Development of a cell-based DIFF-rGFP assay system for generalized discovery of viral protease Inhibitors (preprint)

Viral protease is an attractive target for antiviral therapeutics, but current viral protease inhibitor screening methods still need to be improved. Here, we systematically investigated the sites that may accommodate exogenous short peptides within Enhanced Green Fluorescent Protein (EGFP)and constructed a series of recombinant green fluorescent proteins (rGFPs). Meanwhile, a cell-based, simple and reliable assay system named DIFF-rGFP was developed relying on the co-expression of rGFP and the protease for protease inhibitor screening with the example of 3CLpro, in which the fluorescence intensity increases with the action of the inhibitor. The DIFF-rGFP assay avoided the requirement of a higher biosafety lab and can be performed in a high-throughput manner. For proof of concept, we demonstrated this method to discover novel inhibitors against SARS-CoV-2. We believe the proposed method, in combination with available drug libraries, may accelerate the identification of novel antivirals.

Safety, immunogenicity, and efficacy of the mRNA vaccine CS-2034 as a heterologous booster versus homologous booster with BBIBP-CorV in adults aged ≥18 years: a randomised, double-blind, phase 2b trial.

BACKGROUND: Heterologous boosting is suggested to be of use in populations who have received inactivated COVID-19 vaccines. We aimed to assess the safety and immunogenicity of a heterologous vaccination with the mRNA vaccine CS-2034 versus the inactivated BBIBP-CorV as a fourth dose, as well as the efficacy against the SARS-CoV-2 omicron (BA.5) variant. METHODS: This trial contains a randomised, double-blind, parallel-controlled study in healthy participants aged 18 years or older (group A) and an open-label cohort in participants 60 years and older (group B), who had received three doses of inactivated whole-virion vaccines at least 6 months before enrolment. Pregnant women and people with major chronic illnesses or a history of allergies were excluded. Eligible participants in group A were stratified by age (18-59 years and ≥60 years) and then randomised by SAS 9.4 in a ratio of 3:1 to receive a dose of the mRNA vaccine (CS-2034, CanSino, Shanghai, China) or inactivated vaccine (BBIBP-CorV, Sinopharm, Beijing, China). Safety and immunogenicity against omicron variants of the fourth dose were evaluated in group A. Participants 60 years and older were involved in group B for safety observations. The primary outcome was geometric mean titres (GMTs) of the neutralising antibodies against omicron and seroconversion rates against BA.5 variant 28 days after the boosting, and incidence of adverse reactions within 28 days. The intention-to-treat group was involved in the safety analysis, while all patients in group A who had blood samples taken before and after the booster were involved in the immunogenicity analysis. This trial was registered at the Chinese Clinical Trial Registry Centre (ChiCTR2200064575). FINDINGS: Between Oct 13, and Nov 22, 2022, 320 participants were enrolled in group A (240 in the CS-2034 group and 80 in the BBIBP-CorV group) and 113 in group B. Adverse reactions after vaccination were more frequent in CS-2034 recipients (158 [44·8%]) than BBIBP-CorV recipients (17 [21·3%], p<0·0001). However, most adverse reactions were mild or moderate, with grade 3 adverse reactions only reported by eight (2%) of 353 participants receiving CS-2034. Heterologous boosting with CS-2034 elicited 14·4-fold (GMT 229·3, 95% CI 202·7-259·4 vs 15·9, 13·1-19·4) higher concentration of neutralising antibodies to SARS-CoV-2 omicron variant BA.5 than did homologous boosting with BBIBP-CorV. The seroconversion rates of SARS-CoV-2-specific neutralising antibody responses were much higher in the mRNA heterologous booster regimen compared with BBIBP-CorV homologous booster regimen (original strain 47 [100%] of 47 vs three [18·8%] of 16; BA.1 45 [95·8%] of 48 vs two [12·5%] 16; and BA.5 233 [98·3%] of 240 vs 15 [18·8%] of 80 by day 28). INTERPRETATION: Both the administration of mRNA vaccine CS-2034 and inactivated vaccine BBIBP-CorV as a fourth dose were well tolerated. Heterologous boosting with mRNA vaccine CS-2034 induced higher immune responses and protection against symptomatic SARS-CoV-2 omicron infections compared with homologous boosting, which could support the emergency use authorisation of CS-2034 in adults. FUNDING: Science and Technology Commission of Shanghai, National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Current genetic strategies to investigate gene functions in Trichoderma reesei.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina, Ascomycota) is a well-known lignocellulolytic enzymes-producing strain in industry. To increase the fermentation titer of lignocellulolytic enzymes, random mutagenesis and rational genetic engineering in T. reesei were carried out since it was initially found in the Solomon Islands during the Second World War. Especially the continuous exploration of the underlying regulatory network during (hemi)cellulase gene expression in the post-genome era provided various strategies to develop an efficient fungal cell factory for these enzymes' production. Meanwhile, T. reesei emerges competitiveness potential as a filamentous fungal chassis to produce proteins from other species (e.g., human albumin and interferon α-2b, SARS-CoV-2 N antigen) in virtue of the excellent expression and secretion system acquired during the studies about (hemi)cellulase production. However, all the achievements in high yield of (hemi)cellulases are impossible to finish without high-efficiency genetic strategies to analyze the proper functions of those genes involved in (hemi)cellulase gene expression or secretion. Here, we in detail summarize the current strategies employed to investigate gene functions in T. reesei. These strategies are supposed to be beneficial for extending the potential of T. reesei in prospective strain engineering.

Improvement of stress adaptation and insulin resistance in women with GDM by WeChat group management during novel coronavirus pneumonia.

Aim: To evaluate the improvement of glycemic control and stress adaptation in patients with GDM by mobile phone WeChat management during novel coronavirus pneumonia. Methods: In this study, 75 women with GDM were included, of whom 35 were included in mobile WeChat group management as the GDM-M group and 40 as the GDM group. Results: After mobile WeChat group management for 4 weeks, E and NE were lower. MDA was lower, and SOD was higher. HOMA-IR was lower. E, NE, and cortisol were related to HOMA-IR positively, MDA was positively related to HOMA-IR, and SOD was negatively related to HOMA-IR. E and cortisol were positively related to MDA but negatively related to SOD. Conclusion: The stress adaptation disorder and insulin resistance in patients with GDM who have completed mobile WeChat group management can be improved during novel coronavirus pneumonia. Mobile WeChat management played a positive role in improving the insulin resistance of women with GDM under special circumstances, which may reduce the risk of maternal and fetal complications.

Improvement of stress adaptation and insulin resistance in women with GDM by WeChat group management during novel coronavirus pneumonia

Aim To evaluate the improvement of glycemic control and stress adaptation in patients with GDM by mobile phone WeChat management during novel coronavirus pneumonia. Methods In this study, 75 women with GDM were included, of whom 35 were included in mobile WeChat group management as the GDM-M group and 40 as the GDM group. Results After mobile WeChat group management for 4 weeks, E and NE were lower. MDA was lower, and SOD was higher. HOMA-IR was lower. E, NE, and cortisol were related to HOMA-IR positively, MDA was positively related to HOMA-IR, and SOD was negatively related to HOMA-IR. E and cortisol were positively related to MDA but negatively related to SOD. Conclusion The stress adaptation disorder and insulin resistance in patients with GDM who have completed mobile WeChat group management can be improved during novel coronavirus pneumonia. Mobile WeChat management played a positive role in improving the insulin resistance of women with GDM under special circumstances, which may reduce the risk of maternal and fetal complications.

Mesophilic Argonaute-based isothermal detection of SARS-CoV-2

Coronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method;however, it has some limitations, such as the need for specific recognition sequences and multiple enzymes for multiplex detection. Therefore, research on the exploration and development of novel nucleases helps to promote specific and sensitive diagnoses. Prokaryotic Argonaute (Ago) proteins exert directed nuclease activity that can target any sequence. Recently, thermophilic Agos have been developed as new detection techniques achieving multiplexity for multiple targets using a single enzyme, as well as accurate recognition of single-base differential sequences. In this study, to overcome the requirement for high reaction temperature of thermophilic Ago-based methods, we expanded the mining of mesophilic Agos to achieve CRISPR-like isothermal detection, named mesophilic Ago-based isothermal detection method (MAIDEN). The principle of MAIDEN uses mesophilic Ago cleavage combined with reverse transcription, which can provide single-strand DNA as a substrate and allow cleavage of fluorescence probes to sense SARS-CoV-2 at moderate temperature. We first mined and optimized the mesophilic Ago and the fluorescence reporter system and then selected a compatible reverse transcription reaction. Furthermore, we optimized MAIDEN into a one-step reaction that can detect SARS-CoV-2 RNA at the nanomolar concentration at a constant temperature of 42°C within 60 min. Therefore, MAIDEN shows advantageous portability and easy-to-implement operation, avoiding the possibility of open-lid contamination. Our study was the first attempt to demonstrate that mesophilic Agos can be harnessed as diagnostic tools, and MAIDEN was easily extended to detect other pathogens in a rapid and efficient manner.

Humoral responses after inactivated COVID-19 vaccination in individuals with and without prior SARS-CoV-2 infection: A prospective cohort study.

We evaluated and compared humoral immune responses after inactivated coronavirus disease 2019 (COVID-19) vaccination among naïve individuals, asymptomatically infected individuals, and recovered patients with varying severity. In this multicenter, prospective cohort study, blood samples from 666 participants were collected before and after 2 doses of inactivated COVID-19 vaccination. Among 392 severe acute respiratory syndrome coronavirus 2-naïve individuals, the seroconversion rate increased significantly from 51.8% (median antispike protein pan-immunoglobulins [S-Igs] titer: 0.8 U/ml) after the first dose to 96% (median S-Igs titer: 79.5 U/ml) after the second dose. Thirty-two percent of naïve individuals had detectable neutralizing antibodies (NAbs) against the original strain but all of them lost neutralizing activity against the Omicron variant. In 274 individuals with natural infection, humoral immunity was significantly improved after a single vaccine dose, with median S-Igs titers of 596.7, 1176, 1086.5, and 1828 U/ml for asymptomatic infections, mild cases, moderate cases, and severe/critical cases, respectively. NAb titers also improved significantly. However, the second dose did not substantially increase antibody levels. Although a booster dose is needed for those without infection, our findings indicate that recovered patients should receive only a single dose of the vaccine, regardless of the clinical severity, until there is sufficient evidence to confirm the benefits of a second dose.

Comparison of initial clinic characteristics of hospitalized patients in Suzhou City during the COVID-19 Omicron wave with ancestral variant wave.

BACKGROUND: Recently, the SARS-CoV-2 variant of concern, Omicron (B.1.1.529), was identified as responsible for a novel wave of COVID-19 worldwide. Here, we compared initial clinical features of hospitalized COVID-19 patients during recent wave (Omicron Variant) with those in ancestral variant wave (2020). METHODS: This is a cohort study of electronic health record (EHR) data from a signal center in the China. The clinical data of 116 cases of Omicron hospitalized in 2022 and 87 cases hospitalized in 2020 were collected. The comparisons were performed with the Mann-Whitney U test, Fisher exact test or the chi-square test, and multivariable logistic regression analysis. RESULTS: Clinically, compared with 2020-cohort, Omicron-cohort was more inclined to cluster in younger population and had more nonsymptomatic (25.0%) and nonsevere cases, as well as suffered from comparable extrapulmonary complication. Radiologically, although the major computed tomography (CT) findings of both cohorts were ground-glass opacities (GGOs), crazy-paving pattern was relatively less seen in the Omicron-cohort. Based on multiple logistic regression analysis, Omicron-cohort was associated with a lower risk of complaining with fever, the presence of lung opacity, and increased Sequential Organ Failure Assessment (SOFA) score. CONCLUSION: This study provided the data of different patterns of clinic characteristics and reduced severity from infections that occurred in Omicron variant as compared with the outbreak of the epidemic in 2020 wave (ancestral variant).

Heat Treatment Promotes Ubiquitin-Mediated Proteolysis of SARS-CoV-2 RNA Polymerase and Decreases Viral Load.

Despite extensive efforts, COVID-19 pandemic caused by the SARS-CoV-2 virus is still at large. Vaccination is an effective approach to curb virus spread, but several variants (e.g., delta, delta plus, omicron, and IHU) appear to weaken or possibly escape immune protection. Thus, novel and quickly scalable approaches to restrain SARS-CoV-2 are urgently needed. Multiple evidences showed thermal sensitivity of SARS-CoV-2 and negative correlation between environmental temperature and COVID-19 transmission with unknown mechanism. Here, we reveal a potential mechanism by which mild heat treatment destabilizes the wild-type RNA-dependent RNA polymerase (also known as nonstructural protein 12 (NSP12)) of SARS-CoV-2 as well as the P323L mutant commonly found in SARS-CoV-2 variants, including omicron and IHU. Mechanistically, heat treatment promotes E3 ubiquitin ligase ZNF598-dependent NSP12 ubiquitination leading to proteasomal degradation and significantly decreases SARS-CoV-2 RNA copy number and viral titer. A mild daily heat treatment maintains low levels of both wild-type and P323L mutant of NSP12, suggesting clinical potential. Collectively, this novel mechanism, heat-induced NSP12 degradation, suggests a prospective heat-based intervention against SARS-CoV-2.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

The Impacts of COVID-19 and Policies on Spatial and Temporal Distribution Characteristics of Traffic: Two Examples in Beijing

The global closure policy to limit the spread of the new coronavirus (COVID-19) in 2020 was based on public safety and health considerations. In the implementation of arrangements to prevent the epidemic, the function of the transportation system as a basis for securing cities has been severely affected. After summarizing the domestic and international literature on epidemic policies and travel, this study analyzes the changes of the spatial and temporal distribution characteristics of people’s travel and the impacts in the context of the two epidemic phases in Beijing and abroad. During the epidemic, traffic volume into and out of Beijing showed a downward trend. In our study, we found that total travel volume in Beijing during the Spring Festival in 2020 was down by about 70% year-on-year, the distribution of daily traffic trips during the day was not affected by the outbreak, and six urban areas in the center of Beijing experienced greater declines in travel volume compared to other urban areas. The conclusions of the study can provide a reference for the sustainability and recovery of urban areas and formulation of policies in the subsequent pandemic era in terms of the relationship between public travel and epidemic control.

COVID-19-related diffuse leukoencephalopathy with microbleeds and persistent coma: a case report with good clinical outcome.

A 60-year-old patient presented with respiratory distress, after recently being tested COVID-19 positive and was mechanically ventilated for 15 days. After cessation of sedation, he remained in deep comatose state, without any reaction on pain stimuli (Glasgow Coma Score 3). MRI of the brain showed diffuse leukoencephalopathy and multiple (>50) microbleeds. Diffuse COVID-19-associated leukoencephalopathy with microhaemorrhages is associated with a poor prognosis. However, 3 months later, our patient showed a remarkable recovery and was able to walk independently. This case report shows COVID-related leukoencephalopathy and intracerebral microbleeds, even with persistent comatose state, may have a favourable clinical outcome and prolonged treatment should be considered in individual cases.

Beyond Sociodemographic and COVID-19-Related Factors: The Association Between the Need for Psychological and Information Support from School and Anxiety and Depression.

BACKGROUND In addition to sociodemographic and COVID-19- related factors, the needs of school support, including material, psychological and information support, have seldom been discussed as factors influencing anxiety and depression among college students during the COVID-19 pandemic. MATERIAL AND METHODS In this cross-sectional study, 3351 college students from China were surveyed through questionnaires about their sociodemographic and COVID-19 characteristics, the needs of school support, and their experiences with anxiety and depression. RESULTS Anxiety and depression were reported by 6.88% and 10.50% of students, respectively. Married, higher education, non-medical, and urban students had significantly higher risks of anxiety or depression. Additionally, symptoms such as cough and fever, especially when following a possible contact with suspected individuals, quarantine history of a personal contact, going out 1-3 times a week, not wearing a mask, and spending 2-3 hours browsing COVID-19-related information were significantly associated with the occurrence of anxiety or depression. Those who used methods to regulate their emotional state, used a psychological hotline, and who had visited a psychiatrist showed higher anxiety or depression. Those who used online curricula and books, used preventive methods for COVID-19, and who had real-time information about the epidemic situation of the school showed lower anxiety and depression. CONCLUSIONS In addition to sociodemographic and COVID-19-related aspects, students' needs for psychological assistance and information from schools were also associated with anxiety and depression among college students.

Activation of Nurr1 with Amodiaquine Protected Neuron and Alleviated Neuroinflammation after Subarachnoid Hemorrhage in Rats

Background. Nurr1, a member of the nuclear receptor 4A family (NR4A), played a role in neuron protection, anti-inflammation, and antioxidative stress in multidiseases. We explored the role of Nurr1 on subarachnoid hemorrhage (SAH) progression and investigated the feasibility of its agonist (amodiaquine, AQ) as a treatment for SAH. Methods. SAH rat models were constructed by the endovascular perforation technique. AQ was administered intraperitoneally at 2 hours after SAH induction. SAH grade, mortality, weight loss, neurological performance tests, brain water content, western blot, immunofluorescence, Nissl staining, and qPCR were assessed post-SAH. In vitro, hemin was introduced into HT22 cells to develop a model of SAH. Results. Stimulation of Nurr1 with AQ improved the outcomes and attenuated brain edema. Nurr1 was mainly expressed in neuron, and administration of AQ alleviated neuron injury in vivo and enhanced the neuron viability and inhibited neuron apoptosis and necrosis in vitro. Besides, AQ reduced the amount of IL-1β+Iba-1+ cells and inhibited the mRNA level of proinflammatory cytokines (IL-1β and TNF-α) and the M1-like phenotype markers (CD68 and CD86). AQ inhibited the expression of MMP9 in HT22 cells. Furthermore, AQ reduced the expression of nuclear NF-κB and Nurr1 while increased cytoplasmic Nurr1 in vivo and in vitro. Conclusion. Pharmacological activation of Nurr1 with AQ alleviated the neuron injury and neuroinflammation. The mechanism of antineuroinflammation may be associated with the Nurr1/NF-κB/MMP9 pathway in the neuron. The data supported that AQ might be a promising treatment strategy for SAH.

Clinicopathologic Features of COVID-19: A Case Report and Value of Forensic Autopsy in Studying SARS-CoV-2 Infection.

ABSTRACT: As of August 23, 2020, the 2019 novel coronavirus disease (COVID-19) has infected more than 23,518,340 people and caused more than 810,492 deaths worldwide including 4,717 deaths in China. We present a case of a 53-year-old woman who was admitted to the hospital because of dry coughs and high fever on January 26, 2020, in Wuhan, China. She was not tested for SARS-CoV-2 RNA until on hospital day 11 (illness day 21) because of a significant shortage of test kits at the local hospital. Then, her test was positive for COVID-19 on hospital day 20. Despite intensive medical treatments, she developed respiratory failure with secondary bacterial infection and expired on hospital day 23 (3 days after she was tested positive for SARS-CoV-2 RNA). A systemic autopsy examination, including immunohistochemistry and ultrastructural studies, demonstrates that SARS-CoV-2 can infect multiple organs with profound adverse effect on the immune system, and the lung pathology is characterized by diffuse alveolar damage. Extrapulmonary SARS-CoV-2 RNA was detected in several organs postmortem. The detailed pathological features are described. In addition, this report highlights the value of forensic autopsy in studying SARS-CoV-2 infection and the importance of clinicopathological correlation in better understanding the pathogenesis of COVID-19.

CT imaging of the COVID-19.

COVID-19 pneumonia presented with certain characteristic chest CT imaging features, which are helpful to the radiologist in the early detection and diagnosis of this emerging global health emergency. In this report, we present chest CT findings from five patients with COVID-19. Except for one case with normal lung appearance, all the other four cases had certain characteristics, including ground-glass opacity (GGO), consolidation and atoll sign. The lesions were mainly distributed in the peripheral portion of lung.