The Reduction of SARS-CoV-2 RNA Concentration in the Presence of Sewer Biofilms

Wastewater surveillance has been widely used to track the prevalence of SARS-CoV-2 in communities. Although some studies have investigated the decay of SARS-CoV-2 RNA in wastewater, understanding about its fate during wastewater transport in real sewers is still limited. This study aims to assess the impact of sewer biofilms on the dynamics of SARS-CoV-2 RNA concentration in naturally contaminated real wastewater (raw influent wastewater without extra SARS-CoV-2 virus/gene seeding) using a simulated laboratory-scale sewer system. The results indicated that, with the sewer biofilms, a 90% concentration reduction of the SARS-CoV-2 RNA was observed within 2 h both in wastewater of gravity (GS, gravity-driven sewers) and rising main (RM, pressurized sewers) sewer reactors. In contrast, the 90% reduction time was 8–26 h in control reactors without biofilms. The concentration reduction of SARS-CoV-2 RNA in wastewater was significantly more in the presence of sewer biofilms. In addition, an accumulation of c.a. 260 and 110 genome copies/cm2 of the SARS-CoV-2 E gene was observed in the sewer biofilm samples from RM and GS reactors within 12 h, respectively. These results confirmed that the in-sewer concentration reduction of SARS-CoV-2 RNA in wastewater was likely caused by the partition to sewer biofilms. The need to investigate the in-sewer dynamic of SARS-CoV-2 RNA, such as the variation of RNA concentration in influent wastewater caused by biofilm attachment and detachment, was highlighted by the significantly enhanced reduction rate of SARS-CoV-2 RNA in wastewater of sewer biofilm reactors and the accumulation of SARS-CoV-2 RNA in sewer biofilms. Further research should be conducted to investigate the in-sewer transportation of SARS-CoV-2 and their RNA and evaluate the role of sewer biofilms in leading to underestimates of COVID-19 prevalence in communities.

Convalescent plasma transfusion in severe or critically ill COVID 19 patients: a rapid systematic review

Currently, there is no availability of any proven specific treatment or prevention strategy to fight against COVID-19. Convalescent plasma (CP) therapy is expected to increase survival rates in COVID-19 as in the case of emerging viral infection (SARS-CoV and MERS-CoV). To collect all the studies relevant to CP therapy in critically ill or severe COVID-19 patients and summarize the findings. The systematic review was conducted according to the PRISMA consensus statement. A systematic search was performed in PubMed, Scopus, Web of Science, and Cochrane databases on April 25, 2020. A total of six studies (28 patients) relevant to CP therapy in severe or critical COVID-19 are considered for inclusion. Two authors extracted the data about study characteristics, demographics, symptoms, co-morbidities, clinical classification of COVID-19, drug therapies, oxygen therapy, laboratory results, chest CT, neutralizing antibody titer, SARS-CoV-2 RNA load, aal outcome. The review findings revealed that CP therapy increases lymphocyte count, reduced s serum inflammatory markers (CRP, IL-6, Procalcitonin) and liver enzyme levels (AST or ALT). There was a rise in serum neutralizing antibody titers in 10 of 14 patients after CP transfusion. In 4 of 14 patients, the titer levels remain unchanged after CP transfusion. All 28 cases (100%) achieved negative to the SARS-CoV-2 RNA after CP transfusion. The convalescent plasma transfusion can improve neutralizing antibody titers and reduces the viral load in severe/critical COVID-19 patients. The review recommends a well-controlled trial design is required to give a definite statement on the safety and efficacy of convalescent plasma therapy in severe/critical COVID-19.

PROTEIN SIGNALING MOLECULES AFFECTING THE DEVELOPMENT OF INNATE IMMUNITY MECHANISMS

The principle protein molecules (interferon gene stimulator, adapter proteins, B-cell lymphoma 2 proteins, zinc-finger antiviral protein, and others), mechanisms of apoptosis, necroptosis, perforation of plasma membranes with kinase-like proteins of a mixed line, and ribonucleic acid neutralization, which ensure the development of innate immunity, are described. The main defense mechanisms that viruses have developed at the various stages of evolution are considered. The features of the development of the mechanisms of apoptosis and autophagy in a new coronavirus infection, which are associated with increased secretion of pro-inflammatory cytokines and chemokines, leading to severe damage to host cells, are given. It has been found that serum levels of several proteins formed during autophagy caused by SARS-CoV-2 can be used to predict disease severity. These include a protein associated with microtubules 1A/1B, a protein of sequestoma 1, and a protein of the cellular system of autophagy ― beclin-1. The multifaceted role of interferons in the inhibition of viral infection and the features of the violation of the activating functions of interferons in coronavirus infection are described. The article can be used under the CC BY-NC-ND 4.0 license © Authors, 2022.

SARS-CoV-2 Detection by Extraction-free qRT-PCR for Massive and Rapid COVID-19 Diagnosis During a Pandemic

Introduction/Objective COVID-19 pandemic severely impacted the healthcare and economy on a global scale. It is widely recognized that mass testing is an efficient way to contain the spread of SARS-CoV-2 infection as well as aid in the development of informed policies for disease management. Here we optimized two different protocols for qRT- PCR with direct samples and systematically compared them with the laboratory standard qRT-PCR detection assay. Methods/Case Report RNA samples from 270 subjects collected in two phases at 2020-2021. The groups consisted from positive (n = 240) and negative (n = 30) samples. We compared the performance of qRT-PCR in direct heat- inactivated (95 °C for 5 min, H), heat-inactivated and pelleted (95 °C for 5 min and centrifuged for 10 min at 12,000 g, HC) against standard laboratory protocol for SARS-CoV-2 qRT-PCR (targeting ORF1ab and N genes). Accuracy, sensitivity, and specificity for PCR assays were calculated using caret and epiR packages available in the R software environment for statistical computing. The Wilcoxon matched rank test was used to compare differences in Ct values. Results (if a Case Study enter NA) Our study suggests that HC samples show higher accuracy for SARS-CoV-2 detection PCR assay compared to direct H (89 % (95 % CI: 80–95 %) vs 83 % (95 % CI: 74–91 %) of the detection in RNA). The median ΔCt was lower by 1.55 and 2.29 cycles (Wilcoxon signed-rank test p = 0.0018 and < 0.0001 for ORF1ab and N genes, accordingly) in HC samples compared to H samples. Conclusion Our results suggest that purified RNA provides more accurate results;heat-inactivated and pelleted sample testing with qRT-PCR showed a slight drop in accuracy. However, the latter could also help to significantly increase testing capacity. Switching to the direct sample testing is justified if the number of tests is doubled at least.

Thermal Shock Viral RNA Release in COVID-19 Testing

Introduction/Objective The public health emergency of the COVID-19 pandemic emphasized the crucial role of medical laboratory professionals and scientists in molecular diagnostics laboratories to ensure success in infection control strategies. The demand for laboratory testing using nucleic acid amplification tests to detect SARS-CoV-2 RNA imposed strains in laboratory supplies. Here, we explored an alternative cost-effective solution that will simplify the pre-PCR steps by using a simple heating method to release viral RNA. Methods/Case Report Samples tested using the reference automated extraction method were used:100 samples identified as positive for SARS-CoV-2 RNA and 500 samples tested negative for SARS-CoV-2 RNA were used for the study and sorted with equal distribution according to Ct values of (a) <20, (b) 20–30, and (c) >30.100 ul from swab preserved in Universal Transport Medium was treated with 30 μg of proteinase K, and another set was tested without proteinase K pre-treatment. All samples with or without proteinase K were diluted to minimize PCR inhibitors. The thermal shock protocol was set at (98°C, 5 minutes;4°C, 2 minutes) and screened for purity. Performance and method verification studies were performed. Internal extraction, positive template, and no template controls were markers used for testing quality. The experimental study was performed by qualified testing personnel and all under the same experimental conditions. Results (if a Case Study enter NA) The Ct values from the thermal shock RNA release were compared to the automated extraction method and statistically analyzed.The criteria for acceptability for validation of this new RNA extraction proceeding were set to 100% concordance compared to the commercial kit using an automated extraction. PCR efficiency was at 98% and a slope of -3.3. Within run precision of 2% and limits of detection from 200 to 20,000 copies/uL The method 100% (50/50) concordance on samples previously identified as negative by automated methods and identified 86% (86/100) with a mean difference of 3 Ct. Conclusion Our findings suggest that the thermal shock treatment of nasopharyngeal swabs in viral transport media can successfully extract viral nucleic acid for nucleic acid amplification and is a reasonable alternative for chemical extraction methods when molecular diagnostic laboratories persistently encounter supply chain issues.

RNA Therapeutics: A Healthcare Paradigm Shift.

COVID-19 brought about the mRNA vaccine and a paradigm shift to a new mode of treating and preventing diseases. Synthetic RNA products are a low-cost solution based on a novel method of using nucleosides to act as an innate medicine factory with unlimited therapeutic possibilities. In addition to the common perception of vaccines preventing infections, the newer applications of RNA therapies include preventing autoimmune disorders, such as diabetes, Parkinson's disease, Alzheimer's disease, and Down syndrome; now, we can deliver monoclonal antibodies, hormones, cytokines, and other complex proteins, reducing the manufacturing hurdles associated with these products. Newer PCR technology removes the need for the bacterial expression of DNA, making mRNA a truly synthetic product. AI-driven product design expands the applications of mRNA technology to repurpose therapeutic proteins and test their safety and efficacy quickly. As the industry focuses on mRNA, many novel opportunities will arise, as hundreds of products under development will bring new perspectives based on this significant paradigm shift-finding newer solutions to existing challenges in healthcare.

Safety Profile of Molnupiravir with Significant Effect on COVID-19: A Review

Background: As the COVID era unfolds, researchers reveal that rapid changes in viral genetic material allow viruses to circumvent challenges triggered by the host immune system and resist anti-viral drugs, potentially leading to persistent viral manifestations in host cells. Molnupiravir (RNA-dependent RNA polymerase inhibitor) is a novel anti-viral medicine promising a vital role in coming setbacks.Objectives: This review aims to clarify the safety and efficacy of the molnupiravir molecule in light of existing case studies. As a result, it is intended to explore and discuss the molecular structure, mechanism of action, discovery and development process, preclinical research, clinical investigations, and other subtopics.Methods: A total of 75 publications were searched using multiple engines, such as Google Scholar, PubMed, Web of Science, Embase, Cochrane Library, ClinicalTrials.gov, and others, with a constraint applied to exclude publications published over 11 years ago. Molnupiravir, safety, efficacy, COVID- 19, RdRp, PK-PD, and clinical study were utilized as keywords.Results: Clinical results on molnupiravir are supported by investigations that were recently disclosed in a study on both sex volunteers (male and female) with an age restriction of 19 to 60 years, followed by a Phase-3 Clinical Trial (NCT04575584) with 775 randomly assigned participants and no fatalities reported due to treatment.Conclusion: Molnupiravir proved a high level of safety, allowing it to be tested further. This review supports the safety and efficacy of this molecule based on the established evidence, which claims the most anticipated employment of molnupiravir in COVID protocol.

Persistence and prevalence of clinical symptoms in SARS-CoV-2 infected patients

Coronavirus disease 2019 (COVID19) is the recently reported disease caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID19 spreads among population through the close/direct contact and nasal droplets when an infected person sneezes, coughs, talks or breaths. On an average, COVID19 infected patient develops clinical symptoms 5-6 days after the infection and some develops symptoms even after 14 days of infection. Detailed study on the symptoms shown by COVID19 patients can help in identifying individuals so that proper identification and isolation of patients can be done and transmission of virus can be reduced significantly. In this study, a total of 99 (66 males and 33 female) COVID19 infected symptomatic patients without any comorbidities were included. Nasopharyngeal and oropharyngeal swabs were used for the collection of samples from the suspected patients to extract total RNA and perform real time PCR for the detection of genetic material for SARS-CoV-2. Samples with Ct value = 36 in case of Orf1ab gene and E gene with good sigmoidal curve were reported as positive for novel SARS-CoV-2. Fever, shortness of breath, dry cough and cough with sputum production persisted for longer timer and were more common symptoms reported by the COVID19 infected patients. In conclusion, understanding of the clinical symptoms shown by COVID19 infected patients can help in identification and isolation of patients so that transmission of virus can be reduced significantly.

Using molecular tools to compare various concentration protocols prior to detecting SARS-CoV-2 RNA in wastewater. (Special Issue: Chemistry and global challenges (part A).)

Background: Reports showed presence of SARS-CoV-2 genetic material in wastewater. Wastewater concentration methods are optimized for detection of non-enveloped viruses so need to be adopted for enveloped viruses and their genetic material. Methods: Conventional (cRT-PCR) and quantitative real time RT-PCR (qRT-PCR) were used as readouts to compare 4 water concentration methods namely, (A) filtration on negatively charged membrane followed by extracting RNA from it, (B) adsorbtion-elution method, (C) flocculation with skimmed milk and (D) polyethylene glycol precipitation, to detect SARS-CoV-2 RNA and 229E human coronavirus (229E-HCoV) as a model for spike-containing enveloped virus from fresh and wastewater. Results: On using cRT-PCR: recovery rate of SARS-CoV-2 RNA was better using method A then B for fresh water and method B then D for wastewater. 229E-HCoV recovery from fresh water was better using method C then A and methods B then D for wastewater. On using qRT-PCR, both methods A and B were better for SARS-CoV-2 RNA recovery from both fresh and wastewater. For the 229E-HCoV methods A was the most efficient for fresh water and method B for wastewater. Conclusion: Method B is recommended for SARS-CoV-2 RNA or whole 229E-HCoV recovery from wastewater.

Apropos of menstrual changes and abnormal uterine bleeding after COVID-19 vaccination.

It is news of 28 October 2022 that the Pharmacovigilance Risk Assessment Committee of the European Medicines Agency has recommended to add heavy menstrual bleeding among the side effects of unknown frequency inside the package insert of nucleoside-modified messenger ribonucleic acid vaccines to prevent coronavirus disease 2019 (COVID-19). The decision has been made in the light of the numerous reports of unexpected menstrual changes or abnormal uterine bleeding following COVID-19 vaccination. Here we advance a possible involvement of the particular adenohypophyseal microcirculation in these strange and still unexplained events.

Photoactive conjugated polymer-based strategy to effectively inactivate RNA viruses

To efficiently combat viral infectious diseases, it is important to develop broadly applicable countermeasures, and efficient antiviral systems can be developed by elaborating the relationship of antiviral efficiency with the interactions between antiviral agents and viruses. In the present study, conjugated polymer (CP)-based photodynamic therapy was used to inhibit RNA virus infections. A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudotyped virus composed of an SARS-CoV-2 envelope coated with the S protein and luciferase RNA genome was employed to assess antiviral efficiency. Three cationic CPs with different backbone structures, fluorene-co-phenylene (PFP), thiophene (PMNT), and phenylene vinylene (PPV), exhibit different photoinactivation effects. The highly efficient photoinactivation of PPV and PMNT is derived from the complete photodegradation of spike proteins, nucleocapsid proteins and nucleic acids of SARS-CoV-2 after binding to the viral spike proteins. Although PFP showed the highest efficiency in the photodegradation of spike proteins due to its strong binding affinity, ineffective viral inhibition was observed, which occurred because the viral gene was partially damaged under light irradiation and the process of delivering the viral gene to cells received assistance. This work preliminarily reveals the effect of CP-virus interactions on their photoinactivation activity and should be beneficial for further research on the development of highly efficient antiviral PDT agents.In this work, a photodynamic therapy system based on conjugated polymers (CPs) is developed to inhibit the infection of RNA viruses. Three cationic CPs with different backbone structures fluorene-co-phenylene (PFP), thiophene (PMNT), and phenylene vinylene (PPV) exhibit different photoinactivation effects. PPV and PMNT cause effective inactivation of viruses under light irradiation, while SARS-CoV-2 pseudotyped viruses keep infectious after treated by PFP, which is determined by the interactions between CPs with the proteins and gene of viruses. This work preliminarily reveals the effect of CP-virus interactions on their photoinactivation activity and would be beneficial to develop high-efficient antiviral PDT agents.

RNA extraction alternative method for SARS-CoV-2 molecular diagnosis

The devastating COVID-19 outbreak posed serious challenges for the diagnostics laboratories, facing global shortage of reagents and equipment. This study aimed at evaluating an additional RNA extraction method respect to those already recommended by WHO and CDC. A new protocol for RNA extraction from nasopharyngeal swab was set up, adapting a Qiagen kit, and validated on a set of 96 clinical samples. The analysis showed a sensitivity of 94% and a specificity of 97%, but considering samples with Ct< 36.5, the sensitivity and the specificity increased to 100%. The adapted method was also able to detect samples with very low viral load (Ct> 38), indicating that the two approaches can be considered equivalent for the SARS-CoV-2 diagnostics. This extraction method can help in increasing the throughput for SARS-CoV-2 molecular test, even in a low automation setting.

Dynamic characteristics of specific antibodies IgM, IgG and viral RNA in patients with COVID-19

Objective: To study the changed trend of IgM and IgG specific antibody with chemiluminescent immunoassay (CLIA) and RT-PCR in SARS-CoV-2 infection patients during hospitalization. Methods: A total of 100 hospitalized patients with SARS-CoV-2 infection who admitted to the First People's Hospital of Zhaoqing were divided into vaccinated group and unvaccinated group according to whether they were vaccinated COVID-19 vaccine or not. The unvaccinated group was further divided into severe, normal, mild and asymptomatic groups. The nucleic acid test results, the positive rate of IgM and IgG antibodies measured by CLIA, and the dynamic trend of S/CO values of all SARS-CoV-2 infected patients since admission 0-<8 days, 8-<15 days, 15-<22 days, 22-<29 days, 29-<36 days and36 days were monitored, and the statistical differences between different groups were compared. Results: The positive rate of IgM antibody in the unvaccinated group 55.6% (15/27) and 0 (0/27) were all significantly higher than that in the vaccinated group 68.5% (50/73) and 49.0% (36/73) at 8-<15 days and 36 days of hospitalization (X2=11.048, 20.805, P < 0.05). The positive rate of IgG antibody in the vaccinated group 96.3% (26/27) and 100% (27/27) were all significantly higher than that in the unvaccinated group 45.2% (31/73) and 78.1% (57/73) at 0-< 8 days and 8-<15 days of hospitalization (X2=21.268, 7.576, P < 0.05). The positive rate of RNA in the unvaccinated were all significantly higher than that in the vaccinated group at 8-<15 days 76.7% (56/73) and 29.6% (8/27), 15-<22 days 65.8% (48/73) and 14.8% (4/27), 22-<29 days 42.5%(31/73) and 7.4% (2/27), 29~<36 days 26.0% (19/73) and 7.4% (2/27) of hospitalization (X2=18.694,20.490, 10.957, 4.119, all P < 0.05). The S/CO value of IgG antibody in the vaccinated group were all significantly higher than that in the unvaccinated group at differentperiods of hospitalization (t=2.841, 7.135, all P < 0.05), but there was no significant difference in the S/CO value of IgM antibodyat different periods of hospitalization in pairwise comparison (P > 0.05). The IgM and IgG antibody levels of severe patients in the unvaccinated group were significantly higher than those in the normal, mild, and asymptomatic groups at 22-<29 days and 29-<36 days of hospitalization (F=17.694,15.116, 4.037, 4.115, all P < 0.05). Conclusion: IgM and IgG antibody levels in severe patients are more activated by immune defense during recovery. In the case of vaccination, IgM antibody can well reflect the whole course of SARS-CoV-2 infection.

SARS-CoV-2 morphology, genome, life cycle and our bodies' immune response: a review Ilyes Zatla, Lamia Boublenza, Hafida Hassaine

A novel coronavirus strain has been testing the capabilities of our modern world and suffocating health care systems, while bringing together scientist's researches and governmental powers, to fight off its robust viral disease. A new zoonotic pathogenic member of the human coronaviruses, that was first documented in Wuhan, China, has crossed the species barrier to infect humans and caused an outbreak of viral pneumonia. In this brief review, we'll discuss the virology of SARS-CoV-2, the virus that causes COVID-19, covering the general structure of the virus, its genetics and its process of replication. SARS-CoV-2 gets into the cell through the recognition of the angiotensin-converting enzyme 2 (ACE2) receptors by the spike glycoprotein, with the aid of the priming protein transmembrane serine protease 2 (TMPRSS2), which is important for its activation, and replicates as a result of a complex process that involves RNA synthesis, proofreading and capping.

CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

Polymerase chain reaction (PCR)‐based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR‐mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point‐of‐care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR‐based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics‐target preamplification‐free detection, microRNA (miRNA) testing, non‐nucleic‐acid detection, and SARS‐CoV‐2 detection‐are also covered.

Effectiveness of rapid immunochromatographic tests to the detection of feline Coronavirus

Feline coronavirus (FCoV) belongs among pathogens with common occurrence in the cats population in the whole world. FCoV is ubiquitous in environments with a higher concentration of cats, e.g. in shelters, multicat households and kennels. FCoV primarily attacks the digestive feline tract, replicates in its cells and is excreted in the feces to surroundings of permanently or transiently infected cats. The aim of the study was the detection of FCoV in the feces of newly admitted cats to the shelter by the qPCR method and by means of commercial rapid immunochromatographic (antigen) tests from three different producers. For each of the antigen tests, sensitivity and specifity were determined by comparison with the qPCR analysis result. Out of 70 examined fecal samples, viral RNA was by the qPCR analysis identified in 44 samples (62.9%). Neither the age nor the gender of cats played a significant role in the viral excretion. Found sensitivity of the antigen tests was at a low (< 35%;tests A and C) to a satisfactory level (> 50%, test B). The number of viral particles in the samples determined by the qPCR method did not correlate significantly with the result of the antigen tests. The results of this study suggest that the use of rapid antigen tests for routine screening of FCoV shedding in feline shelters is limited due to the high rate of false-negative results.

STING gene knockout pig alveolar macrophage cell model constructed by CRISPR/Cas9 technology and its effect on type I interferon transcription. (Special Issue: Swine industry science.)

Objective: To elucidate the mechanism of interferon gene stimulating factor (STING) in the anti-pathogenic microbial infection of pigs, so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis, epidemic diarrhea and porcine pseudorabies. Method: High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/ Cas9 technology. The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers, packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells. Monoclonal cell lines were obtained by puromycin screening and limited dilution method. The knockout efficiencies of the STING gene were identified by PCR amplification, Sequencing and Western blotting;The effect of STING gene knockout on the expression of type I interferon was verified by real-time fluorescent quantitative PCR. Result: When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector, the editing effect of STING eukaryotic expression carrier could be detected in cells, and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency. Thus, the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were transfected 293T cells to package lentivirus, and then infected 3D4/21 cells with lentivirus. The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained. The STING protein was not observed by Western blotting, indicating that the STING gene knockout 3D4/21 cells(3D4/ 21-STING-/-)were successfully constructed. The transcription level of IFN-beta in 3D4/21-STING-/- cells decreased significantly (P<0.05) compared with parental cells when stimulated by transfection of Haemophilusparasuis DNA. Conclusion : By applying CRISPR/Cas9 technology, STING gene is successfully knock out in 3D4/21 cells, resulting in loss of function of STING gene;STING knockout leads to the transcription disorder of type I interferon when cells are stimulated by DNA, which also suggests that STING gene may be a key factor in the anti-pathogenic microbial infection of pigs.

SARS-CoV-2 RNA abundance in wastewater as a function of distinct urban sewershed size

During the COVID-19 pandemic, wastewater-based epidemiology has emerged as a promising approach for monitoring SARS-CoV-2 prevalence on a community-level. Despite much being known about the utility of making these measurements in large wastewater treatment plants, little is known about the correlation with finer geographic resolution, such as those obtained through sewershed sub-area catchments. This study aims to identify community wastewater surveillance characteristics between sewershed areas that affect the strength of the association of SARS-CoV-2 RNA detection in a metropolitan area. For this, wastewater from 17 sewershed areas were sampled in Louisville/Jefferson County, Kentucky (USA), from August 2020 to April 2021 (N = 727), which covered approximately 97% of the county's households. Solids were collected from the treatment plants from November 2020 to December 2020 (N = 42). Our results indicate that the sewersheds differ in SARS-CoV-2 trends;however, high pairwise correlation spatial trends were not observed, and the mean SARS-CoV-2 RNA concentrations of smaller upstream community sewershed areas did not differ from their respective treatment centers. Solid samples could only be collected at treatment plants, therefore not allowing us to evaluate SARS-CoV-2 abundance as a function of the sewershed scale. The population size sensitivity of SARS-CoV-2 concentration detection is non-linear: at low population levels the measures are either too sensitive and generate a high level of variability, or at high population levels the estimates are dampened making small changes in community infection levels more difficult to discern. Our results suggest selecting sampling sites that include a wide population range. This study and its findings may inform other system-wide strategies for sampling wastewater for estimating non-SARS-CoV-2 targets.

Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.