Identification of an immunodominant neutralizing epitope of porcine Deltacoronavirus spike protein.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that, because of its broad host range, poses a potential threat to public health. Here, to identify the neutralizing B-cell epitopes within the S1-CTD protein, we generated three anti-PDCoV monoclonal antibodies (mAbs). Of these, the antibody designated 4E-3 effectively neutralized PDCoV with an IC50 of 3.155 µg/mL. mAb 4E-3 and one other, mAb 2A-12, recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 4E-3 was mapped to 280FYSDPKSAV288 and designated S280-288, the minimal fragment recognized by mAb 2A-12 was mapped to 506TENNRFTT513, and designated S506-513. Subsequently, alanine (A)-scanning mutagenesis indicated that Asp283, Lys285, and Val288 were the critical residues recognized by mAb 4E-3. The S280-288 epitope induces PDCoV specific neutralizing antibodies in mice, demonstrating that it is a neutralizing epitope. Of note, the S280-288 coupled to Keyhole Limpet Hemocyanin (KLH) produces PDCoV neutralizing antibodies in vitro and in vivo, in challenged piglets it potentiates interferon-γ responses and provides partial protection against disease. This is the first report about the PDCoV S protein neutralizing epitope, which will contribute to research of PDCoV-related pathogenic mechanism, vaccine design and antiviral drug development.

The safety of digestive tract cancer surgery during COVID-19: A living systematic review and meta-analysis.

Surgery is the primary curative treatment of solid cancers. However, its safety has been compromised by the outbreak of COVID-19. Therefore, it is necessary to evaluate the safety of digestive tract cancer surgery in the context of COVID-19. We used the Review Manager software (v.5.4) and Stata software (version 16.0) for meta-analysis and statistical analysis. Sixteen retrospective studies involving 17,077 patients met the inclusion criteria. The data indicates that performing digestive tract cancer surgery during the COVID-19 pandemic led to increased blood loss(MD = -11.31, 95%CI:-21.43 to -1.20, P = 0.03), but did not increase postoperative complications(OR = 1.03, 95%CI:0.78 to1.35, P = 0 0.86), anastomotic leakage (OR = 0.96, 95%CI:0.52 to1.77, P = 0 0.89), postoperative mortality (OR = 0.65, 95%CI:0.40 to1.07, P = 0 0.09), number of transfusions (OR = 0.74, 95%CI:0.30 to 1.80, P = 0.51), number of patients requiring ICU care(OR = 1.37, 95%CI:0.90 to 2.07, P = 0.14), postoperative 30-d readmission (OR = 0.94, 95%CI:0.82 to 1.07, P = 0 0.33), total hospital stay (MD = 0.11, 95%CI:-2.37 to 2.59, P = 0.93), preoperative waiting time(MD = - 0.78, 95%CI:-2.34 to 0.79, P = 0.33), postoperative hospital stay(MD = - 0.44, 95%CI:-1.61 to 0.74, P = 0.47), total operation time(MD = -12.99, 95%CI:-28.00 to 2.02, P = 0.09) and postoperative ICU stay (MD = - 0.02, 95%CI:-0.62 to 0.57, P = 0.94). Digestive tract cancer surgery can be safely performed during the COVID-19.

Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)-related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV-related hepatocarcinogenesis and their clinical significance. METHODS: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell-based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. RESULTS: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinases (MMPs)-induced extracellular matrix (ECM) degradation and NETs-mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs-aroused HCC growth and metastasis. In addition, HBV-induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll-like receptor (TLR4)/receptor for advanced glycation end products (RAGE)-reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV-related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. CONCLUSIONS: Our findings indicated that activation of RAGE/TLR4-ROS signaling by HBV-induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV-related HCC.

Nervous system manifestations related to COVID-19 and their possible mechanisms.

In December 2019, the novel coronavirus disease (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection broke. With the gradual deepening understanding of SARS-CoV-2 and COVID-19, researchers and clinicians noticed that this disease is closely related to the nervous system and has complex effects on the central nervous system (CNS) and peripheral nervous system (PNS). In this review, we summarize the effects and mechanisms of SARS-CoV-2 on the nervous system, including the pathways of invasion, direct and indirect effects, and associated neuropsychiatric diseases, to deepen our knowledge and understanding of the relationship between COVID-19 and the nervous system.

Extended SARS-CoV-2 RBD booster vaccination induces humoral and cellular immune tolerance in mice.

The repetitive applications of vaccine boosters have been brought up in face of continuous emergence of SARS-CoV-2 variants with neutralization escape mutations, but their protective efficacy and potential adverse effects remain largely unknown. Here, we compared the humoral and cellular immune responses of an extended course of recombinant receptor binding domain (RBD) vaccine boosters with those from conventional immunization strategy in a Balb/c mice model. Multiple vaccine boosters after the conventional vaccination course significantly decreased RBD-specific antibody titers and serum neutralizing efficacy against the Delta and Omicron variants, and profoundly impaired CD4+ and CD8+T cell activation and increased PD-1 and LAG-3 expressions in these T cells. Mechanistically, we confirmed that extended vaccination with RBD boosters overturned the protective immune memories by promoting adaptive immune tolerance. Our findings demonstrate potential risks with the continuous use of SARS-CoV-2 vaccine boosters, providing immediate implications for the global COVID-19 vaccination enhancement strategies.

Effect of human aminopeptidase N (hAPN) on porcine deltacoronavirus infecting HEK293 cells

HEK293 cells were used as the cell model to investigate the role of human aminopeptidase N (hAPN) in the invasion of porcine deltacoronavirus (PDCoV) into human cells. The proliferation of PDCoV on HEK293 cells was firstly identified by RT-qPCR/RT-PCR. And then, hAPN knockout cell line was constructed by CRISPR/Cas9 technology and cell viability of HEK293 hAPN knockout and wild-type cells was verified by CCK-8 assay. Effect of hAPN knockout and overexpression on PDCoV replication was detected by RT-qPCR and Western blot. Meanwhile, interaction of PDCoV S protein and hAPN protein was analyzed by homology modeling and molecular docking. Results showed that PDCoV virus copies rapidly increased at 12-36 h and reached peak level at 36 h, it could propagate at least for passage 2 on HEK293 cells. There was no significant difference in cell viability between hAPN knockout cells and wild-type cells. Knockout of hAPN inhibit PDCoV replication and overexpression of hAPN enhance PDCoV replication. Homology modeling and molecular docking analysis showed S1 protein could bind hAPN domain II. Residues TYR92, THR51, THR48, PHE16 and MET14of S1 protein receptor binding motif 1 (RBM1) can form hydrogen bonds with residues PHE490, GLN531, ARG528 and SER529 of hAPN. This study indicates that hAPN plays a critical role in HEK293 cells during PDCoV infection, which provides new theoretical evidence for further studies on the mechanism of PDCoV entry into host cells and cross-species transmission.

Research on the Public Opinion Guidance Mechanism of Major Public Health Incidents

Public opinion guidance plays a crucial role in the management of major public health incidents, and thus, exploring its mechanism is conducive to the comprehensive governance of social security. This study conducts a case study on the anti-pandemic public opinion guidance and analyzes the public opinion representation and the internal mechanism of public opinion guidance in the context of the COVID-19 in China. The findings suggest that the public opinion on the COVID-19 manifested a three-stage progressive and stable tendency and witnessed the strength of China, specifically, benefiting from the systematic and complete integration and release mechanism for anti-pandemic information, the three-dimensional mechanism for the dissemination of knowledge related to pandemic prevention and health, the innovative disclosure mechanism for precise information, and diversified channels for international public opinion guidance. The guidance mechanism proposed in this study provides significant suggestions for the public opinion guidance of global major public health incidents in future.

Analysis of Clinical Manifestations and Imaging of COVID-19 Patients in Intensive Care.

Objective: The study aims to summarize and analyze the clinical and CT findings of severe COVID-19 patients. Methods: From February 11 to March 31, 2020, 61 COVID-19 patients in intensive care in the E1-3 ward of Tongji Hospital were analyzed retrospectively. Results: The main clinical manifestations were cough, expectoration in 56 cases (91.8%), shortness of breath, chest tightness in 48 cases (78.7%), fever in 61 cases (100%), muscle ache and weakness in 40 cases (65.6%), diarrhea or vomiting in 8 cases (13.1%), and headache in 4 cases (6.6%). After admission, the leukocyte count was normal in 40 cases (57.7%), higher in 9 cases (15.4%), and lower in 12 cases (26.9%). The lymphocyte count decreased in 53 cases (86.9%). CRP was increased in 29 cases (47.5%); PCT was increased in 15 cases (24.6%); ESR was increased in 38 cases (62.3%); D-dimer increased in 39 cases (63.9%); ALT/AST increased in 40 cases (65.6%); CK/CK-MB increased in 8 cases (13.1%); troponin I increased in 6 cases (9.8%); NT-proBNP increased in 35 cases (57.4%); IL-1 increased in 5 cases (8.2%); IL-2 receptor increased in 28 cases (45.9%); IL-6 increased in 23 cases (37.7%); IL-8 increased in 15 cases (24.6%); IL-10 increased in 12 cases (19.7%); and NTF increased in 22 cases (36.1%). The chest CT images showed that 38 cases (65.5%) of right lung lesions were more extensive than those of left lung lesions, 20 cases (34.5%) of left lung lesions were more extensive than those of right lung lesions, 42 cases (72.5%) of lower lobe lesions were more extensive than those of upper lobe lesions, 6 cases (10.3%) of upper lobe lesions were more extensive than those of lower lobe lesions, and 10 cases (17.2%) of upper and lower part lesions were roughly the same. Ground-glass opacity (GGO) was found in 12 cases (20.7%); GGO with focal consolidation in 38 cases (65.5%); small patchy edge fuzzy density increased in 24 cases (41.4%); large consolidation in 20 cases (34.5%); reticular or fibrous cord in 54 cases (93.1%); and air bronchogram in 8 cases (13.8%). Conclusions: COVID-19 patients in intensive care have no specific clinical manifestation and CT findings. However, analysis and summary of relevant data can help us assess the severity of the disease, decide the timing of treatment, and predict prognosis.

Susceptibility of cell lines from different animal species to Porcine Deltacoronavirus

[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR,RT-q PCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero,PAM,PK15,ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM,PK15,ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15,LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15,LLC-PK1, and ST cells at P1,P3, and P9. Therefore, only three cell lines (PK15,LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 107.11,107.00, and 107.37 TCID50/mL at P9,respectively. After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.

Phylogeny, Evolution, and Transmission Dynamics of Canine and Feline Coronaviruses: A Retro-Prospective Study

Canine coronavirus (CCoV) and feline coronavirus (FCoV) are endemic in companion animals. Due to their high mutation rates and tendencies of genome recombination, they pose potential threats to public health. The molecular characteristics and genetic variation of both CCoV and FCoV have been thoroughly studied, but their origin and evolutionary dynamics still require further assessment. In the present study, we applied a comprehensive approach and analyzed the S, M, and N genes of different CCoV/FCoV isolates. Discriminant analysis of principal components (DAPC) and phylogenetic analysis showed that the FCoV sequences from Chinese isolates were closely related to the FCoV clusters in Netherlands, while recombination analysis indicated that of S N-terminal domain (NTD) was the most susceptible region of mutation, and recombination of this region is an important cause of the emergence of new lineages. Natural selection showed that CCoV and FCoV subgenotypes were in selection constraints, and CCoV-IIb was in strong positive selection. Phylodynamics showed that the mean evolution rate of S1 genes of CCoV and FCoV was 1.281 × 10–3 and 1.244 × 10–3 subs/site/year, respectively, and the tMRCA of CCoV and FCoV was about 1901 and 1822, respectively. Taken together, our study centered on tracing the origin of CCoV/FCoV and provided ample insights into the phylogeny and evolution of canine and feline coronaviruses.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.

Differential health and economic impacts from the COVID-19 lockdown between the developed and developing countries: Perspective on air pollution.

It is enlightening to determine the discrepancies and potential reasons for the degree of impact from the COVID-19 control measures on air quality as well as the associated health and economic impacts. Analysis of air quality, socio-economic factors, and meteorological data from 447 cities in 46 countries indicated that the COVID-19 control measures had significant impacts on the PM2.5 (particulate matter with an aerodynamic diameter less than 2.5 µm) concentrations in 20 (reduced PM2.5 concentrations of -7.4-29.1 µg m-3) of the selected 46 countries. In these 20 countries, the robustly distinguished changes in the PM2.5 concentrations caused by the control measures differed between the developed (95% confidence interval (CI): -2.7-5.5 µg m-3) and developing countries (95% CI: 8.3-23.2 µg m-3). As a result, the COVID-19 lockdown reduced death and hospital admissions change from the decreased PM2.5 concentrations by 7909 and 82,025 cases in the 12 developing countries, and by 78 and 1214 cases in the eight developed countries. The COVID-19 lockdown reduced the economic cost from the PM2.5 related health burden by 54.0 million dollars in the 12 developing countries and by 8.3 million dollars in the eight developed countries. The disparity was related to the different chemical compositions of PM2.5. In particular, the concentrations of primary PM2.5 (e.g., BC) in cities of developing countries were 3-45 times higher than those in developed countries, so the mass concentration of PM2.5 was more sensitive to the reduced local emissions in developing countries during the COVID-19 control period. The mass fractions of secondary PM2.5 in developed countries were generally higher than those in developing countries. As a result, these countries were more sensitive to the secondary atmospheric processing that may have been enhanced due to reduced local emissions.

Severity-associated markers and assessment model for predicting the severity of COVID-19: a retrospective study in Hangzhou, China.

BACKGROUND: The severity of COVID-19 associates with the clinical decision making and the prognosis of COVID-19 patients, therefore, early identification of patients who are likely to develop severe or critical COVID-19 is critical in clinical practice. The aim of this study was to screen severity-associated markers and construct an assessment model for predicting the severity of COVID-19. METHODS: 172 confirmed COVID-19 patients were enrolled from two designated hospitals in Hangzhou, China. Ordinal logistic regression was used to screen severity-associated markers. Least Absolute Shrinkage and Selection Operator (LASSO) regression was performed for further feature selection. Assessment models were constructed using logistic regression, ridge regression, support vector machine and random forest. The area under the receiver operator characteristic curve (AUROC) was used to evaluate the performance of different models. Internal validation was performed by using bootstrap with 500 re-sampling in the training set, and external validation was performed in the validation set for the four models, respectively. RESULTS: Age, comorbidity, fever, and 18 laboratory markers were associated with the severity of COVID-19 (all P values < 0.05). By LASSO regression, eight markers were included for the assessment model construction. The ridge regression model had the best performance with AUROCs of 0.930 (95% CI, 0.914-0.943) and 0.827 (95% CI, 0.716-0.921) in the internal and external validations, respectively. A risk score, established based on the ridge regression model, had good discrimination in all patients with an AUROC of 0.897 (95% CI 0.845-0.940), and a well-fitted calibration curve. Using the optimal cutoff value of 71, the sensitivity and specificity were 87.1% and 78.1%, respectively. A web-based assessment system was developed based on the risk score. CONCLUSIONS: Eight clinical markers of lactate dehydrogenase, C-reactive protein, albumin, comorbidity, electrolyte disturbance, coagulation function, eosinophil and lymphocyte counts were associated with the severity of COVID-19. An assessment model constructed with these eight markers would help the clinician to evaluate the likelihood of developing severity of COVID-19 at admission and early take measures on clinical treatment.

Mild Breakthrough Infection in a Healthcare Professional Working in the Isolation Area of a Hospital Designated for Treating COVID-19 Patients - Shaanxi Province, China, March, 2021.

WHAT IS ALREADY KNOWN ABOUT THIS TOPIC?: Healthcare workers are at high risk of acquiring COVID-19 from occupational exposure to COVID-19 virus during their daily medical service work. Excellent infection prevention and control measures and adequate personal protective equipment (PPE) are essential to reduce the risk of hospital-acquired COVID-19. WHAT IS ADDED BY THIS REPORT?: On March 17, 2021, a female healthcare professional who already received both doses of the COVID-19 vaccination and was working in the isolation area of a designated COVID-19 hospital was diagnosed with COVID-19 in Xi'an city. Her exposure likely occurred five days before illness onset when she obtained nasopharyngeal and oropharyngeal swabs from the two imported cases that were identified as belonging to the B.1.1.7 lineage, the variant first detected in the United Kingdom. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICES?: Since the healthcare worker had been fully vaccinated and had mild symptomatology, it is considered a mild breakthrough infection. All vaccines are associated with breakthrough infections. In addition to rigorous adherence to infection prevention and control measures, use of adequate PPE, and using good clinical practices, the potential role of chronic upper respiratory infection in acquiring COVID-19 during medical procedures deserves further consideration.

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

Establishment and application of indirect ELISA for detection of bovine coronavirus antibody

[Background] Bovine coronavirus(BCoV) is one of the main causes of neonatal calf death, and effective detection is the prerequisite to prevent and control the disease. [Objective] At present, BCoV ELISA detection method has some defects, such as low sensitivity, instability and so on. This study aims to improve these defections to establish indirect ELISA detection method. [Methods] The method of indirect ELISA was established by using the soluble recombinant N protein of the epidemic BCoV-CD strain as an antigen. The epitope of the N protein was predicted by DNAStar soft, and was prepared by prokaryotic expression in the non-denatured condition. The seroepidemiological investigation of BCoV infection in Heilongjiang province in recent 5 years was carried out by using this method. [Results] The optimum working conditions of the ELISA method were as follows: the coating solution was 50 mmol/L pH 9.6 carbonate, and the antigen coating concentration was 2.5 μg/mL;The sample diluent was PBST, the dilution concentration was 1 μg/mL, and incubated at 37 °C for 1.5 h;The dilution concentration of HRP-labeled secondary antibody was 1:7 500, and incubated at 37 °C for 1.0 h;The blocked condition was 1% gelatin at 37 °C for 30 minutes. The negative-positive cut off value was 0.225. The method had no cross-reaction with positive serum of bovine rotavirus, bovine viral diarrhea virus, bovine respiratory syncytial body, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 and Escherichia coli. The intra-and inter-assay coefficient of variation was less than 10%, and the coincident rate with virus neutralization test was 93.5%. The results showed that the positive rate of BCoV antibody was 98.84% in 603 serum samples of cows in some areas of Heilongjiang Province. [Conclusion] The ELISA method established in this study has strong specificity, high sensitivity and good stability, which provides a technical basis for the further development of ELISA kit.