The Alpha-1 Subunit of the Na+/K+-ATPase (ATP1A1) Is a Host Factor Involved in the Attachment of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.

Precautions against COVID-19 reduce respiratory virus infections among children in Southwest China.

Acute respiratory tract infections pose a serious threat to the health of children worldwide, with viral infections representing a major etiology of this type of disease. Protective measures such as mask-wearing, social distancing, and hand hygiene can be effective in curbing the spread of severe acute respiratory syndrome coronavirus 2. These precautions may also have an impact on the spread of other respiratory viruses. In this study, we retrospectively compared the respiratory virus infections of children in Southwest China before and after the outbreak of COVID-19. Nasopharyngeal swabs were collected from 1578 patients under 14 years old with acute respiratory tract infection symptoms before and after COVID-19 pandemic. Nine common respiratory viruses including human bocavirus, human rhinoviruses, human coronaviruses, human adenoviruses, human metapneumovirus, respiratory syncytial virus, influenza A virus, influenza B virus, and parainfluenza virus were measured by advanced fragment analysis. The respiratory virus infection rates among children of all ages and genders in Southwest China under the precautions against COVID-19 pandemic were significantly lower than that of the same period before the pandemic. Our findings indicate that public health measures implemented during the COVID-19 pandemic, including strict mask-wearing, social distancing, and hand hygiene, may be effective in preventing the transmission of other respiratory viruses in children, thereby controlling the spread of infections.

Precautions against COVID-19 reduce respiratory virus infections among children in Southwest China

Acute respiratory tract infections pose a serious threat to the health of children worldwide, with viral infections representing a major etiology of this type of disease. Protective measures such as mask-wearing, social distancing, and hand hygiene can be effective in curbing the spread of severe acute respiratory syndrome coronavirus 2. These precautions may also have an impact on the spread of other respiratory viruses. In this study, we retrospectively compared the respiratory virus infections of children in Southwest China before and after the outbreak of COVID-19. Nasopharyngeal swabs were collected from 1578 patients under 14 years old with acute respiratory tract infection symptoms before and after COVID-19 pandemic. Nine common respiratory viruses including human bocavirus, human rhinoviruses, human coronaviruses, human adenoviruses, human metapneumovirus, respiratory syncytial virus, influenza A virus, influenza B virus, and parainfluenza virus were measured by advanced fragment analysis. The respiratory virus infection rates among children of all ages and genders in Southwest China under the precautions against COVID-19 pandemic were significantly lower than that of the same period before the pandemic. Our findings indicate that public health measures implemented during the COVID-19 pandemic, including strict mask-wearing, social distancing, and hand hygiene, may be effective in preventing the transmission of other respiratory viruses in children, thereby controlling the spread of infections.

A protein vaccine with Alum/c-GAMP/poly(I:C) rapidly boosts robust immunity against SARS-CoV-2 and variants of concern.

Adjuvants are important components in vaccines to increase the immunogenicity of proteins and induce optimal immunity. In this study, we designed a novel ternary adjuvant system Alum + c-GAMP + poly(I:C) with STING agonist 3,3'-c-GAMP (c-GAMP) and TLR3 agonist poly(I:C) co-adsorbed on the conventional adjuvant aluminum gel (Alum), and further constructed an S1 protein vaccine. Two doses of vaccination with the ternary adjuvant vaccine were sufficient to induce a balanced Th1/Th2 immune response and robust humoral and cellular immunity. Additionally, the ternary adjuvant group had effective neutralizing activity against live virus SARS-CoV-2 and pseudovirus of all variants of concern (alpha, beta, gamma, delta and omicron). These results indicate that the ternary adjuvants have a significant synergistic effect and can rapidly trigger potent immune responses; the combination of the ternary adjuvant system with S1 protein is a promising COVID-19 vaccine candidate.

RBD conjugate vaccine with a built-in TLR1/2 agonist is highly immunogenic against SARS-CoV-2 and variants of concern.

The coronavirus 2019 (COVID-19) pandemic is causing serious impacts in the world, and safe and effective vaccines and medicines are the best methods to combat the disease. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in interacting with the angiotensin-converting enzyme 2 (ACE2) receptor, and is regarded as an important target of vaccines. Herein, we constructed the adjuvant-protein conjugate Pam3CSK4-RBD as a vaccine candidate, in which the N-terminal of the RBD was site-selectively oxidized by transamination and conjugated with the TLR1/2 agonist Pam3CSK4. This demonstrated that the conjugation of Pam3CSK4 significantly enhanced the anti-RBD antibody response and cellular response. In addition, sera from the Pam3CSK4-RBD immunized group efficiently inhibited the binding of the RBD to ACE2 and protected cells from SARS-CoV-2 and four variants of concern (alpha, beta, gamma and delta), indicating that this adjuvant strategy could be one of the effective means for protein vaccine development.

SARS-CoV-2-Reactive Mucosal B Cells in the Upper Respiratory Tract of Uninfected Individuals.

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.

Detection and Neutralization of SARS-CoV-2 Using Non-conventional Variable Lymphocyte Receptor Antibodies of the Evolutionarily Distant Sea Lamprey.

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.

Computational and Experimental Studies Reveal That Thymoquinone Blocks the Entry of Coronaviruses Into In Vitro Cells.

INTRODUCTION: Since December 2019, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic in China and worldwide. New drugs for the treatment of COVID-19 are in urgent need. Considering the long development time for new drugs, the identification of promising inhibitors from FDA-approved drugs is an imperative and valuable strategy. Recent studies have shown that the S1 and S2 subunits of the spike protein of SARS-CoV-2 utilize human angiotensin-converting enzyme 2 (hACE2) as the receptor to infect human cells. METHODS: We combined molecular docking and surface plasmon resonance (SPR) to identify potential inhibitors for ACE2 from available commercial medicines. We also designed coronavirus pseudoparticles that contain the spike protein assembled onto green fluorescent protein or luciferase reporter gene-carrying vesicular stomatitis virus core particles. RESULTS: We found that thymoquinone, a phytochemical compound obtained from the plant Nigella sativa, is a potential drug candidate. SPR analysis confirmed the binding of thymoquinone to ACE2. We found that thymoquinone can inhibit SARS-CoV-2, SARS-CoV, and NL63 pseudoparticles infecting HEK293-ACE2 cells, with half-maximal inhibitory concentrations of 4.999, 7.598, and 6.019 µM, respectively. The SARS-CoV-2 pseudoparticle inhibition had half-maximal cytotoxic concentration of 35.100 µM and selection index = 7.020. CONCLUSION: Thymoquinone is a potential broad-spectrum inhibitor for the treatment of coronavirus infections.

Risk factors and prevention strategies for the occupational exposure of medical staff during the coronavirus disease-19 pandemic (Review).

During the sudden epidemic of this novel coronavirus-induced pneumonia, a number of medical staff were infected and even succumbed to coronavirus disease-19 (COVID-19). Based on the experience of medical professionals from The Hubei 672 Orthopedics Hospital of Integrated Chinese and Western Medicine on this COVID-19 pandemic, the present review summarizes the risk factors associated with the occupational exposure of front-line medical staff. Challenges encountered include insufficient understanding, lack of early protection, environmental factors and routine procedures and the lack of adequate prevention strategies. Overcoming these challenges can potentially enhance awareness of COVID-19 prevention and control among medical staff, in addition to strengthening the personal protection of front-line medical staff, rational area layout, regular disinfection, standardization of daily procedures, reasonable scheduling and early psychological intervention. The present article may serve as a referencing point for the prevention and control of this epidemic.

A study of solid phase competition ELISA for detection of A-type foot-and-mouth disease virus antibody

In order to evaluate the immune levels of type A FMD vaccine, the monoclonal antibody (MAb) 3D9 of FMDV prepared in our lab was used as the capture antibody, and the HRP- labeled MAb 9A9 was used as the detection antibody. A solidphase competition ELISA method (SPCE) for the detection of antibody against FMDV serotype A based on monoclonal antibodies was developed and its conditions were optimized. The results showed that the optimal concentration of MAb 3D9 was 1.16 g/mL, the optimal dilution of A- type FMDV antigen was 1:5, and the optimal dilution of HRP- labeled MAb 9A9 was 1:5000. When the serum diluted at 1:32, the critical value of the assay was determined to be 35%. The method was used to detect positive serum of type A FMDV, type O FMDV, BCV, BRV, PRRSV, PCV and CSFV, respectively. The results showed that the solid phase competition ELISA method could specifically detect type A FMDV, but had no cross- reaction with other viruses. It showed that the method established in this study has strong specificity. The sensitivity test was performed on 3 positive sera samples by VNT (virus neutralization test). The sensitivity is 1:1 024, 1:256, 1:128, respectively, indicating the method has high sensitivity. Repeatability results showed the coefficient of variation of the intra-assay and inter-assay repeated tests were less than 10%, indicating that the repeatability is good. This method was used to detect 112 serum samples along with the liquid- phase blocking ELISA method (LPBE) and VNT and analyze the correlation. The results showed the correlation coefficient between the method established in this study and LPBE and VNT was 0.901 and 0.916, respectively. It indicated that this method had a good reliability and has a higher correlation with VNT. Using the SPCE and Korean Jeno A FMDV ELISA antibody detection diagnosis kitto detect 470 clinical samples (90 sheep serum samples, 170 cow serum samples and 210 pig serum samples). The results showed the overall coincidence rate between this method and Jeno kit is 90.3%, 91.8% and 89.0%, respectively, indicating that the detection result of this method is relatively accurate. This study laid a foundation for the establishment of a method for the detection of immunity level of type A foot-and-mouth disease vaccine.

Development of solid phase competition elisa for detection ofO-type foot-and-mouth disease virus antibodies based onmonoclonal antibody

In order to detect antibodies against foot-and-mouth disease virus (FMDV), the monoclonal antibody (MAb) 3D9was used as the capture antibody, and the HRP-labeled MAb 8E8 was used as the detection antibody. After several conditions wereoptimized, the critical value of the detection was established. A solid phase competition ELISA (SPCE) method for the detecting oftype O FMDV antibodies based on monoclonal antibodies. Specific, sensitive, and reproducible tests were performed on this method. The correlation between this method and the liquid phase blocking ELISA (LPBE) method and the virus neutralization test (VNT)was compared. The results showed that the optimal dilution of MAb 3D9 was 1:25 000, the optimal dilution of inactivated type OFMDV antigen was 1:3, and the optimal dilution of MAb 8E8 was 1:5 000. When the serum was diluted at 1:32, the cut-off value of the assay was determined to be 45%. The method was used to detect reference positive serum of type A FMDV, bovinecoronavirus, bovine rotavirus, porcine reproductive and respiratory syndrome virus, porcine circovirus 2, and classical swine fevervirus, respectively. The test results were all negative and no cross-reaction occurred. After testing, when the dilution of the positivestandard serum is 1:512, the method still has good sensitivity;the coefficient of variation of the intra-assay and inter-assay repeatedtests is less than 10%, indicating that the repeatability is better. The correlation between this method and VNT and LPBE was 0.923and 0.896, respectively. This study established a new method for the detection of domestic type O FMDV antibodies in China.