Disease X Testing: The results of an international external quality assessment exercise

The United Nations Secretary-General Mechanism (UNSGM) for investigation of the alleged use of chemical and biological weapons is the only established international mechanism of this type under the UN. The UNGSM may launch an international investigation, relying on a roster of expert consultants, qualified experts, and analytical laboratories nominated by the member states. Under the framework of the UNSGM, we organized an external quality assurance exercise for nominated laboratories, named the Disease X Test, to improve the ability to discover and identify new pathogens that may cause possible epidemics and to determine their animal origin. The "what-if" scenario was to identify the etiological agent responsible for an outbreak that has tested negative for many known pathogens, including viruses and bacteria. Three microbes were added to the samples, Dabie bandavirus, Mammarenavirus, and Gemella spp., of which the last two have not been taxonomically named or published. The animal samples were from Rattus norvegicus, Marmota himalayana, New Zealand white rabbit, and the tick Haemaphysalis longicornis. Of the 11 international laboratories that participated in this activity, six accurately identified pathogen X as a new Mammarenavirus, and five correctly identified the animal origin as R. norvegicus. These results showed that many laboratories under the UNSGM have the capacity and ability to identify a new virus during a possible international investigation of a suspected biological event. The technical details are discussed in this report.Copyright © 2022

Historical perspective: other human coronavirus infectious diseases, SARS and MERS

Alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1) are common causes of upper respiratory tract infection in humans. SARS-CoV-1 and MERS-CoV emerged in 2002 and 2012, respectively, with the potential of causing severe and lethal disease in humans, termed SARS and MERS, respectively. Bats appear to be the common natural source of SARS-like coronaviruses including SARS-CoV-1, but their role in MERS-CoV is less clear. Civet cats and dromedary camels are the intermediary animal sources for SARS-CoV-1 and MERS-CoV, respectively. Nosocomial outbreaks are hallmarks of SARS and MERS. MERS patients with comorbidities or immunosuppression tend to progress more rapidly to respiratory failure and have a higher case fatality rate than SARS patients. SARS has disappeared since 2004, while there are still sporadic cases of MERS in the Middle East. Continued global surveillance is essential for SARS-like coronaviruses and MERS-CoV to monitor changing epidemiology due to viral variants.Copyright © ERS 2021.

Human coronaviruses in the 'One Health' context

Seven human coronaviruses have been identified so far: four seasonal coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1) and three novel coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2). While seasonal coronaviruses cause only mild symptoms, novel coronaviruses cause severe and potentially fatal infections. All known coronaviruses originated in animals. Bats are considered as an origin for the majority of coronaviruses capable of infecting humans;however, rodents are proposed as natural hosts for HCoV-OC43 and HCoV-HKU1. Different animal species could serve as intermediate hosts including alpacas (HCoV-229E), livestock (HCoV-OC43), civet cats (SARS-CoV), camels (MERS-CoV), and pangolins (SARS-CoV-2). In Croatia, SARS-CoV-2 was detected in humans, pet animals, wildlife, and the environment. The COVID-19 pandemic has highlighted the role of the 'One Health' approach in the surveillance of zoonotic diseases.Copyright © 2022, University Hospital of Infectious Diseases. All rights reserved.

Dry-spun carbon nanotube ultrafiltration membranes tailored by anti-viral metal oxide coatings for human coronavirus 229E capture in water.

Although waterborne virus removal may be achieved using separation membrane technologies, such technologies remain largely inefficient at generating virus-free effluents due to the lack of anti-viral reactivity of conventional membrane materials required to deactivating viruses. Here, a stepwise approach towards simultaneous filtration and disinfection of Human Coronavirus 229E (HCoV-229E) in water effluents, is proposed by engineering dry-spun ultrafiltration carbon nanotube (CNT) membranes, coated with anti-viral SnO2 thin films via atomic layer deposition. The thickness and pore size of the engineered CNT membranes were fine-tuned by varying spinnable CNT sheets and their relative orientations on carbon nanofibre (CNF) porous supports to reach thicknesses less than 1 µm and pore size around 28 nm. The nanoscale SnO2 coatings were found to further reduce the pore size down to ∼21 nm and provide more functional groups on the membrane surface to capture the viruses via size exclusion and electrostatic attractions. The synthesized CNT and SnO2 coated CNT membranes were shown to attain a viral removal efficiency above 6.7 log10 against HCoV-229E virus with fast water permeance up to ∼4 × 103 and 3.5 × 103 L.m-2.h-1.bar-1, respectively. Such high performance was achieved by increasing the dry-spun CNT sheets up to 60 layers, orienting successive 30 CNT layers at 45°, and coating 40 nm SnO2 on the synthesized membranes. The current study provides an efficient scalable fabrication scheme to engineer flexible ultrafiltration CNT-based membranes for cost-effective filtration and inactivation of waterborne viruses to outperform the state-of-the-art ultrafiltration membranes.

Therapeutic Effect of Gegentang Granules on Mouse Model with hCoV-229E Pneumonia and Hanshi Yidu Xifei Syndrome.

Objective:To determine the therapeutic effect of Gegentang granules on a disease-syndrome mouse model combining human coronavirus 229EhCoV-229Epneumonia with Hanshi Yidu Xifei syndrome in vivo. Method(s): Mice were randomly divided into normal group,infection group,cold-dampness group,model group,chloroquine phosphate group0.18 g.kg-1,interferon-alpha2bIFN-alpha2bgroup1.83x106 U.kg-1, Gegentang granules high-dose and low-dose groups6.6,3.3 g.kg-1with 10 mice in each group. Cold-dampness environment and hCoV-229E infection were used for modeling,and the general status and lung index of mice in each group were observed. The viral load in lung tissue was detected by real-time fluorescent quantitative polymerase chain reactionReal-time PCR,the pathological changes in lung tissue were evaluated by hematoxylin-eosinHEstaining,the levels of serum gastrointestinal hormones and inflammatory factors in lung tissue were detected by enzyme-linked immunosorbent assayELISA,and the percentage of peripheral blood lymphocytes was detected by flow cytometry. Result(s):Comparing with model group,Gegentang granules could significantly alleviate the physical signs of Hanshi Yidu Xifei syndrome,including listlessness,weakness of limbs,sticky stool,etc. Comparing with model group,Gegentang granules high-dose group significantly reduced lung index,histopathological score of interstitial lung and bronchus,and the level of serum motilinP< 0.05,P<0.01,two doses of Gegentang granules could significantly increase the level of serum gastrinP< 0.05,P<0.01,the percentage of CD4+ ,CD8+ T lymphocytes in peripheral bloodP<0.05,P<0.01,and the level of tumor necrosis factor-alphaTNF-alphain lung tissue was significantly decreasedP<0.01,and the level of interleukin-6IL-6showed decreasing tendency. Conclusion(s): Gegentang granules has therapeutic effect on model mice. It can improve the appearance and behavior characterization,regulate the level of gastrointestinal hormones,decrease lung index and histopathological score,and possibly play an immunomodulatory role by inhibiting the expression of inflammatory cytokines in lung tissue and restoring the percentage of peripheral blood lymphocytes.Copyright © 2022, China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. All rights reserved.

RNA-sequencing unravels unique host-response to infection with SARS-CoV-2

Introduction/Aim: As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Method(s): Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses (n = 4). After infection following apical application of coronaviruses at low dose (MOI 0.1), cells were harvested for bulk RNA sequencing. Gene were considered significant with a fold change (FC) > 2 and false discovery rate of FDR < 0.05. Inhibitor experiments were conducted on CALU-3 cells using DIM-C-pPhOH 10 muM (NR4A1 antagonist), Sp600125 10 muM (JNK inhibitor), T-5224 10 muM (AP-1 transcription factor inhibitor) and Cytosporone B (CsB 5 muM;NR4A1 agonist) preincubated for 1 h with these compounds and subsequently infected with SARS-CoV-2 or MERS-CoV (MOI of 1). Samples were collect 24 h later for PCR. Result(s): PCR and RNA-Seq demonstrated that all tested coronaviruses efficiently infected ALI-PBEC and replicated over 72 h (p < 0.05). RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1 (FC > 1, FDR < 0.05), no such increase was observed following SARS-CoV-2 infection. Further, we found that an NR4A1 antagonist reduced viral replication of MERS and SARs-CoV-2 100-fold in Calu-3 cells. Conclusion(s): In conclusion, these data suggest that SARS-CoV-2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

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.

Proteome2virus: Shotgun mass spectrometry data analysis pipeline for virus identification

Objectives: Shotgun proteomics is a generic method enabling detection of multiple viral species in one assay. The reliable and accurate identification of these viral species by analyzing peptides from MS-spectra is a challenging task. The aim of this study was to develop an easy accessible proteome analysis approach for the identification of viruses that cause respiratory and gastrointestinal infections. Method(s): For this purpose, a shotgun proteomics based method and a web application, 'proteome2virus', were developed. Identified peptides were searched in a database comprising proteomic data of 46 viruses known to be infectious to humans. Result(s): The method was successfully tested for cultured viruses and eight fecal samples consisting of ten different viral species from seven different virus families, including SARS-CoV-2. The samples were prepared with two different sample preparation methods and were measured with two different mass spectrometers. Conclusion(s): The results demonstrate that the developed web application is applicable to different MS data sets, generated from two different instruments, and that with this approach a high variety of clinically relevant viral species can be identified. This emphasizes the potential and feasibility for the diagnosis of a wide range of viruses in clinical samples with a single shotgun proteomics analysis.Copyright © 2023

Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome.

Objective:To determine the therapeutic effect of in vitro cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method: BALB/c mice were randomly divided into six groups according to their weight grade:normal group,HCoV-229E infection group,cold and damp group,a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome,and high and low dose group of in vitro cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. In vitro cultivation of bezoar (0.128,0.064 g.kg-1 )was administrated by gavage for 3 days from the day of infection. The observation indexes included:general state observation of mice,inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the viral load in the lung tissues of mice. Serum levels of motilin(MTL),gastrin(GAS),and cytokines interleukin(IL)-10,IL-6, tumor necrosis factor-alpha(TNF-alpha)and interferon-gamma(IFN-gamma)in lung tissue of mice were determined by enzyme-linked immunosorbent assay(ELISA). The percentages of CD4+ T lymphocytes,CD8+ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of in vitro cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group, model group mice lung index increased significantly(P<0.01), nucleic acids significantly increased expression of lung tissue in mice(P<0.01),significantly higher serum MTL content in mice,GAS content significantly decreased(P<0.05,P<0.01),lung tissue cells in the immune factor TNF-alpha,IL-10 and IL-6 were significantly increased(P<0.01),peripheral blood lymphocyte CD4+ T cells in mice,The percentages of CD8+ T cells and B cells were significantly decreased(P<0.01). Compared with model group, in vitro cultivation bezoar mice lung index of high and low dose group were significantly lower(P<0.01),the lung tissue of mice express nucleic acid decreased significantly(P<0.01),MTL content decreased significantly(P< 0.01),the lung tissue of mice in the IL-6,IL-10,the TNF-alpha,IFN-gamma levels were significantly lower(P<0.01), in vitro cultivation bezoar high dose group can significantly increase the CD4+ T cell percentage(P<0.05),in vitro cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation,pulmonary interstitial edema,as well as blood stasis symptoms. Conclusion:In vitro cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice,improving the pathological damage of the lung tissue,adjusting the immune effective and inhibiting the clearing of inflammatory factors,and to provide a laboratory basis for clinical medication.Copyright © 2021, China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. All rights reserved.

Humoral response against SARS-CoV-2 and other endemic corona viruses

In a study of two Hospitals in Saxony (Chemnitz and Leipzig), we analyzed the antibody development towards SARS-CoV-2 and against a variety of endemic coronaviruses. Here we analyzed 760 sera from a Saxonian cohort for antibody reactivity against: Common cold coronaviruses, HCoV-229E, HCoV-HKU 1, HCoV-NL63 and HCoV-OC43, MERS-CoV and SARS-CoV. For the SARS CoV-2 immune response we tested the following antigens: Spike, S1, S2, RBD and nucleocapsid. These 11 antigen determinants were tested in a commercial multiplex Luminex based assay. We tested sera from 544 individuals (347 females and 197 males;498 SARS-CoV-2 PCR positive and 262 SARS-CoV-2 PCR negative) between May 2020 and March 2022. We observed up to 10% reactivity against the MERS virus in both the PCR positive and negative group. Against the common cold corona viruses 80%-90 % of the individuals in both groups show detectable antibodies. Regarding the antibody response against SARS-CoV a significant difference was observe. Only 19% of COVID-19 infected individuals show antibodies against the virus, while 81% of the PCR-positive individuals produced antibodies. The presence of antibodies against the SARS-CoV-2 is positively correlated with those against SARS-CoV (p = 0.001). No changes in endemic antibody responses were see in the two groups. The antibody status after first immunization event (infection/ vaccination) shows differences in nucleocapsiddirected antibody production, found in the natural infection group (about 60%). In the vaccination group, more individuals (up to 95%) show an immune response against Spike, S1 and RBD compare with natural infection. In summary, the examined cohort shows a general immunization up to 90% against most endemic corona viruses. Correlation analyses show cross-reactivity between SARS-CoV-2 and SARS-CoV. Longitudinal antibody analyses are under way, as also correlations of humoral response with immunogenetic factors.

LASU quarantine dye: Antimicrobial Phthalocyanine Activated by Indoor Light

Novel safe and stable teracationic Zinc phthalocyanine is efficient against bacteria, fungi and viruses also under indoor light Recently a novel photosensitizer with outstanding properties, phthalocyanine LASU has being developed. The compound possesses unprecedented stability and antimicrobial activity. It can be activated by a weak indoor light of 270 lux and shows the activity against G+ and G- bacteria as well as fungi and viruses. Over 3 log inactivation of bacteria and fungi on the surface of a LASU-impregnated material can be achieved in 1/2-1 h of illumination with a regular indoor and/or natural light. A cotton filter impregnated with 0.1 g/m2 of LASU eradicates on its surface the coronavirus HCoV-229E by 3.5 log in 30 minutes under indoor/natural light of 500 lux. The molecule is photostable and remains active for weeks with no significant bleaching. Another remarkable property is its ability to bind to cellulose support. It readily attaches to a fiber substrate through electrostatic interactions, moreover, the size of LASU ring matches the pitch of cellulose polymer, making the conjugate unusually strong. Hence the compound does not leech to water and is stable against temperature and surfactants. The toxicology studies also reveals that substance is non-irritating for human skin, and is non-mutagenic, which makes it suitable for human-wearable items.Copyright © 2023

Multiple detection of 13 kinds of viruses in 500 children with acute respiratory tract infection in Hami of Xinjiang province

OBJECTIVE: To investigate and analyze multiple detection of 13 kinds of viruses in 500 children with acute respiratory tract infection in Hami of Xinjiang. METHODS: A total of 500 children with acute respiratory tract infection treated in the hospital between Jan 2018 and Jan 2021 were enrolled. Thirteen kinds of respiratory infection viruses including human respiratory syncytial virus(RSV), human rhinovirus(hRV), respiratory adenovirus(AdV), influenza A and B viruses(Inf A, Inf B), parainfluenza virus(PIV 1/2/3), human enterovirus(hEV), human metapneumovirus(hMPV), human coronavirus(hCoV 229E/OC43) and human Boca virus(hBoV) were detected by multiple reverse transcription polymerase chain reaction(RT-PCR) amplification and capillary electrophoresis. And the results were compared with those by direct sequencing method. RESULTS: Of the 500 samples, 379 samples were positive(75.80%), and the top three detection rates were RSV(19.40%), hRV(16.00%) and Inf B(12.60%). The differences in positive rates of the respiratory virus among <1 year group, 1-3 years group and >3 years group were significant(84.97%, 77.47%, 65.45%)(P<0.05). The detection rate of RSV was the highest in <1 year group, and the detection rates of Inf A and Inf B were the highest in >3 years group. The differences in positive rates of respiratory viruses among the spring group, summer group, autumn group and winter group were significant(74.05%, 63.73%, 77.24%, 84.03%)(P<0.05). The detection rates of RSV, PIV 3, and hMPV were the highest in the winter group, and detection rate of AdV was the highest in spring group. CONCLUSION: RSV is the main infection virus in children with acute respiratory infection in Hami of Xinjiang. The distribution of respiratory viruses is related to age and onset season in children.

The Circulation of Common Respiratory Viruses and Their Co-infection with Severe Acute Respiratory Syndrome Coronavirus 2 Before and After Coronavirus Disease of 2019 Vaccination

Background: Respiratory viruses play important roles in respiratory tract infections;they are the major cause of diseases such as the common cold, bronchiolitis, pneumonia, etc., in humans that circulate more often in the cold seasons. During the COVID-19 pandemic, many strict public health measures, such as hand hygiene, the use of face masks, social distancing, and quarantines, were implemented worldwide to control the pandemic. Besides controlling the COVID-19 pandemic, these introduced measures might change the spread of other common respiratory viruses. Moreover, with COVID-19 vaccination and reducing public health protocols, the circulation of other respiratory viruses probably increases in the community. Objective(s): This study aims to explore changes in the circulation pattern of common respiratory viruses during the COVID-19 pan-demic. Method(s): In the present study, we evaluated the circulation of seven common respiratory viruses (influenza viruses A and B, rhi-novirus, and seasonal human Coronaviruses (229E, NL63, OC43, and HKU1) and their co-infection with SARS-CoV-2 in suspected cases of COVID-19 in two time periods before and after COVID-19 vaccination. Clinical nasopharyngeal swabs of 400 suspected cases of COVID-19 were tested for SARS-CoV-2 and seven common respiratory viruses by reverse transcription real-time polymerase chain reaction. Result(s): Our results showed common respiratory viruses were detected only in 10% and 8% of SARS-CoV-2-positive samples before and after vaccination, respectively, in which there were not any significant differences between them (P-value = 0.14). Moreover, common viral respiratory infections were found only in 12% and 32% of SARS-CoV-2-negative specimens before and after vaccination, respectively, in which there was a significant difference between them (P-value = 0.041). Conclusion(s): Our data showed a low rate of co-infection of other respiratory viruses with SARS-CoV-2 at both durations, before and after COVID-19 vaccination. Moreover, the circulation of common respiratory viruses before the COVID-19 vaccination was lower, probably due to non-pharmaceutical interventions (NPI), while virus activity (especially influenza virus A) was significantly in-creased after COVID-19 vaccination with reducing strict public health measures.Copyright © 2023, Author(s).

SARS-CoV-2 in Terms of Medical Microbiology Laboratory.

Coronaviruses (CoV), which are in the Coronaviridae family, cause different severity of gastrointestinal, respiratory and systemic diseases in wild and domestic animals, and can lead to different clinical manifestations, ranging from colds to pneumonia, depending on immunity. To date, seven types of coronavirus have been identified as infectious agents in humans;of these, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43 typically cause cold symptoms in immunocompetent individuals, while SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus) is zoonotic and cause severe respiratory diseases and deaths. SARS-CoV-2, the causative agent of COVID-19, is the seventh coronavirus identified as an infection agent in humans, which started in December 2019 in Wuhan, Hubei Province of China and was identified as a pandemic in a short time. Since the World Health Organization (WHO) defines SARS-CoV-2-sourced COVID-19 as a pandemic, and because of the increasing number of cases and deaths worldwide, structure of the novel virus and viral diagnosis methods gained importance respectively for vaccine studies and for controlling the outbreak caused by the virus.Copyright © 2020 Ankara Pediatric Hematology Oncology Training and Research Hospital. All rights reserved.

Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome.

Objective:To determine the therapeutic effect of in vitro cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method: BALB/c mice were randomly divided into six groups according to their weight grade:normal group,HCoV-229E infection group,cold and damp group,a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome,and high and low dose group of in vitro cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. In vitro cultivation of bezoar (0.128,0.064 g.kg-1 )was administrated by gavage for 3 days from the day of infection. The observation indexes included:general state observation of mice,inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the viral load in the lung tissues of mice. Serum levels of motilin(MTL),gastrin(GAS),and cytokines interleukin(IL)-10,IL-6, tumor necrosis factor-alpha(TNF-alpha)and interferon-gamma(IFN-gamma)in lung tissue of mice were determined by enzyme-linked immunosorbent assay(ELISA). The percentages of CD4+ T lymphocytes,CD8+ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of in vitro cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group, model group mice lung index increased significantly(P<0.01), nucleic acids significantly increased expression of lung tissue in mice(P<0.01),significantly higher serum MTL content in mice,GAS content significantly decreased(P<0.05,P<0.01),lung tissue cells in the immune factor TNF-alpha,IL-10 and IL-6 were significantly increased(P<0.01),peripheral blood lymphocyte CD4+ T cells in mice,The percentages of CD8+ T cells and B cells were significantly decreased(P<0.01). Compared with model group, in vitro cultivation bezoar mice lung index of high and low dose group were significantly lower(P<0.01),the lung tissue of mice express nucleic acid decreased significantly(P<0.01),MTL content decreased significantly(P< 0.01),the lung tissue of mice in the IL-6,IL-10,the TNF-alpha,IFN-gamma levels were significantly lower(P<0.01), in vitro cultivation bezoar high dose group can significantly increase the CD4+ T cell percentage(P<0.05),in vitro cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation,pulmonary interstitial edema,as well as blood stasis symptoms. Conclusion:In vitro cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice,improving the pathological damage of the lung tissue,adjusting the immune effective and inhibiting the clearing of inflammatory factors,and to provide a laboratory basis for clinical medication.Copyright © 2021, China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. All rights reserved.

Effect and mechanism of Qingre Xiaoyanning Tablet in treatment of coronavirus infection.

Objective To study the anti-coronavirus effect of Qingre Xiaoyanning Tablet (), and provide experimental basis for evaluating its prevention and treatment of coronavirus infection. Methods A total of 96 BALB/c mice with half male and half female were randomly divided into control group, model group, Lianhua Qingwen Capsules (, 0.546 g/kg) group and Qingre Xiaoyanning Tablet (8.72, 17.44, 34.89 g/kg) groups with 16 mice in each group. BALB/c mice were infected with ip cyclophosphamide combined with HCoV-229E coronavirus to establish a model of coronavirus infection. The therapeutic effect of Qingre Xiaoyanning Tablet was evaluated by body weight, lung index, viral load, hemagglutination titer and pathological changes in lung tissue of mice;Levels of interleukin-1beta (IL-1beta), IL-4, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and vascular cell adhesion molecule-1 (VCAM-1) in alveolar lavage fluid were detected by ELISA;The proportion of macrophages, lymphocytes (CD3+, CD4+) and NK cells in lung tissue was detected by flow cytometry;Western blotting was used to detect Toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MYD88), inhibitor kappa B kinase-beta (IKK-beta), inhibitor kappa B (IkappaB) and p-IkappaB protein expressions in lung tissue. Results Compared with model group, Qingre Xiaoyanning Tablet significantly increased the body weight of virus infected mice (P < 0.05, 0.01), decreased lung index and hemagglutination titer (P < 0.01), improved lung disease (P < 0.05), and significantly inhibited viral mRNA expression (P < 0.01);TNF-alpha, IL-1 beta and VCAM-1 levels in alveolar lavage fluid were decreased (P < 0.05, 0.01), IFN-gamma level was increased (P < 0.05);The percentage of macrophages was significantly decreased (P < 0.05, 0.01), percentage of CD3+, CD4+ lymphocytes and NK cells was increased (P < 0.01);MYD88, TLR4, IkappaB and IKK-beta protein expressions in lung tissue were significantly down regulated (P < 0.05, 0.01). Conclusion Qingre Xiaoyanning Tablet can inhibit the replication of coronavirus in vivo, reduce inflammatory reaction, protect lung tissue, and has obvious anti-coronavirus effect in vivo. Its mechanism may be related to the regulation of TLR4/MyD88/IKK/IkappaB signal pathway and improving immunity.Copyright © 2023 Editorial Office of Chinese Traditional and Herbal Drugs. All rights reserved.

RNA sequencing unravels unique host transcriptional responses to infection with SARS-CoV-2 in well differentiated primary human bronchial epithelial cells compared to other coronaviruses

As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses. After infection following apical application of coronaviruses at low dose, cells were harvested for bulk RNA sequencing. Results demonstrated that all tested coronaviruses efficiently infected ALI-PBEC. RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1, no such increase was observed following SARS-CoV-2 infection. Further, preliminary experiments indicated that an NR4A1 antagonist reduced viral replication in Calu-3 cells. In conclusion, these data suggest that SARS-CoV2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

Delayed immunological reaction of Human Microvascular Lung Endothelium in response to Human Coronavirus 229E infection

Introduction: In the natural conditions first and major target for respiratory viruses (RVs) are epithelial cells. Nonetheless, recently we have demonstrated that RVs are able to infect not only epithelium, but also Human Microvascular Lung Endothelial Cells (HMVEC-L) which increased network is observed during severe asthma due to increased angiogenesis. Furthermore, on the surface of HMVEC-L we observed intense expression of aminopeptidase N (AP-N)- an entry receptor for Human Coronavirus 229E (HCoV-229E). Due to the facts, that possibility of being infected by HCoV-229E should be considered and there is no research based on this model the aim of this study was to assess the vulnerability of HMVEC-L to HCoV-229E infection. Method(s): HMVEC-L was incubated with HCoV-229E (MOI 0,1;1,0;3,0) for 3 hours, 3x PBS washed and cultured for 120 hours. In relevant time points (5;24;48;72;96 and 120h) viral copy number and mRNA expression of inflammatory, anti-viral and receptor factors were evaluated in Real-Time PCR. Confocal microscopy (CM) and flow cytometry (FACS) were used to measure AP-N surface expression. Result(s): FACS and CM confirmed intense surface expression of AP-N on HMVEC-L. HCoV-229E efficiently infected HMVEC-L (604 945,5 +/-194 930,2 viral copies/mul) in 48h cultures (MOI 0,1) and induced relatively late (between 72- 96h) mRNA expression of RANTES (1181,12);IL-6 (89,6);IFN-beta (53,7);OAS-1 (64,3);PKR (11,4) and TLR-3 (42,4). Increased mRNA expression was also accompanied by protein release to the supernatants. Conclusion(s): HCoV-229E may efficiently infect HMVEC-L and induce delayed inflammatory and anti-viral response.

Screening for Potential Traditional Herbal Inhibitors Against 3-Chymo-trypsin-like Main Protease (3CLpro) from Four Different Coronaviruses: An in silico Approach

Background: The recent outbreak of the COVID-19 pandemic has raised a global health concern due to the unavailability of any vaccines or drugs. The repurposing of traditional herbs with broad-spectrum anti-viral activity can be explored to control or prevent a pandemic. Objective(s): The 3-chymotrypsin-like main protease (3CLpro), also referred to as the "Achilles' heel" of the coronaviruses (CoVs), is highly conserved among CoVs and is a potential drug target. 3CLpro is essential for the virus' life cycle. The objective of the study was to screen and identify broad--spectrum natural phytoconstituents against the conserved active site and substrate-binding site of 3CLpro of HCoVs. Method(s): Herein, we applied the computational strategy based on molecular docking to identify potential phytoconstituents for the non-covalent inhibition of the main protease 3CLpro from four different CoVs, namely, SARS-CoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Result(s): Our study shows that natural phytoconstituents in Triphala (a blend of Emblica officinalis fruit, Terminalia bellerica fruit, and Terminalia chebula fruit), namely chebulagic acid, chebulinic acid, and elagic acid, exhibited the highest binding affinity and lowest dissociation constants (Ki), against the conserved 3CLpro main protease of SARSCoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Besides, phytoconstituents of other herbs like Withania somnifera, Glycyrrhiza glabra, Hyssopus officinalis, Camellia sinensis, Prunella vulgaris, and Ocimum sanctum also showed good binding affinity and lower Ki against the active site of 3CLpro. The top-ranking phyto-constituents' binding interactions clearly showed strong and stable interactions with amino acid residues in the catalytic dyad (CYS-HIS) and substrate-binding pocket of the 3CLpro main proteases. Conclusion(s): This study provides a valuable scaffold for repurposing traditional herbs with anti--CoV activity to combat SARS-CoV-2 and other HCoVs until the discovery of new therapies.Copyright © 2021 Bentham Science Publishers.

The Genomes of SARS-CoV-2 and Related Coronaviruses: Are Accessory Proteins Key for Pathogenesis?

Objective: In January 2020, scientists deciphered the first genome of SARS-CoV-2 that has created a ravage in the world by infecting over 30 million people worldwide with above 0.95 million deaths as of mid-September 2020. With no potent therapeutics against COVID-19, research-ers around the world are relentlessly working for the development of a vaccine that can ease the pain the world is suffering today, both in terms of economic and psychological instability. Understanding the genome of SARS-CoV-2 is essential to decipher the keys that would help scientists to develop drugs or vaccines to prevent the disease. Method(s): Coronaviruses are not unknown to the human as other than SARS-CoV-2, at least six ad-ditional coronaviruses (SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) are known that causes mild to severe diseases in human. We have compared the se-quences of these seven coronaviruses to identify the key regions which are responsible for pathoge-nesis. Result(s): The genomes of the seven coronaviruses that are known to infect humans differ signifi-cantly, especially in the regions of accessory genes. Conclusion(s): The analysis of these virus genomes is the key to find out targets for the development of a potent drug or vaccine against COVID-19.Copyright © 2021 Bentham Science Publishers.