A versatile integrated tube for rapid and visual SARS-CoV-2 detection.

The coronavirus disease 2019 (COVID-19) caused by novel severe acute respiratory coronavirus 2 (SARS-CoV-2) has been rapidly spreading worldwide. Rapid and widespread testing is essential to promote early intervention and curb the ongoing COVID-19 pandemic. Current gold standard reverse transcription-polymerase chain reaction (RT-PCR) for detecting SARS-CoV-2 is restricted to professional laboratories and well-trained personnel, thus, limiting its widespread use in resource-limited conditions. To overcome these challenges, we developed a rapid and convenient assay using a versatile integrated tube for the rapid and visual detection of SARS-CoV-2. The reaction conditions of the method were optimized using SARS-CoV-2 RNA standards and the sensitivity and specificity were further determined. Finally, it was verified on clinical specimens. The assay was completed within 40 min, and the result was visible by the naked eye. The limits of detection (LODs) for the target ORF1ab and N genes were 50 copies/µl. No cross-reactivity was observed with the RNA standard samples of four respiratory viruses or clinical samples of common respiratory viral infections. Ninety SARS-CoV-2 positive and 30 SARS-CoV-2 negative patient specimens were analyzed. We compared these results to both prior and concurrent RT-PCR evaluations. As a result, the overall sensitivity and specificity for detection SARS-CoV-2 were 94.5 and 100.0%, respectively. Conclusion: The integrated tube assay has the potential to provide a simple, specific, sensitive, one-pot, and single-step assay for SARS-CoV-2.

Development of SARS-CoV-2 animal vaccines using a stable and efficient NDV expression system.

With the continuation of the coronavirus disease 2019 pandemic and the emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants, the control of the spread of the virus remains urgent. Various animals, including cats, ferrets, hamsters, nonhuman primates, minks, tree shrews, fruit bats, and rabbits, are susceptible to SARS-CoV-2 infection naturally or experimentally. Therefore, to avoid animals from becoming mixing vessels of the virus, vaccination of animals should be considered. In the present study, we report the establishment of an efficient and stable system using Newcastle disease virus (NDV) as a vector to express SARS-CoV-2 spike protein/subunit for the rapid generation of vaccines against SARS-CoV-2 in animals. Our data showed that the S and S1 protein was sufficiently expressed in rNDV-S and rNDV-S1-infected cells, respectively. The S protein was incorporated into and displayed on the surface of rNDV-S viral particles. Intramuscular immunization with rNDV-S was found to induce the highest level of binding and neutralizing antibodies, as well as strong S-specific T-cell response in mice. Intranasal immunization with rNDV-S1 provoked a robust T-cell response but barely any detectable antibodies. Overall, the NDV-vectored vaccine candidates were able to induce profound humoral and cellular immunity, which will provide a good system for developing vaccines targeting both T-cell and antibody responses.

A versatile integrated tube for rapid and visual SARS-CoV-2 detection

The coronavirus disease 2019 (COVID-19) caused by novel severe acute respiratory coronavirus 2 (SARS-CoV-2) has been rapidly spreading worldwide. Rapid and widespread testing is essential to promote early intervention and curb the ongoing COVID-19 pandemic. Current gold standard reverse transcription-polymerase chain reaction (RT-PCR) for detecting SARS-CoV-2 is restricted to professional laboratories and well-trained personnel, thus, limiting its widespread use in resource-limited conditions. To overcome these challenges, we developed a rapid and convenient assay using a versatile integrated tube for the rapid and visual detection of SARS-CoV-2. The reaction conditions of the method were optimized using SARS-CoV-2 RNA standards and the sensitivity and specificity were further determined. Finally, it was verified on clinical specimens. The assay was completed within 40 min, and the result was visible by the naked eye. The limits of detection (LODs) for the target ORF1ab and N genes were 50 copies/μl. No cross-reactivity was observed with the RNA standard samples of four respiratory viruses or clinical samples of common respiratory viral infections. Ninety SARS-CoV-2 positive and 30 SARS-CoV-2 negative patient specimens were analyzed. We compared these results to both prior and concurrent RT-PCR evaluations. As a result, the overall sensitivity and specificity for detection SARS-CoV-2 were 94.5 and 100.0%, respectively. Conclusion The integrated tube assay has the potential to provide a simple, specific, sensitive, one-pot, and single-step assay for SARS-CoV-2.

Establishing an integrated prevention and control system to ensure zero nosocomial infections – an analysis of the logistics support in controlling nosocomial infections during the hospital's fight against the COVID-19 epidemic Aufbau eines integrierten Präventions- und Kontrollsystems zur Gewährleistung der Null-Strategie zur Prävention nosokomialer Infektionen – eine Analyse der logistischen Unterstützung zur Prävention nosokomialer Infektionen während des Kampfes des Krankenhauses gegen die COVID-19-Epidemie

Aim: To analyze the role of the logistics support services in nosocomial infection control during emergency periods, with a focus on job responsibilities including the organization of vehicle parking, supply of hospital meals, washing of medical bedding and clothing, disposal and management of medical sewage and waste, elevator services, disinfection of air conditioning systems, disinfection and cleaning of ambulances, management of hospital buildings, storage of sterilization supplies, reception and delivery of oxygen cylinders and protection of staff health as examples. Methods: The adjustment and optimization of the emergency support system and working mode as part of hospitals' response to major public emergencies were summarized, and the vital supporting role of the logistics support services in nosocomial infection control was analyzed. Results: The logistics support services played a crucial role in ensuring the high-performance operations of the hospitals and control of nosocomial infections, resulting in the excellent outcome of "zero infection” among hospital staff. Conclusion: Establishing a safe, flexible and efficient system for the logistics support services is important in ensuring an effective response by hospitals to health emergencies.

Virtual screening and molecular dynamics simulation for identification of natural antiviral agents targeting SARS-CoV-2 NSP10.

New variations of SARS-CoV-2 continue to emerge in the global pandemic, which may be resistant to at least some vaccines in COVID-19, indicating that drug and vaccine development must be continuously strengthened. NSP10 plays an essential role in SARS-CoV-2 viral life cycle. It stimulates the enzymatic activities of NSP14-ExoN and NSP16-O-MTase by the formation of NSP10/NSP14 and NSP10/NSP16 complexes. Inhibiting NSP10 can block the binding of NSP10 to NSP14 and NSP16. This study has identified potential natural NSP10 inhibitors from ZINC database. The protein druggable pocket was identified for screening candidates. Molecular docking of the selected compounds was performed and MM-GBSA binding energy was calculated. After ADMET assessment, 4 hits were obtained for favorable druggability. The analysis of site interactions suggested that the hits all had excellent binding. Molecular dynamics studies revealed that selected natural compounds stably bind to NSP10. These compounds were identified as potential leads against NSP10 for the development of strategies to combat SARS-CoV-2 replication and could serve as the basis for further studies.

Ultralight, Safe, Economical, and Portable Oxygen Generators with Low Energy Consumption Prepared by Air-Breathing Electrochemical Extraction.

Pure oxygen is vital in medical treatment, first aid, and chemical synthesis. Hypoxia can cause severe damage to the organ systems such as respiratory, digestive, and nervous systems and even directly cause death. Notably, the severe Coronavirus disease 2019 (COVID-19) pandemic has exacerbated the shortage of medical oxygen in the world. Hence, a safe, economical, and portable oxygen supply device is urgently needed. Here, we have successfully prepared a device with air-breathing electrochemical extraction of pure oxygen (ABEEPO) with light weight and high energy efficiency. By renovating the structure of the electrolytic cell, the components bipolar plate and end plate are replaced with a plastic membrane, and the component current collector is replaced with a highly conductive graphene composite membrane electrode. Due to the use of the plastic membrane and graphene composite membrane electrode, the weight of the electrolytic cell is reduced from 1319.4 to 1.6 g, and the flexibility of the electrolytic cell is successfully realized. Through optimizing anode catalysts, working area, and operating voltage, a high flow rate per mass (234 mL h-1 g-1) was achieved at a voltage of 1.2 V. The device exhibits high stability in 2 h. The new portable oxygen production device would be effective for hypoxia treatment.

Design of SARS-CoV-2 Mpro, PLpro dual-target inhibitors based on deep reinforcement learning and virtual screening.

Background: Since December 2019, SARS-CoV-2 has continued to spread rapidly around the world. The effective drugs may provide a long-term strategy to combat this virus. The main protease (Mpro) and papain-like protease (PLpro) are two important targets for the inhibition of SARS-CoV-2 virus replication and proliferation. Materials & methods: In this study, deep reinforcement learning, covalent docking and molecular dynamics simulations were used to identify novel compounds that have the potential to inhibit both Mpro and PLpro. Results & conclusion: Three compounds were identified that can effectively occupy the Mpro protein cavity with the PLpro protein cavity and form high-frequency contacts with key amino acid residues (Mpro: His41, Cys145, Glu166; PLpro: Cys111). These three compounds can be further investigated as potential lead compounds for SARS-CoV-2 inhibitors.

Clustering cases of Chlamydia psittaci pneumonia mimicking COVID-19 pneumonia.

BACKGROUND: The onset symptoms of people infected by Chlamydia psittaci can mimic the coronavirus disease 2019 (COVID-19). However, the differences in laboratory tests and imaging features between psittacosis and COVID-19 remain unknown. AIM: To better understand the two diseases and then make an early diagnosis and treatment. METHODS: Six patients from two institutions confirmed as psittacosis by high-throughput genetic testing and 31 patients confirmed as COVID-19 were retrospectively included. The epidemiology, clinical characteristics, laboratory tests and computed tomography (CT) imaging features were collected and compared between the two groups. The follow-up CT imaging findings of patients with psittacosis were also investigated. RESULTS: The white blood cell count (WBC), neutrophil count and calcium were more likely to be decreased in patients with COVID-19 but were increased in patients with psittacosis (all P = 0.000). Lymphocyte count and platelet count were higher in patients with psittacosis than in those with COVID-19 (P = 0.044, P = 0.035, respectively). Lesions in patients with psittacosis were more likely to be unilateral (P = 0.001), involve fewer lung lobes (P = 0.006) and have pleural effusions (P = 0.002). Vascular enlargement was more common in patients with COVID-19 (P = 0.003). Consolidation in lung CT images was absorbed in all 6 patients. CONCLUSION: Psittacosis has the potential for human-to-human transmission. Patients with psittacosis present increased WBC count and neutrophil count and have specific CT imaging findings, including unilateral distribution, less involvement of lung lobes and pleural effusions, which might help us to differentiate it from COVID-19.

MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation.

The highly pathogenic Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a severe respiratory virus. Recent reports indicate additional central nervous system (CNS) involvement. In this study, human DPP4 transgenic mice were infected with MERS-CoV, and viral antigens were first detected in the midbrain-hindbrain 4 days post-infection, suggesting the virus may enter the brainstem via peripheral nerves. Neurons and astrocytes throughout the brain were infected, followed by damage of the blood brain barrier (BBB), as well as microglial activation and inflammatory cell infiltration, which may be caused by complement activation based on the observation of deposition of complement activation product C3 and high expression of C3a receptor (C3aR) and C5a receptor (C5aR1) in neurons and glial cells. It may be concluded that these effects were mediated by complement activation in the brain, because of their reduction resulted from the treatment with mouse C5aR1-specific mAb. Such mAb significantly reduced nucleoprotein expression, suppressed microglial activation and decreased activation of caspase-3 in neurons and p38 phosphorylation in the brain. Collectively, these results suggest that MERS-CoV infection of CNS triggers complement activation, leading to inflammation-mediated damage of brain tissue, and regulating of complement activation could be a promising intervention and adjunctive treatment for CNS injury by MERS-CoV and other coronaviruses.

Summary of global surveillance data of infectious diseases in June 2021

In June 2021, a total of 67 infectious diseases were reported globally, affecting 225 countries and regions. Except for influenza, the top five infectious diseases affecting greatest number of countries and regions were COVID-19 (225), dengue fever (29), measles (19), poliomyelitis (12) and chikungunya (12). The top five infectious diseases with highest case fatality rates were Ebola virus disease (52.2%), Middle East respiratory syndrome (34.4%), lassa fever (20.0%), plague (11.1%) and meningitis (5.6%). The top five infectious diseases with greatest number of deaths were COVID-19, malaria, measles, dengue fever and cholera. The prevalent infectious diseases in Asia were COVID-19 and dengue fever, the prevalent infectious diseases in Africa were COVID-19, cholera, plague, yellow fever and lassa fever, the prevalent infectious diseases in America were COVID-19, dengue fever and chikungunya, the prevalent infectious disease in Europe was COVID-19.

Generation and Characterization of a Nanobody Against SARS-CoV.

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Community Transmission via Indirect Media-To-Person Route: A Missing Link in the Rapid Spread of COVID-19.

To prevent the spread of coronavirus disease 2019 (COVID-19), stringent quarantine measures have been implemented so that healthy people and virus carriers have isolated themselves in the same community owing to the limit capacity of healthcare facilities. With the exponential growth of the infected population, the residential environment is contaminated by fomites from the infected residents and consequently threating the health of susceptible residents. Till now, little has been acknowledged on this indirect transmission route and its role on community transmission. Here we address the impact of self-isolated virus carriers on the residential environment and elucidate the potential transmission pathways via contaminated environment in communities. We urge further investigation on the superspreading cases in communities and hope to arouse the attention to evaluate the potential risk of indirect transmission route as well as the corresponding control measures.

Single-cell analysis reveals cell communication triggered by macrophages associated with the reduction and exhaustion of CD8+ T cells in COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has become an ongoing pandemic. Understanding the respiratory immune microenvironment which is composed of multiple cell types, together with cell communication based on ligand-receptor interactions is important for developing vaccines, probing COVID-19 pathogenesis, and improving pandemic control measures. METHODS: A total of 102 consecutive hospitalized patients with confirmed COVID-19 were enrolled in this study. Clinical information, routine laboratory tests, and flow cytometry analysis data with different conditions were collected and assessed for predictive value in COVID-19 patients. Next, we analyzed public single-cell RNA-sequencing (scRNA-seq) data from bronchoalveolar lavage fluid, which offers the closest available view of immune cell heterogeneity as encountered in patients with varying severity of COVID-19. A weighting algorithm was used to calculate ligand-receptor interactions, revealing the communication potentially associated with outcomes across cell types. Finally, serum cytokines including IL6, IL1ß, IL10, CXCL10, TNFα, GALECTIN-1, and IGF1 derived from patients were measured. RESULTS: Of the 102 COVID-19 patients, 42 cases (41.2%) were categorized as severe. Multivariate logistic regression analysis demonstrated that AST, D-dimer, BUN, and WBC were considered as independent risk factors for the severity of COVID-19. T cell numbers including total T cells, CD4+ and CD8+ T cells in the severe disease group were significantly lower than those in the moderate disease group. The risk model containing the above mentioned inflammatory damage parameters, and the counts of T cells, with AUROCs ranged from 0.78 to 0.87. To investigate the molecular mechanism at the cellular level, we analyzed the published scRNA-seq data and found that macrophages displayed specific functional diversity after SARS-Cov-2 infection, and the metabolic pathway activities in the identified macrophage subtypes were influenced by hypoxia status. Importantly, we described ligand-receptor interactions that are related to COVID-19 serverity involving macrophages and T cell subsets by communication analysis. CONCLUSIONS: Our study showed that macrophages driving ligand-receptor crosstalk contributed to the reduction and exhaustion of CD8+ T cells. The identified crucial cytokine panel, including IL6, IL1ß, IL10, CXCL10, IGF1, and GALECTIN-1, may offer the selective targets to improve the efficacy of COVID-19 therapy. TRIAL REGISTRATION: This is a retrospective observational study without a trial registration number. Video Abstract.

Sensitive and Easy-Read CRISPR Strip for COVID-19 Rapid Point-of-Care Testing.

Rapid and clinically sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) play an important role in the contact tracing and containment of the COVID-19 pandemic. A recently developed field-deployable clustered regularly interspaced short palindromic repeats (CRISPR) detection assay with lateral flow strips shows promise for point-of-care detection of SARS-CoV-2. However, the limit of detection of paper strip-based assays (10-100 copies/µL) is much lower than that of fluorescence-based detection methods. In this study, we developed an easy-readout and sensitive enhanced (ERASE) strip to visualize the results of CRISPR detection and improve the sensitivity to 1 copy/µL with an unambiguous easy-read result. Using 649 clinical samples from blind specimens collected from patients in China, we validated our ERASE assay for SARS-CoV-2 RNA detection with 90.67% positive predictive agreement and 99.21% negative predictive agreement. In conclusion, our study provided a customized CRISPR strip for use in a simple, rapid, ultrasensitive, and highly specific assay for SARS-CoV-2 detection. (Clinical Trial Registration number: 2020-008-01; [2020]IEC(ZD01); PJ-NBEY-2020-009-01; 2020#34).

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.

In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers.

Rapid and accurate diagnosis of COVID-19 plays an essential role in the current epidemic prevention and control. Despite the promise of nucleic acid and antibody tests, there is still a great challenge to reduce the misdiagnosis, especially for asymptomatic individuals. Here we report a generalizable method for highly specific and ultrasensitive detection of serum COVID-19-associated antigens based on an aptamer-assisted proximity ligation assay. The sensor is based on binding two aptamer probes to the same protein target that brings the ligation DNA region into close proximity, thereby initiating ligation-dependent qPCR amplification. Using this system, serum nucleocapsid protein has been detected quantitatively by converting protein recognition into a detectable qPCR signal using a simple, homogeneous and fast detection workflow in ∼2 hours. In addition, this system has also been transformed into a universal platform for measuring specific interactions between spike S1 and its receptor ACE2, and more importantly demonstrated the feasibility for screening and investigation of potential neutralizing aptamers. Since in vitro selection can obtain aptamers selective for many COVID-19-associated antigens, the method demonstrated here will serve as an important tool for the diagnosis and therapeutics of COVID-19.

Clinical characteristics and outcomes of discharged COVID-19 patients with reoccurrence of SARS-CoV-2 RNA.

AIM: Data are limited on clinical characteristics and outcomes of recovered the 2019 coronavirus disease (COVID-19) patients with the reoccurrence of SARS-CoV-2 RNA. PATIENTS & METHODS: Discharged patients in our hospital were included, who had recovered from COVID-19 with the reoccurrence of SARS-CoV-2 RNA. RESULTS: Six patients were redetectable and positive for SARS-CoV-2 RNA after discharge from 3 to 15 days. The main symptoms, although no fever, included fatigue, dry cough and pharyngeal or chest discomfort, which were generally milder in the repositive period compared with the period of initial infection. Their laboratory indexes were significantly improved compared with the initial infection, and the pulmonary lesions were continuously improving. All close contacts were SARS-CoV-2 RNA-negative. CONCLUSION: No worsening outcomes or active transmission to close contacts were found for the repositive COVID-19 patients.