Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.

In vivo antiviral effect of plant essential oils against avian infectious bronchitis virus.

BACKGROUND: Infectious bronchitis virus (IBV) leads to huge economic losses in the poultry industry worldwide. The high levels of mutations of IBV render vaccines partially protective. Therefore, it is urgent to explore an effective antiviral drug or agent. The present study aimed to investigate the in vivo anti-IBV activity of a mixture of plant essential oils (PEO) of cinnamaldehyde (CA) and glycerol monolaurate (GML), designated as Jin-Jing-Zi. RESULTS: The antiviral effects were evaluated by clinical signs, viral loads, immune organ indices, antibody levels, and cytokine levels. The infection rates in the PEO-M (middle dose) and PEO-H (high dose) groups were significantly lower than those in the prevention, positive drug, and PEO-L (low dose) groups. The cure rates in the PEO-M and PEO-H groups were significantly higher than those in the prevention, positive drug, and PEO-L groups, and the PEO-M group had the highest cure rate of 92.31%. The symptom scores and IBV mRNA expression levels were significantly reduced in the PEO-M group. PEO significantly improved the immune organ indices and IBV-specific antibody titers of infected chickens. The anti-inflammatory factor levels of IL-4 and IFN-γ in the PEO-M group maintained high concentrations for a long time. The IL-6 levels in the PEO-M group were lower than those in prevention, positive drug, and PEO-L groups. CONCLUSION: The PEO had remarkable inhibition against IBV and the PEO acts by inhibiting virus multiplication and promoting immune function, suggesting that the PEO has great potential as a novel anti-IBV agent for inhibiting IBV infection.

Pharmaceutical services and monitoring strategies for patients with new type of Coronavirus pneumonia complicated by diabetes

New coronavirus pneumonia is menacing, and patients with new coronavirus pneumonia combined with other underlying diseases are more at risk. Glycemic control level directly affects the body's immune response and body state. Its low immune status is extremely likely to increase the risk of illness. Infected patients are more likely to aggravate the infection and further cause cytokine storms. Therefore, patients with new type of coronavirus infection and type 2 diabetes need better blood glucose control and management while treating new type of coronavirus infection. This article combines the research on the rational use of diabetes and analyzes the characteristics of the existing clinical data of COVID-19 to explore the pharmacological practice mode and medication monitoring strategy of this special patient. It is hoped to provide COVID-19 patients with diabetes with a more optimized and reasonable medication regimen and improve the clinical medication level.

Estimated Spike Evolution and Impact of Emerging SARS-CoV-2 Variants (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has been mutating and thus variants emerged. This suggests that SARS-CoV-2 could mutate at an unsteady pace. Supportive evidence comes from the accelerated evolution which was revealed by tracking mutation rates of the genomic location of Spike protein. This process is sponsored by a small portion of the virus population but not the largest viral clades. Moreover, it generally took one to six months for current variants that caused peaks of COVID-19 cases and deaths to survive selection pressure. Based on this statistic result and the above speedy Spike evolution, another upcoming peak would come around July 2021 and disastrously attack Africa, Asia, Europe, and North America. This is the prediction generated by a mathematical model on evolutionary spread. The reliability of this model and future trends out of it comes from the comprehensive consideration of factors mainly including mutation rate, selection course, and spreading speed. Notably, if the prophecy is true, then the new wave will be the first determined by accelerated Spike evolution.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Prediction, identification and preliminary study on immune efficacy of s1 protein epitope-polypeptides of avian infectious bronchitis virus

For the sake of verifying the immunogenicity of candidate epitope-polypeptide, the B and T cell epitopes of S1 protein of avian infectious bronchitis virus (IBV) H120 strain were predicted and the corresponding epitope-polypeptides were synthesized, and then were used to immunize mice, the immune effect was analyzed. Five epitope-polypeptides against S1 protein of IBV H120 strain were selected by epitope prediction software and acquired by chemical synthesis, then were immunized to mice. The specific antibodies, neutralizing antibodies and T lymphocyte subsets induced by each epitope-polypeptides were analyzed by indirect ELISA, neutralization test and flow cytometry. The ELISA results showed that the five epitope-polypeptides had good reactivity. The antibody titers of antisera induced by the five epitope-polypeptides sorted from high to low as follows: Pep76-106, Pep240-257, Pep511-537, Pep403-421, Pep135-172. The neutralization test results showed that the neutralization titers of antisera induced by the five epitope-polypeptides groups in mice were higher than that of the blank control group, and the order of neutralization titers was Pep240-257 = Pep403-421 = Pep511-537 > Pep76-106 = Pep135-172. The flow cytometry results showed that the percentages of CD3+, CD4+CD8- and CD8+CD4- T lymphocytes in all the five epitope-polypeptides groups were significantly higher (P<0.01) than those in the blank control group. The number of the CD3+ and CD4+CD8- T lymphocytes sorted from large to small as follows: Pep403-421, Pep240-257, Pep76-106, Pep511-537 and Pep135-172. The number of the CD8+CD4- T lymphocytes sorted from large to small as follows: Pep403-421, Pep76-106, Pep511-537, Pep240-257 and Pep135-172. In conclusion, Pep240-257, Pep76-106 and Pep403-421 could induce humoral immunity among the five epitope-polypeptides, while Pep403-421 could induce cellular immunity. Thus, peptide of Pep403-421 could induce cellular immunity and humoral immunity. This study laid a foundation for further understanding the immunological characteristics of the S1 protein and the development of diagnostic reagents and effective epitope vaccines.

A Randomized Controlled Study of Muscle Training Exercise Based Pulmonary Rehabilitation on Activity of Daily Living in Patients with Coronavirus Disease 2019 (preprint)

Background: Hospitalized patients with Coronavirus Disease 2019 (COVID-19) pneumonia showed a severve loss of muscle mass and strength over admission. Therefore, early physical interventions might be conducive to prevent disability and fasten recovery. Methods: We designed a prospective, randomized controlled trial to identify the effectiveness and safety of pulmonary rehabilitation based on muscle exercise in COVID-19 patients. The study was conducted between February 7th and March 31st 2020 in Union Hospital. Patients were randomly assigned to the pulmonary rehabilitation exercise group or the control group. Primary outcome was improvement of activity of daily living (ADL). Secondary outcome was changes of muscle strength assessed by manual muscle test (MMT) and arterial blood gas analysis. Length of hospital staying (LOS) and adverse events related to physical activity were also observed.Results: A total of sixty patients were in analysis, and thirty patients were in PR group. Patients had a mean age of 54.43±10.57 years. A statistically and clinically significant increase in ADL was observed in PR group (75.00 [66.25,90.00] to 100.00 [100.00,100.00], p<0.001). We also found that the improvement of ADL was related to younger age and higher PaO2 (p<0.01). Both groups had MMT improvement and there was no statistical difference between groups. There was a significant increase in PaO2 (80.23±6.49 vs. 90.47±7.82, respectively, p<0.001) between two groups before discharge. There was no statically significant difference in LOS between study group and control group (p=0.62). None of these patients had severe complications during the study.Conclusions: The protocal of pulmonary rehabilitation based on muscle training exercise was feasible in COVID-19 patients and it might accelerate recovery in ADL as compared with the spontaneous recovery in the control group.Clinical Trial Registration: ChiCTR, ChiCTR2000032457. Registered 29 April 2020- Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=52925.

In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations (preprint)

To curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

Combining Visible Light and Infrared Imaging for Efficient Detection of Respiratory Infections such as COVID-19 on Portable Device (preprint)

Coronavirus Disease 2019 (COVID-19) has become a serious global epidemic in the past few months and caused huge loss to human society worldwide. For such a large-scale epidemic, early detection and isolation of potential virus carriers is essential to curb the spread of the epidemic. Recent studies have shown that one important feature of COVID-19 is the abnormal respiratory status caused by viral infections. During the epidemic, many people tend to wear masks to reduce the risk of getting sick. Therefore, in this paper, we propose a portable non-contact method to screen the health condition of people wearing masks through analysis of the respiratory characteristics. The device mainly consists of a FLIR one thermal camera and an Android phone. This may help identify those potential patients of COVID-19 under practical scenarios such as pre-inspection in schools and hospitals. In this work, we perform the health screening through the combination of the RGB and thermal videos obtained from the dual-mode camera and deep learning architecture.We first accomplish a respiratory data capture technique for people wearing masks by using face recognition. Then, a bidirectional GRU neural network with attention mechanism is applied to the respiratory data to obtain the health screening result. The results of validation experiments show that our model can identify the health status on respiratory with the accuracy of 83.7\% on the real-world dataset. The abnormal respiratory data and part of normal respiratory data are collected from Ruijin Hospital Affiliated to The Shanghai Jiao Tong University Medical School. Other normal respiratory data are obtained from healthy people around our researchers. This work demonstrates that the proposed portable and intelligent health screening device can be used as a pre-scan method for respiratory infections, which may help fight the current COVID-19 epidemic.