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.

Fructus aurantii extracts ameliorate intestinal inflammation and regulate the intestinal barrier through Fc epsilon RI signaling pathway

Fructus Aurantii (FA) is the dry and immature fruit of Citrus aurantium L. and its rutaceous cultivars. FA has been widely used to treat digestive system diseases since ancient China, and it promotes gastrointestinal (GI) motility in functional dyspepsia (FD), but its potential therapeutic mechanisms remain unclear. We examined the effects of FA ethanol extracts in an iodoacetamide (IA)-induced FD rat model. Firstly, key FA therapy targets for FD were gathered using systematic pharmacology. Combined with systemic pharmacological analyses, plasma metabolomics based on UPLC-QTOF-MS were conducted. Then, MetaboAnalyst was used to jointly analyze systemic pharmacology targets and metabolomic metabolites to select key metabolic pathways. Finally, the key path is verified by experiments. FA exerted distinct therapeutic effects in anti-inflammation and promoting gastrointestinal motility in our IA-induced FD rat model. When compared with the model group, FA down-regulated the inflammatory factors interleukin 1beta and tumor necrosis factor-a. At the same time, FA up-regulated tight junction proteins in the intestinal epithelial barrier. Through the integrated analysis of metabolomics and systemic pharmacology, we conducted experimental verification on Fc epsilon RI signaling pathway. When compared with the model group, FA down-regulatedphospho-mitogen activated protein kinase, phospho-extracellular signal regulated kinase1/2, myosin light chain kinase, and phospho-myosin regulatory light chain protein levels. Thus, FA ameliorated FD by regulating the Fc epsilon RI signaling pathway. Our integrated strategy identified underlying FA mechanisms toward FD treatment and provided a foundation for FA development as a clinical agent for FD.

Remove of triclosan from aqueous solutions by nanoflower MnO2: insight into the mechanism of oxidation and adsorption

Triclosan (TCS) has been proved to have a harmful effect on human health and ecological environment, especially during the COVID-19 epidemic, when plentiful antibacterial hand sanitizers were discharged. Manganese dioxide (MnO2) showed a good effect on the removal of TCS. The morphology of MnO2 was regulated in this study to increase the active sites for removing TCS and improve the removal effect. The results showed that nanoflower T-MnO2 exhibited best removal efficiency due to its high oxygen vacancy, high Mn3+ content, easily released lattice oxygen and unique tunnel structure which make its Mn-O bond easier to activate. Further study of the mechanism revealed that the process of removing TCS by MnO2 was the first adsorption and then oxidation process and the detailed reaction process was clarified. 3-chlorophenol and 2,4-dichlorophenol were proved to be their oxidative product. Additionally, it was verified that oxidation dominated in the removal of TCS by MnO2 rather than adsorption through Density functional theory (DFT) calculations analysis. It is determined that nanoflower MnO2 was a promising material for removing TCS.

Characteristics of the Severe Acute Respiratory Syndrome Coronavirus 2 Omicron BA.2 Subvariant in Jilin, China from March to May 2022

Background and Objectives In the midst of the pandemic, new coronavirus mutants continue to emerge;the most relevant variant worldwide is omicron. Here, patients who recovered from the disease living in Jilin Province were analyzed to identify factors affecting the severity of omicron infection and to provide insights into its spread and early indication. Methods In this study, 311 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were divided into two groups. Data on the patients' demographic characteristics and laboratory tests, including platelet count (PLT), neutrophil count (NE), C-reactive protein (CRP), serum creatinine (SCR), and neutrophil-to-lymphocyte ratio (NLR), were collected. The biomarkers for moderate and severe coronavirus disease 2019 (COVID-19) and factors affecting the incubation period and time to subsequent negative nucleic acid amplification test (NAAT) were also investigated. Results Age, gender, vaccination, hypertension, stroke, chronic obstructive pulmonary disease (COPD)/chronic bronchitis/asthma, and some laboratory tests were statistically different between the two groups. In the receiver operating characteristic (ROC) analysis, PLT and CRP had higher area under the ROC curve values. In the multivariate analysis, age, hypertension, COPD/chronic bronchitis/asthma, and CRP were correlated with moderate and severe COVID-19. Moreover, age was correlated with longer incubation. In the Kaplan-Meier curve analysis, gender (male), CRP, and NLR were associated with longer time to subsequent negative NAAT. Conclusions Older patients with hypertension and lung diseases were likely to have moderate or severe COVID-19, and younger patients might have a shorter incubation. A male patient with high CRP and NLR levels might take more time to turn back negative in the NAAT.

SARS-CoV-2 RdRp uses NDPs as a substrate and is able to incorporate NHC into RNA from diphosphate form molnupiravir.

The coronavirus disease 2019 has been ravaging throughout the world for three years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 employs the viral RNA dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Systematic characterization of RdRp will undoubtedly aid in the development of antiviral drugs targeting RdRp. Here, our research reveals that RdRp can recognize and utilize nucleoside diphosphates as a substrate to synthesize RNA with an efficiency of about two thirds of using nucleoside triphosphates as a substrate. Nucleoside diphosphates incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate ß-d-N4-hydroxycytidine into RNA while using diphosphate form molnupiravir as a substrate. This incorporation results in genome mutation and virus death. It is also observed that diphosphate form molnupiravir is a better substrate for RdRp than the triphosphate form molnupiravir, presenting a new strategy for drug design.

Host protection against Omicron BA.2.2 sublineages by prior vaccination in spring 2022 COVID-19 outbreak in Shanghai.

The Omicron family of SARS-CoV-2 variants are currently driving the COVID-19 pandemic. Here we analyzed the clinical laboratory test results of 9911 Omicron BA.2.2 sublineages-infected symptomatic patients without earlier infection histories during a SARS-CoV-2 outbreak in Shanghai in spring 2022. Compared to an earlier patient cohort infected by SARS-CoV-2 prototype strains in 2020, BA.2.2 infection led to distinct fluctuations of pathophysiological markers in the peripheral blood. In particular, severe/critical cases of COVID-19 post BA.2.2 infection were associated with less pro-inflammatory macrophage activation and stronger interferon alpha response in the bronchoalveolar microenvironment. Importantly, the abnormal biomarkers were significantly subdued in individuals who had been immunized by 2 or 3 doses of SARS-CoV-2 prototype-inactivated vaccines, supporting the estimation of an overall 96.02% of protection rate against severe/critical disease in the 4854 cases in our BA.2.2 patient cohort with traceable vaccination records. Furthermore, even though age was a critical risk factor of the severity of COVID-19 post BA.2.2 infection, vaccination-elicited protection against severe/critical COVID-19 reached 90.15% in patients aged ≽ 60 years old. Together, our study delineates the pathophysiological features of Omicron BA.2.2 sublineages and demonstrates significant protection conferred by prior prototype-based inactivated vaccines.

Primary assessment of the diversity of Omicron sublineages and the epidemiologic features of autumn/winter 2022 COVID-19 wave in Chinese mainland.

With the recent ongoing autumn/winter 2022 COVID-19 wave and the adjustment of public health control measures, there have been widespread SARS-CoV-2 infections in Chinese mainland. Here we have analyzed 369 viral genomes from recently diagnosed COVID-19 patients in Shanghai, identifying a large number of sublineages of the SARS-CoV-2 Omicron family. Phylogenetic analysis, coupled with contact history tracing, revealed simultaneous community transmission of two Omicron sublineages dominating the infections in some areas of China (BA.5.2 mainly in Guangzhou and Shanghai, and BF.7 mainly in Beijing) and two highly infectious sublineages recently imported from abroad (XBB and BQ.1). Publicly available data from August 31 to November 29, 2022 indicated an overall severe/critical case rate of 0.035% nationwide, while analysis of 5706 symptomatic patients treated at the Shanghai Public Health Center between September 1 and December 26, 2022 showed that 20 cases (0.35%) without comorbidities progressed into severe/critical conditions and 153 cases (2.68%) with COVID-19-exacerbated comorbidities progressed into severe/critical conditions. These observations shall alert healthcare providers to place more resources for the treatment of severe/critical cases. Furthermore, mathematical modeling predicts this autumn/winter wave might pass through major cities in China by the end of the year, whereas some middle and western provinces and rural areas would be hit by the upcoming infection wave in mid-to-late January 2023, and the duration and magnitude of upcoming outbreak could be dramatically enhanced by the extensive travels during the Spring Festival (January 21, 2023). Altogether, these preliminary data highlight the needs to allocate resources to early diagnosis and effective treatment of severe cases and the protection of vulnerable population, especially in the rural areas, to ensure the country's smooth exit from the ongoing pandemic and accelerate socio-economic recovery.

Class-Aware Attention Network for infectious keratitis diagnosis using corneal photographs.

Infectious keratitis is one of the common ophthalmic diseases and also one of the main blinding eye diseases in China, hence rapid and accurate diagnosis and treatment for infectious keratitis are urgent to prevent the progression of the disease and limit the degree of corneal injury. Unfortunately, the traditional manual diagnosis accuracy is usually unsatisfactory due to the indistinguishable visual features. In this paper, we propose a novel end-to-end fully convolutional network, named Class-Aware Attention Network (CAA-Net), for automatically diagnosing infectious keratitis (normal, viral keratitis, fungal keratitis, and bacterial keratitis) using corneal photographs. In CAA-Net, a class-aware classification module is first trained to learn class-related discriminative features using separate branches for each class. Then, the learned class-aware discriminative features are fed into the main branch and fused with other feature maps using two attention strategies to assist the final multi-class classification performance. For the experiments, we have built a new corneal photograph dataset with 1886 images from 519 patients and conducted comprehensive experiments to verify the effectiveness of our proposed method. The code is available at https://github.com/SWF-hao/CAA-Net_Pytorch.

Rapidly accelerated development of neutralizing COVID-19 antibodies by reducing cell line and CMC development timelines.

Since the Coronavirus Disease 2019 (COVID-19) outbreak, unconventional cell line development (CLD) strategies have been taken to enable development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies at expedited speed. We previously reported a novel chemistry, manufacturing, and control (CMC) workflow and demonstrated a much-shortened timeline of 3-6 months from DNA to investigational new drug (IND) application. Hereafter, we have incorporated this CMC strategy for many SARS-CoV-2-neutralizing antibody programs at WuXi Biologics. In this paper, we summarize the accelerated development of a total of seven antibody programs, some of which have received emergency use authorization  approval in less than 2 years. Stable pools generated under good manufacturing practice (GMP) conditions consistently exhibited similar productivity and product quality at different scales and batches, enabling rapid initiation of phase I clinical trials. Clones with comparable product quality as parental pools were subsequently screened and selected for late-stage development and manufacturing. Moreover, a preliminary stability study plan was devised to greatly reduce the time required for final clone determination and next-generation sequencing-based viral testing was implemented to support rapid conditional release of the master cell bank for GMP production. The successful execution of these COVID-19 programs relies on our robust, fit for purpose, and continuously improving CLD platform. The speed achieved for pandemic-related biologics development may innovate typical biologics development timelines and become a new standard in the industry.

Characteristics of the Severe Acute Respiratory Syndrome Coronavirus 2 Omicron BA.2 Subvariant in Jilin, China from March to May 2022.

Background and Objectives: In the midst of the pandemic, new coronavirus mutants continue to emerge; the most relevant variant worldwide is omicron. Here, patients who recovered from the disease living in Jilin Province were analyzed to identify factors affecting the severity of omicron infection and to provide insights into its spread and early indication. Methods: In this study, 311 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were divided into two groups. Data on the patients' demographic characteristics and laboratory tests, including platelet count (PLT), neutrophil count (NE), C-reactive protein (CRP), serum creatinine (SCR), and neutrophil-to-lymphocyte ratio (NLR), were collected. The biomarkers for moderate and severe coronavirus disease 2019 (COVID-19) and factors affecting the incubation period and time to subsequent negative nucleic acid amplification test (NAAT) were also investigated. Results: Age, gender, vaccination, hypertension, stroke, chronic obstructive pulmonary disease (COPD)/chronic bronchitis/asthma, and some laboratory tests were statistically different between the two groups. In the receiver operating characteristic (ROC) analysis, PLT and CRP had higher area under the ROC curve values. In the multivariate analysis, age, hypertension, COPD/chronic bronchitis/asthma, and CRP were correlated with moderate and severe COVID-19. Moreover, age was correlated with longer incubation. In the Kaplan-Meier curve analysis, gender (male), CRP, and NLR were associated with longer time to subsequent negative NAAT. Conclusions: Older patients with hypertension and lung diseases were likely to have moderate or severe COVID-19, and younger patients might have a shorter incubation. A male patient with high CRP and NLR levels might take more time to turn back negative in the NAAT.

Predictive value of C-reactive protein for disease severity and survival in COVID-19 patients: a systematic review and meta-analysis.

Coronavirus disease 2019 (COVID-19) is an infectious disease that can develop multiple complications and even be life-threatening. The aim of this study is to summarize current evidence of C-reactive protein's (CRP) predictive value for disease severity and survival of COVID-19 patients, focusing on curing patients and reducing the risk of death. We systematically searched related studies from four large databases: Web of Science, PubMed, China National Knowledge Infrastructure (CNKI), and Wanfang Database, all published between December 2019 and June 2021. Then, we implemented meta-analysis using random-effects models through STATA 15.1 and Review Manager 5.3. We also implemented sensitivity analysis and used funnel plots to check publication bias. From the systematic search of the four databases, we were able to identify 18 studies containing a total of 3052 patients. Meta-analysis results showed that 1) CRP levels were lower in non-severe patients than in severe patients (Standardized Mean Difference (SMD) = - 0.87 mg/L, 95% Confidence Interval (CI) = [ - 1.27, - 0.47], p < 0.001); 2) CRP levels were lower in non-intensive care unit (ICU) patients than in ICU patients (SMD = - 1.39 mg/L, 95% CI = [- 1.68, - 1.11], p < 0.001), and 3) CRP levels were lower in survivors than in non-survivors (SMD =- 1.32 mg/L, 95% CI = [- 1.95, - 0.69], p < 0.001). Sensitivity analysis showed these results were stable. Funnel plots indicated no publication bias. The CRP level may timely reflect disease severity and predict survival of COVID-19 patients and may be worthy of further popularization and application in clinic practice.

Neutrophil infiltration and myocarditis in patients with severe COVID-19: A post-mortem study

Aims To investigate cardiac pathology in critically ill patients with coronavirus disease 2019 (COVID-19) and identify associations between pathological changes and clinical characteristics. Methods The present autopsy cohort study included hearts from 26 deceased patients hospitalized in intensive care units due to COVID-19, and was conducted at four sites in Wuhan, China. Cases were divided into a neutrophil infiltration group and a no-neutrophil group based on the presence or absence of histopathologically identified neutrophilic infiltrates. Results Among the 26 patients, histopathological examination identified active myocarditis in four patients. All patients with myocarditis exhibited extensive accompanying neutrophil infiltration, and all patients without myocarditis did not. The neutrophil infiltration group exhibited significantly higher rates of detection of interleukin-6 (100 vs. 4.6%) and tumor necrosis factor-alpha (100 vs. 31.8%) than the no-neutrophil group (both p < 0.05). On admission, four patients with neutrophil infiltration in myocardium had significantly higher baseline levels of aspartate aminotransferase, D dimer, and high-sensitivity C reactive protein than the other 22 patients (all p < 0.05). During hospitalization, patients with neutrophil infiltration had significantly higher maximum creatine kinase-MB (median 280.0 IU/L vs. 38.7 IU/L, p = 0.04) and higher troponin I (median 1.112 ng/ml vs. 0.220 ng/ml, p = 0.56) than patients without neutrophil infiltration. Conclusion Active myocarditis was frequently associated with neutrophil infiltration in the hearts of deceased patients with severe COVID-19. Patients with neutrophil-infiltrated myocarditis had a series of severely abnormal laboratory test results on admission, and high maximum creatine kinase-MB during hospitalization. The role of neutrophils in severe heart injury and systemic conditions in patients with COVID-19 should be emphasized.

Two cases of intestinal perforation due to mesenteric artery embolism during extracorporeal membrane oxygenation and intra-aortic balloon pumping.

The patient in case 1 was a 50-year-old man who presented to the emergency department of the local hospital with chest pain and syncope for 3 hours due to acute myocardial infarction. He underwent cardiopulmonary resuscitation (CPR) followed by extracorporeal membrane oxygenation (ECMO), and intestinal perforation was detected on day 9. The patient in case 2 was a 58-year-old man who was admitted to the hospital with abdominal pain lasting for 3 days. He also required CPR and ECMO for cardiogenic shock, and intestinal perforation was identified on day 7 of ECMO. We believe that this case report will be important to alert clinicians to the possibility of this complication and to encourage early detection and intervention to improve prognosis. Conventionally, the gastrointestinal tract has received secondary attention in patients receiving ECMO support because the vital organs tend to be considered first. However, this case report illustrates the importance of monitoring gastrointestinal function in patients undergoing ECMO.

Immunogenicity and safety of an egg culture-based quadrivalent inactivated non-adjuvanted subunit influenza vaccine in subjects ≥3 years: A randomized, multicenter, double-blind, active-controlled phase III, non-inferiority trial.

Subunit influenza vaccine only formulated with surface antigen proteins has better safety profiles relative to split-virion influenza vaccine. Compared to the traditional quadrivalent split-virion influenza vaccine, a novel quadrivalent subunit influenza vaccine is urgently needed in China. We completed a phase 3, randomized, double-blind, active-controlled, non-inferiority clinical study at two sites in Henan Province, China. Eligible volunteers were split into four age cohorts (3-8 years, 9-17 years, 18-64 years, and ≥ 65 years, based on their dates of birth) and randomly assigned (1:1) to the subunit and the split-virion ecNAIIV4 groups. All volunteers were intramuscularly administered a single vaccine dose at baseline, and children aged 3-8 years received a boosting dose at day 28. And the immune response was evaluated by measuring hemagglutinin-inhibition antibody titers against the four vaccine strains in blood samples. Safety profiles had nonsignificant differences between the study groups in ≥ 3 years cohort. Most adverse reactions post-vaccination, both local and systemic, were mild to moderate and resolved within 3 days. And no serious adverse events occurred. The immunogenicity of the trial vaccine was non-inferior to the comparator. Further, a two-dose vaccine series can provide better seroprotection than that of a one-dose series in children aged 3-8 years, with clinically acceptable safety profiles. Clinical Trials Registration. ChiCTR2100049934.

Opsonizing antibodies mediated SARS-CoV-2 entry into monocytes leads to inflammation

Opsonizing antibodies and CD16 permit the entry of severe acute respiratory syndrome coronavirus 2 into monocytes/macrophages, which further activate NLRP3 inflammasomes, leading to pyroptosis and the release of inflammatory cytokines.

Effect of the COVID-19 Pandemic on Serum Vitamin D Levels in People Under Age 18 Years: A Systematic Review and Meta-Analysis.

BACKGROUND During the COVID-19 pandemic the implementation of a range of measures to suppress transmission, such as social distancing and home confinement resulted in limited sunlight exposure and physical inactivity in people under age 18 years, which can elevate the risk of vitamin D deficiency and insufficiency. The aim of this study was to systemically evaluate the effect of the COVID-19 pandemic on serum vitamin D levels in people under age 18 years. MATERIAL AND METHODS Following the PRISMA recommendations, we searched PubMed, Embase, and the Cochrane Database for trials from inception to November 3, 2021. All trials assessing the effects of the COVID-19 pandemic on serum vitamin D levels in people under age 18 years were included and analyzed. Mean differences (MDs) of serum 25-hydroxyvitamin D (25[OH] D) levels before and during the COVID-19 pandemic were calculated and pooled using a random-effects model. Risk differences were used to assess changes in the proportions of people under age 18 years with vitamin D deficiency. RESULTS Our analysis included 5 studies comprising 4141 people under age 18 years. The combined result MD of serum 25(OH)D levels before and during the COVID-19 pandemic as 3.28 ng/mL, 95% CI=0.95-5.62 ng/mL, P<0.01] indicated serum 25(OH)D levels were significantly lower during the COVID-19 pandemic. The decreased serum 25(OH)D level was not observed among infants (age under 1 year) (P=0.28). CONCLUSIONS During the COVID-19 pandemic, the serum vitamin D levels of people under age 18 years were significantly lower and vitamin D supplementation for people under age 18 years might reduce the risk of COVID-19. More research is needed to validate the present findings.

Development of the TOA-Related Models for PM2.5 Prediction Pre- and Post-COVID-19 Outbreak over Yangtze River Delta Region of China

The lockdown and the strict regulation measures implemented by Chinese government due to the outbreak of the COVID-19 pandemic not only decelerated the spread of the virus but also brought a positive effect on the nationwide atmospheric quality. In this study, we extended our previous research on remotely sensed estimation of PM2.5 concentrations in Yangtze River Delta region (i.e., YRD) of China from 2019 to the strict regulation period of 2020 (i.e., 24 Jan, 2020-31 Aug, 2020). Unlike the method using aerosol optical depth (AOD) developed in previous studies, we validated the possibility of moderate resolution imaging spectroradiometer (MODIS) top-of-atmosphere (TOA) reflectance (i.e., MODIS TOA) at 21 bands in estimating the PM2.5 concentrations in YRD region. Two random forests (i.e., TOA-sig RF and TOA-all RF) incorporated with different MODIS TOA datasets were developed, and the results showed that the TOA-sig RF model performed better with R2 of 0.81 (RMSE=8.07 μg/m3) than TOA-all RF model with R2 of 0.79 (RMSE=9.13 μg/m3). The monthly averaged PM2.5 exhibited the highest value of 50.81 μg/m3 in YRD region in January 2020 and sharply decreased from February to August 2020. The annual mean PM2.5 concentrations derived by TOA-sig RF model were 47.74, 32.14, and 21.04 μg/m3 in winter, spring, and summer in YRD during the strict regulation period of 2020, respectively, showing much lower values than those in 2019. Our research demonstrated that the PM2.5 concentrations could be effectively estimated by using MODIS TOA reflectance at 21 bands and the random forest.