity were key markers of SARS-CoV-2 susceptibility. Conclusion(s): Lipid and metabolic profiles differ in 'nonsusceptible' compared to individuals susceptible to SARS-CoV-2. Our study suggests that lipid profiles are relevant actors during SARS-CoV-2 pathogenesis and highlight certain lipids relevant to understand SARS-CoV-2 pathogenesis. (Figure Presented).

Surfactant Protein B Reduces Respiratory Viral Infectivity in Human Lungs

Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.

A novel isothermal digital amplification system and its application for absolute quantification of respiratory infectious virus

Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023

Overview of SARS-CoV-2 and Possible Targets for the Management of COVID-19 Infections

COVID-19 is a respiratory infection caused by a newer strain of coronavirus known as SARS-CoV-2. The major problem of COVID-19 infections is the ARDS, followed by respiratory failure, organ failure, and even death with multiple organ dysfunction, including cardiovascular collapse. Moreover, it affects the old age population with co-morbid conditions. The deficiency of diet, micronutrients, and vitamins also plays a key role in diminishing the immune power, and increases the rate of viral infectivity. The possible reasons and management methods are discussed in this review. The management methods enhance the host immune system via multi-functional and multi-targeted actions. The global rate of COVID-19 outbreak necessitates the need to develop newer medicines. The drug discovery process is based on the exposure of viral proteins, genome sequence, replication mechanisms, pathophysiological mechanisms, and host cell components (as a target) reactions. This article highlights the overview of coronavirus components, the replications process, and possible targets for the management of coronavirus infections. It may lead to the rapid development of newer medicines for the treatment of coronavirus in-fections.Copyright © 2022 Bentham Science Publishers.

Proteomic determinants of disease, mortality and sequelae in COVID-19 patients requiring invasive mechanical ventilation

Introduction: The molecular mechanisms linked to the pathology of severe COVID-19 and its outcomes are poorly described. Aim(s): To analyze the proteomic profile of bronchial aspirates (BAS) samples from critically ill COVID-19 patients in order to identify factors associated with the disease and its prognosis. Method(s): Multicenter study including 74 critically ill non-COVID-19 and COVID-19 patients. BAS was obtained by bronchoaspiration after invasive mechanical ventilation (IMV) initiation. Proximity extension assay (PEA) technology was used for proteomic profiling. Random forest (RF) statistical models were used to predict the variable importance. Result(s): After adjusting for confounding factors, CST5, NADK, SRPK2 and TGF-alpha showed differences between COVID-19 and non-COVID-19 patients. Reduced levels of ENTPD2 and PTN were observed in non-survivors, even after adjustment. AGR2, NQO2, IL-1alpha, OSM and TRAIL, were the top five strongest predictors for ICU mortality and were used to build a prediction model. PTN (HR=4.00) ENTPD2 (HR=2.14) and the prediction model (HR=6.25) were associated with higher risk of death. In survivors, FCRL1, NTF4 and THOP1 correlated with lung function (DLCO levels) 3-months after hospital discharge. Similar findings were observed for Flt3L and THOP1 and radiological features (TSS). The proteins identified are expressed in immune and non-immune lung cells. A poor control of viral infectivity and an inappropriate reparative response seems to be linked to the disease and fatal outcomes, respectively. Conclusion(s): In critically ill COVID-19 patients, specific proteomic profiles are associated with the pathology, mortality and lung sequelae.

Food safety concerns in "COVID-19 era"

In March 2020, the World Health Organization (WHO) declared that the COVID-19 outbreak can be characterized as a pandemic. Human-to-human transmission of the SARS-CoV-2 virus may initially be blamed as the first cause of spread, but can an infection be contracted by ingestion of contaminated food or touching contaminated food surfaces? Recently cold-chain food contamination has been indicated as a possible source of many human cases in China. However, the risk of a food-related COVID-19 infection is still debated since the virus may reach people through a fresh product or packaging, which have been touched/sneezed on by infected people. This review summarizes the most recent evidence on the zoonotic origin of the pandemic, reports the main results regarding the transmission of SARS-CoV-2 through food or a food chain, as well as the persistence of the virus at different environmental conditions and surfaces. Emphasis is also posed on how to manage the risk of food-related COVID-19 spread and potential approaches that can reduce the risk of SARS-CoV-2 contamination.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Etiology, clinical features, infection control and therapy of SARS-CoV-2 Omicron variant.

The Omicron variant of SARS-CoV-2 is a new variant of concern after Alpha, Beta, Gamma and Delta variants. The amino acid mutations in the viral antigens, especially in the receptor binding region (RBD) of spike protein, were significantly more than those of other variants, which lead to the significant increase of infectivity, transmissibility and immune escape of Omicron variant. In addition, those spike mutations impaired the protective effect of vaccination. When compared to the infection of other variants, the latency of Omicron variant infection was significantly shortened, and the pathogenicity decreased markedly, which is in consistence with the fact that the vast majority of infected individuals showed no symptoms or only mild disease. Exacerbations in patients infected by Omicron variant were often associated with the progress of underlying disease. Early detection and medical isolation of infected persons, careful personal protection measures to cut off transmission routes, and active vaccination to protect susceptible people are key measures to prevent the spread of Omicron variant epidemic. A small number of patients infected with Omicron variant may develop so-called long COVID-19, post-COVID-19 syndrome, or post-COVID-19 condition, which means that long-term follow-up is needed in those patients. Effective anti-Omicron variant therapy can shorten the course of infection, promote the recovery from infection, and also contribute to the control of infection. Therefore, the development of antiviral drugs with ideal cost-benefit ratio and convenient administration is one of the research hotspot in the future.Copyright © 2022 Authors. All rights reserved.

Impact and Inescapable Effects of Coronavirus: History to Modern Pandemic Episode

Coronavirus disease 2019 (COVID-19) is an acute respiratory tract infection causing a pandemic that emerged in 2019 initially in China involving 13.8% cases with severe, and 6.1% with critical course and later throughout the globe. Vaccines or antiviral medications are yet to be used to prevent or treat infections of Human Coronavirus (HCoV). The much-discovered HCoV found in 2003, SARS-COVID-19, which caused respiratory syndrome, has special pathogenesis as it causes respiratory tract infection. The coronavirus spike protein's association with its host cell receptor complement is crucial in deciding the virus infectivity, tissue tropism and species variety. SARS, COVID-19, infects human cells by binding to angiotensin-converting enzyme 2 (ACE2) receptor and uses the TMPRSS2 cell protease to activate it. Lungs are most affected by COVID-19 as host cells are accessed by the virus through ACE2, which is most abundant in alveolar cells of the lungs. Special attention and efforts should be given in reducing transmission in vulnerable populations, including infants, health care providers and the elderly. COVID 19, is the main causative agent of potentially lethal disease and is of significant concern for global public health and in pandemics which was highlighted in this review.Copyright © 2021 Bentham Science Publishers.

Virology, clinical features and diagnosis of covid 19: Review analysis

COVID-19 requires unprecedented mobilization of the health systems to prevent the rapid spread of this unique virus, which spreads via respiratory droplet and causes respiratory disease. There is an urgent need for an accurate and rapid test method to quickly identify many infected patients and asymptomatic carriers to prevent virus transmission and assure timely treatment of the patients. This article aims as an outcome of review of the evidence on viral load and its virulence of SARS-CoV2,so that it will help in further understanding the fact useful for investigating and managing the COVID-19 cases. A search of available evidence was conducted in pub-med "COVID-19 viral load and virulence" and its associated characters world-wide and Google Scholar to capture the most recently published articles. The WHO and Centre for Disease Control and Prevention (CDC) database of publications on novel coronavirus were also screened for relevant publications. s of 55 articles were screened by two authors and 15 were included in this study based on the inclusion criteria. SARS-coV2, the causative agent of COVID-19 falls under the coronavirus family but it has higher infectivity compared to SARS and MERS with higher reproduction numbers(Ro). Virulence has been found to be different throughout the world,however lower compared to SARS and MERS,till date. The most common clinical features have been found to be cough and fever. RT - PCR remains the most sensitive and specific method for the diagnosis of COVID-19 although it is time consuming, costly and requires highly skilled human resources. Hence, newer modalities like RT-LAMP can be alternative for point of care diagnosis as this is both cost effective and requires less skilled human resources. Despite recent advances in disease diagnosis and treatment outcomes using latest technological advances in molecular biology, the global pandemic COVID-19 remains a major headache for governments across the world due to limited testing capacity and lack of appropriate treatment and vaccine. Copyright © 2020, Kathmandu University. All rights reserved.

Genetic risk score for risk stratification of patients positive for SARS-CoV-2 virus. The CARGENCORS study

Background: The disease presentation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from asymptomatic to fatal. COVID-19 patients with pre-existing coronary artery disease (CAD) risk factors or overt cardiovascular disease are at particular risk of severe disease. We hypothesised that a specific genetic risk score (GRS) based on single nucleotide polymorphisms (SNPs) allele count to score COVID-19 severity might include SNPs counts related to CAD incidence and to thrombosis, inflammation, and viral infectivity determinants involved in the severity of SARS-CoV-2. Such GRS could improve the early risk stratification of COVID-19 patients and optimize treatment strategies. Purpose: To evaluate the capacity of a genetic risk score (GRS) with candidate genes to predict COVID-19 severity. Methods: We conducted an age- and sex-matched case-control study with 1:2 ratio recruitment involving 2454 patients from Catalan hospitals and primary care. Cases were hospitalized severe (requiring at least oxygen treatment) or fatal COVID-19 patients;and controls were moderate-symptom and asymptomatic patients treated at home. Standard parametric and non-parametric methods, as required, were used to compare patient characteristics by severity. Individual genotypes for 33 CAD, 14 thrombosis, 22 inflammation, 15 viral infectivity SNPs and 2 COVID-19 SNPs already published were tested for association with severity with Cochran-Armitage statistics and p-values corrected for multiple comparisons. GRS was computed as the unweighted count of adverse alleles (0, 1 or 2). The odds ratio of severe COVID-19 was analysed for GRS (and its component SNPs) with logistic regression models adjusted for potential confounding factors. Area under the curve (AUC) improvement and net reclassification index (NRI) for GRS was estimated from a basic model including CAD and COVID-19 severity risk factors. Models' performance was measured with the Akaike information criterion. Results: SNPs identifications are not shown to prevent patent conflict. Cases and control characteristics are compared in Table 1. Cases had a more adverse cardiovascular and anthropometric risk profile. After correcting for multiple testing by Benjamini-Hochberg criteria, we observed 13 SNPs to be significantly associated with severity. After excluding the close SNPs in linkage disequilibrium, 7 were retained in the GRS model, which yielded the discrimination and reclassification characteristics described in Table 2. Conclusion: A GRS with 7 SNPs related to CAD, thrombosis and inflammation significantly improves the severe COVID-19 risk assessment done with age, sex, comorbidity, and anthropometry alone. (Figure Presented).

Use of Immune-Viral Dynamics Modeling to Understand Molnupiravir Drug Effect for COVID-19

Background. Molnupiravir (MOV) is an orally administered ribonucleoside prodrug of β-D-N4-hydroxycytidine (NHC) against SARS-CoV-2. Here we present viral dynamics analysis of Phase 2 clinical virology data to inform MOV Phase 3 study design and development strategy. Methods. An Immune-Viral Dynamics Model (IVDM) was developed with mechanisms of SARS-CoV-2 infection, replication, and induced immunity, which together describe the dynamics of viral load (VL) during disease progression. Longitudinal virology data from ferret studies (Cox, et al. Nat. Microbiol 2021:6-11) were used to inform IVDM, which was further translated to human by adjusting parameter values to capture clinical data from MOVe-IN/MOVe-OUT studies. Different placements of drug effects (on viral infectivity vs. productivity) and representations of immune response were explored to identify the best ones to describe data. A simplified 95% drug effect was implemented to represent a highly effective dose of MOV. Results. IVDM showed data were best described when MOV acts on viral infectivity, consistent with the error catastrophe mechanism of action. A cascade of innate and adaptive immune response and a basal level activation enabled durable immunity and continued viral decay after treatment end. IVDM reasonably describes VL and viral titer data from animals and humans. Influence of MOV start time was explored using simulations. Consistent with the ferret studies, simulations showed when treatment is started within the first week post infection, MOV reduces viral growth, resulting in a lower and shortened duration of detectable VL. When started later (e.g. >7 days since symptom onset), the magnitude of drug effect is substantially diminished in a typical patient with an effective immune response which reduces VL prior to treatment start. Further work is needed to model response in patients with longer term infection, where MOV drug effects may have more persistent utility. Conclusion. A COVID-19 IVDM developed using multiscale MOV virology data supports drug action on viral infectivity and importance of interplay of treatment and immune response and can describe infection time course and drug effect. IVDM provided mechanistic interpretations for VL drug effect in clinical studies.

CAR-NK Cells Targeting Sars-Cov-2 Glycosites As COVID-19 Treatment

Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6;Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6;Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3;Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3;Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK;65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05;78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03;n=6;Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. [Formula presented] Disclosures: Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding;BMS: Research Funding;Kiadis Pharma: Rese rch Funding.

Systematic review of the effects of environmental factors on virus inactivation: implications for coronavirus disease 2019.

Environmental factors such as temperature and relative humidity can affect the inactivation and transmission of coronaviruses. By reviewing medical experiments on virus survival and virus transmission between infected and susceptible species in different temperature and humidity conditions, this study explores the influence of temperature and relative humidity on the survival and transmission of viruses, and provides suggestions, with experimental evidence, for the environmental control measures of Coronavirus Disease 2019. The results indicated that (1) virus viability and infectivity is increased at a low temperature of 5 â„ƒ and reduced at higher temperatures. (2) Virus survival and transmission is highly efficient in a dry environment with low relative humidity, and also in a wet environment with high relative humidity, and it is minimal at intermediate relative humidity. Therefore, in indoor environments, the lack of heating in winter or overventilation, leading to low indoor temperature, can help virus survival and help susceptible people being infected. On the contrary, modulating the indoor relative humidity at an intermediate level is conducive to curb epidemic outbreaks.

SARS-CoV-2 in a stream running through an underprivileged, underserved, urban settlement in São Paulo, Brazil: A 7-month follow-up.

COVID-19 pandemic has led to concerns on the circulation of SARS-CoV-2 in the environment, its infectivity from the environment and, the relevance of transmission via environmental compartments. During 31 weeks, water samples were collected from a heavily contaminated stream going through an urban, underprivileged community without sewage collection. Our results showed a statistically significant correlation between cases of COVID-19 and SARS in the community, and SARS-CoV-2 concentrations in the water. Based on the model, if the concentrations of SARS-CoV-RNA (N1 and N2 target regions) increase 10 times, there is an expected increase of 104% [95%CI: (62-157%)] and 92% [95%CI: (51-143%)], respectively, in the number of cases of COVID-19 and SARS. We believe that differences in concentration of the virus in the environment reflect the epidemiological status in the community, which may be important information for surveillance and controlling dissemination in areas with vulnerable populations and poor sanitation. None of the samples were found infectious based cultures. Our results may be applicable globally as similar communities exist worldwide.

Gamma irradiation-mediated inactivation of enveloped viruses with conservation of genome integrity: Potential application for SARS-CoV-2 inactivated vaccine development.

Radiation inactivation of enveloped viruses occurs as the result of damages at the molecular level of their genome. The rapidly emerging and ongoing coronavirus disease 2019 (COVID-19) pneumonia pandemic prompted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now a global health crisis and an economic devastation. The readiness of an active and safe vaccine against the COVID-19 has become a race against time in this unqualified global panic caused by this pandemic. In this review, which we hope will be helpful in the current situation of COVID-19, we analyze the potential use of γ-irradiation to inactivate this virus by damaging at the molecular level its genetic material. This inactivation is a vital step towards the design and development of an urgently needed, effective vaccine against this disease.

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications - A systematic review.

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009-2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 µM; incubation on ice or at room temperature (20 - 25 °C) for 5-120 min; and dye activation using LED or high light (500-800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever.