Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.
Animals ; Enzyme-Linked Immunosorbent Assay/methods* ; Swine ; Antibodies, Viral/blood* ; Recombinant Proteins/genetics* ; Deltacoronavirus/isolation & purification* ; Coronavirus Infections/virology* ; Swine Diseases/diagnosis* ; Coronavirus Nucleocapsid Proteins ; Sensitivity and Specificity

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

Adolescent ; Adult ; Aged ; COVID-19/virology* ; COVID-19 Nucleic Acid Testing/methods* ; Child ; Coronavirus Nucleocapsid Proteins/genetics* ; Feces/virology* ; Female ; Humans ; Male ; Middle Aged ; Phosphoproteins/genetics* ; RNA, Viral/genetics* ; SARS-CoV-2/isolation & purification* ; Young Adult

OBJECTIVE: To investigate the clinical outcome of patients with moderate type of coronavirus disease 2019 (COVID-19) after discharge by retesting viral nucleic acid. METHODS: Seven patients with moderate COVID-19 met the discharge criteria enacted by National Health Commission were quarantined in hospital for 7 days, then continuously quarantined at home for 4 weeks after discharged. During the quarantined period, the symptoms and signs were documented, and sputum or nasal swab and feces samples were collected to test SARS-CoV-2 nucleic acid by RT-PCR method. RESULTS: There was no symptoms and signs during the quarantine period in all 7 patients. However, respiratory swabs from 3 patients were confirmed positive of SARS-CoV-2 nucleic acid at 5 to 7 days after they met the discharge criteria. CONCLUSIONS There is a relatively high incidence of positive viral nucleic acid in patients met the discharge criteria, and it is suggested that patients met the current discharge criteria should be quarantined in hospital for another 7 days and the follow-up viral testing is necessary.
Betacoronavirus ; isolation & purification ; Coronavirus Infections ; diagnosis ; Feces ; chemistry ; virology ; Follow-Up Studies ; Humans ; Pandemics ; Patient Discharge ; statistics & numerical data ; Pneumonia, Viral ; diagnosis ; Quarantine ; statistics & numerical data ; RNA, Viral ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVE: To analysis the medication characteristics of the prescriptions issued via open channel by the National and Provincial Health Committee and the State Administration of Traditional Chinese Medicine in treating coronavirus disease 2019 (COVID-19). METHODS: We collected the data of traditional Chinese medicine related to treatment plans published by the National and Provincial Health Committee and the State Administration of Traditional Chinese Medicine from the start of COVID-19 outbreak to February 19, 2020. The frequency analysis, cluster analysis and association analysis were performed. RESULTS: The study collected 4 national and 34 regional prevention and treatment plans, 578 items, 84 traditional Chinese formulations, 60 Chinese patent medicines, and 230 Chinese herbs. The high frequently used herbs were , and . The commonly used traditional formulations included decoction, powder, and decoction. The Chinese patent drugs included pill, injection, and capsule. The most common paired medications were and and Two core combinations and one novel formula were discovered in the study. CONCLUSIONS powder and decoction are the basic formulations for syndrome of COVID-19. In addition, decoction, powder, decoction and decoction are the basic formulations for syndrome of COVID-19. The main medication characteristics are clearing heat, entilating lung, removing toxicity and removing turbidity. It shows that removing toxicity and eliminating evil are the prescription thought in treating epidemic disease of traditional Chinese medicine.
Betacoronavirus ; isolation & purification ; Coronavirus Infections ; therapy ; Data Mining ; Drugs, Chinese Herbal ; chemistry ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; Pandemics ; Pneumonia, Viral ; therapy

Since the coronavirus disease 2019 (COVID-19) affects the cardio-pulmonary function of pregnant women, the anesthetic management and protection of medical staff in the cesarean section is significantly different from that in ordinary surgical operation. This paper reports a case of cesarean section for a woman with COVID-19, which was successfully performed in the First Affiliated Hospital of Zhejiang University School of Medicine on February 8, 2020. Anesthetic management, protection of medical staff and psychological intervention for the pregnant woman during the operation were discussed. Importance has been attached to the preoperative evaluation of pregnant women with COVID-19 and the implementation of anesthesia plan. For moderate patients, intraspinal anesthesia is preferred in cesarean section, and try to reduce its influence in respiration and circulation in both maternal and infant; general anesthesia with endotracheal intubation should be adopted for severe or critically ill patients. Ensure the safety of medical environment, and anesthetists should carry out level-Ⅲ standard protection. Special attention and support should be paid to maternal psychology: fully explanation before operation to reduce anxiety; relieve the discomfort during operation, so as to reduce tension; avoid the bad mood due to pain after operation.
Anesthesia ; Betacoronavirus ; isolation & purification ; Cesarean Section ; methods ; Coronavirus Infections ; complications ; Female ; Humans ; Infant ; Injections, Spinal ; Pandemics ; Pneumonia, Viral ; complications ; Pregnancy

OBJECTIVE: To explore the feasibility of radical resection for cancer patients complicated with coronavirus disease 2019 (COVID-19). METHODS: The management and clinical outcome of a sigmoid cancer patient with COVID-19 were analyzed. RESULTS: The inflammation indicators and fever of this patient were effectively controlled and the lung lesions remained stable after active anti-viral treatment, then the radical colorectomy was performed after the viral negative conversion for twice. CONCLUSIONS The case indicates that radical resection can be performed in SARS-CoV-2 patients with twice-negative SARS-CoV-2 nucleic acid testing results.
Betacoronavirus ; isolation & purification ; Colonic Neoplasms ; complications ; surgery ; Coronavirus Infections ; complications ; therapy ; Disease Management ; Humans ; Pandemics ; Pneumonia, Viral ; complications ; therapy ; Treatment Outcome

OBJECTIVE: To analyze the clinical characteristics and pregnancy outcomes of pregnant women complicated with coronavirus disease 2019 (COVID-19). METHODS: The clinical data of 3 pregnant women with COVID-19 admitted to the First Affiliated Hospital of Zhejiang University School of Medicine from January 19 to February 10, 2020 were retrospectively analyzed. RESULTS: There was one case in the first-trimester pregnancy (case 1), one in the second-trimester pregnancy (case 2) and one in third-trimester pregnancy (case 3). Cough, fever, fatigue, lung imaging changes were the main manifestations. The white cell count, lymphocyte percentage had no significantly changes in case 1 and case 3, while the levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), IL-6 and IL-10 elevated. The lymphocyte count and lymphocyte percentage decreased and the inflammatory indicators significantly increased in case 2. All patients were treated with antiviral, antitussive, oxygen inhalation; case 3 received glucocorticoids, case 2 with severe illness received glucocorticoids and additionally gamma globulin. All three cases were cured and discharged. Case 1 with early pregnancy chose to terminate pregnancy after discharge; case 2 chose to continue pregnancy without obstetric complications; and case 3 had cesarean section delivery due to abnormal fetal heart monitoring. CONCLUSIONS The report shows that COVID-19 in pregnancy women could be cured with active treatment, and the maternal and fetal outcomes can be satisfactory.
Antiviral Agents ; therapeutic use ; Betacoronavirus ; isolation & purification ; Cesarean Section ; Coronavirus Infections ; complications ; drug therapy ; Female ; Glucocorticoids ; therapeutic use ; Humans ; Oxygen ; therapeutic use ; Pandemics ; Pneumonia, Viral ; complications ; drug therapy ; Pregnancy ; Pregnancy Complications, Infectious ; drug therapy ; pathology ; Pregnancy Outcome ; Retrospective Studies ; Treatment Outcome ; gamma-Globulins ; therapeutic use

Acute respiratory failure due to acute hypoxemia is the major manifestation in severe coronavirus disease 2019 (COVID-19). Rational and effective respiratory support is crucial in the management of COVID-19 patients. High-flow nasal cannula (HFNC) has been utilized widely due to its superiority over other non-invasive respiratory support techniques. To avoid HFNC failure and intubation delay, the key issues are proper patients, timely application and improving compliance. It should be noted that elder patients are vulnerable for failed HFNC. We applied HFNC for oxygen therapy in severe and critical ill COVID-19 patients and summarized the following experiences. Firstly, to select the proper size of nasal catheter, to locate it at suitable place, and to confirm the nose and the upper respiratory airway unobstructed. Secondly, an initial ow of 60 L/min and 37℃ should be given immediately for patients with obvious respiratory distress or weak cough ability; otherwise, low-level support should be given first and the level gradually increased. Thirdly, to avoid hypoxia or hypoxemia, the treatment goal of HFNC should be maintained the oxygen saturation (SpO) above 95% for patients without chronic pulmonary disease. Finally, patients should wear a surgical mask during HFNC treatment to reduce the risk of virus transmission through droplets or aerosols.
Aged ; Betacoronavirus ; isolation & purification ; Cannula ; Coronavirus Infections ; therapy ; Humans ; Oxygen ; administration & dosage ; Pandemics ; Pneumonia, Viral ; therapy