Chestnut inner shell extract inhibits viral entry of porcine epidemic diarrhea virus and other coronaviruses in vitro.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes acute diarrhea in suckling piglets. Although vaccines are able to reduce the incidence of PEDV infection, outbreaks of PEDV continue to be reported worldwide and cause serious economic losses in the swine industry. To identify novel antiviral sources, we identified the chestnut (Castanea crenata) inner shell (CIS) as a natural material with activity against PEDV infection in vitro. The ethanol fractions of CIS extracts potently inhibited PEDV infection with an IC90 of 30 µg/ml. Further investigation of the virus lifecycle demonstrated that CIS extract particularly targeted the early stages of PEDV infection by blocking viral attachment and membrane fusion at rates of 80~90%. In addition, CIS extract addition reduced the viral entry of other members of the Coronaviridae family. Our data demonstrated that CIS extract inhibited PEDV infection by blocking cell entry in vitro and suggest that CIS extract is a new prophylactic and therapeutic agent against PEDV and other coronavirus infections.

Evaluation of Four Rapid Antigen Tests for the Detection of SARS-CoV-2 Infection with Nasopharyngeal Swabs.

Owing to the high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, the capacity of testing systems based on the gold standard real-time reverse transcription-polymerase chain reaction (rRT-PCR) is limited. Rapid antigen tests (RATs) can substantially contribute to the prevention of community transmission, but their further assessment is required. Here, using 1503 nasopharyngeal swabs, we compared the diagnostic performance of four RAT kits (Abbott Panbio™ COVID-19 Ag Rapid Test, SD Biosensor Standard™ Q COVID-19 Ag Test, Humasis COVID-19 Ag Test, and SG Medical Acrosis COVID-19 Ag Test) to the cycle threshold (Ct) values obtained from rRT-PCR. The precision values, area under the curve values, SARS-CoV-2 variant detection ability, and non-SARS-CoV-2 specificity of all four kits were similar. An assay using the Acrosis kit had a significantly better positive detection rate with a higher recall value and cut-off value than that using the other three RAT kits. During the current COVID-19 pandemic, the Acrosis kit is an effective tool to prevent the spread of SARS-CoV-2 in communities.

Resistance of Field-Isolated Porcine Epidemic Diarrhea Virus to Interferon and Neutralizing Antibody.

Variant porcine epidemic diarrhea virus (PEDV), belonging to the genogroup G2b, has higher pathogenicity and mortality than classical PEDV, belonging to the genogroup G1a. To understand the pathogenesis of the G2b PEDV, we examined the resistance of the G2b PEDV to interferon (IFN) and neutralizing antibodies, which are important for controlling PEDV infection. We found that the G2b PEDV showed higher resistance to IFN than G1a PEDV. The G1a PEDV could replicate in IFN-deficient Vero cells, but not in IFN-releasing porcine alveolar macrophages, whereas the G2b PEDV showed similar infectivity in both types of cells. We also found that G2b PEDV was not effectively blocked by neutralizing antibodies, unlike G1a PEDV, suggesting differences in the antigenicity of the two strains. These results provide an understanding of the occurrence of variant PEDV and its pathogenesis.

Performance Analysis of Self-Collected Nasal and Oral Swabs for Detection of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic human coronavirus and is rapidly transmitted by infected individuals regardless of their symptoms. During the COVID-19 pandemic, owing to the dearth of skilled healthcare workers (HCWs) to collect samples for early diagnosis, self-collection emerged as a viable alternative. To evaluate the reliability of self-collection, we compared the virus detection rate using 3990 self-collected swabs and HCW-collected swabs, procured from the same individuals and collected immediately after the self-collection. The results of multiplex reverse-transcription quantitative polymerase chain reaction revealed that the viral load in the HCW-collected swabs was marginally (18.4–28.8 times) higher than that in self-collected swabs. Self-collection showed no significant difference in sensitivity and specificity from HCW-collection (κ = 0.87, McNemar’s test;p = 0.19), indicating a comparable performance. These findings suggest that self-collected swabs are acceptable substitutes for HCW-collected swabs, and that their use improved the specimen screening efficiency and reduced the risk of SARS-CoV-2 infection among HCWs during and after the COVID-19 pandemic.

Performance Analysis of Self-Collected Nasal and Oral Swabs for Detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic human coronavirus and is rapidly transmitted by infected individuals regardless of their symptoms. During the COVID-19 pandemic, owing to the dearth of skilled healthcare workers (HCWs) to collect samples for early diagnosis, self-collection emerged as a viable alternative. To evaluate the reliability of self-collection, we compared the virus detection rate using 3990 self-collected swabs and HCW-collected swabs, procured from the same individuals and collected immediately after the self-collection. The results of multiplex reverse-transcription quantitative polymerase chain reaction revealed that the viral load in the HCW-collected swabs was marginally (18.4-28.8 times) higher than that in self-collected swabs. Self-collection showed no significant difference in sensitivity and specificity from HCW-collection (κ = 0.87, McNemar's test; p = 0.19), indicating a comparable performance. These findings suggest that self-collected swabs are acceptable substitutes for HCW-collected swabs, and that their use improved the specimen screening efficiency and reduced the risk of SARS-CoV-2 infection among HCWs during and after the COVID-19 pandemic.

Chestnut inner shell extract inhibits viral entry of porcine epidemic diarrhea virus and other coronaviruses in vitro

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes acute diarrhea in suckling piglets. Although vaccines are able to reduce the incidence of PEDV infection, outbreaks of PEDV continue to be reported worldwide and cause serious economic losses in the swine industry. To identify novel antiviral sources, we identified the chestnut (Castanea crenata) inner shell (CIS) as a natural material with activity against PEDV infection in vitro. The ethanol fractions of CIS extracts potently inhibited PEDV infection with an IC90 of 30 μg/ml. Further investigation of the virus lifecycle demonstrated that CIS extract particularly targeted the early stages of PEDV infection by blocking viral attachment and membrane fusion at rates of 80~90%. In addition, CIS extract addition reduced the viral entry of other members of the Coronaviridae family. Our data demonstrated that CIS extract inhibited PEDV infection by blocking cell entry in vitro and suggest that CIS extract is a new prophylactic and therapeutic agent against PEDV and other coronavirus infections.

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is still rapidly spreading as of March 2022. An accurate and rapid molecular diagnosis is essential to determine the exact number of confirmed cases. Currently, the viral transport medium (VTM) required for testing is in short supply due to a sharp increase in the laboratory tests performed, and alternative VTMs are needed to alleviate the shortage. Guanidine thiocyanate-based media reportedly inactivate SARS-CoV-2 and are compatible with quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays, but the compatibility and the viral detection capacity have not been fully validated. To evaluate the guanidine thiocyanate-based Gene Transport Medium (GeneTM) as an alternative VTM, we prepared 39 SARS-CoV-2-positive and 7 SARS-CoV-2-negative samples in GeneTM, eNAT™, and phosphate-buffered saline (PBS). The cycle threshold (Ct) values of three SARS-CoV-2 targets (the S, RdRP, and N genes) were analyzed using RT-qPCR testing. The comparison of Ct values from the positive samples showed a high correlation (R 2= 0.95-0.96) between GeneTM and eNAT™, indicating a comparable viral detection capacity. The delta Ct values of the SARS-CoV-2 genes in each transport medium were maintained for 14 days at cold (4°C) or room (25°C) temperatures, suggesting viral samples were stably preserved in the transport media for 14 days. Together, GeneTM is a potential alternative VTM with comparable RT-qPCR performance and stability to those of standard media.

Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell - cell fusion.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell - cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell - cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell - cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446RYVR449 and 543AVRR546. We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell - cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.

A chimeric MERS-CoV virus-like particle vaccine protects mice against MERS-CoV challenge.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. METHODS: In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. RESULTS: Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. CONCLUSIONS: Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.

Correlation between Stress and Anxiety to Viral Epidemics (SAVE) and Burnout among Korean Dental Hygienists during the COVID-19 Pandemic: A Cross-Sectional Study.

This study aimed to investigate the correlations among Stress and Anxiety to Viral Epidemics (SAVE), job stress (JS), and burnout among Korean dental hygienists during the COVID-19 pandemic and to identify the moderating effect of JS. As a cross-sectional study, a self-reporting questionnaire was used to survey 204 clinical dental hygienists to measure the levels of SAVE, JS, and burnout, along with their demographic characteristics as the control variables. Pearson correlation analysis and hierarchical multiple regression analysis were performed to analyse the correlations among burnout, SAVE, and JS, including the moderating effect of JS. With education level and subjective health controlled, JS (ß = 1.05, p < 0.001), SAVE (ß = 0.69, p = 0.020) and the interaction between SAVE and JS (ß = -0.93, p = 0.050) were identified as significant influencing factors of burnout. The adjusted explanatory power of the model was found to be 52.4%. In summary, both SAVE and JS were significant influencing factors of burnout among dental hygienists, while a moderating effect of JS was also identified. Therefore, it is necessary to create a work environment that can relieve SAVE and JS to reduce burnout among dental hygienists.

Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations.

Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.

Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37-3.15 × 101, 0.41-3.62 × 101, and 0.33-1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3-100% sensitivity and 100% specificity. Bland-Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

Effect of Oral Health Education Using a Mobile App (OHEMA) on the Oral Health and Swallowing-Related Quality of Life in Community-Based Integrated Care of the Elderly: A Randomized Clinical Trial.

This study investigated the effect of oral health education using a mobile app (OHEMA) on the oral health and swallowing-related quality of life (SWAL-QoL) of the elderly population in a community-based integrated care project (CICP). Forty elderly individuals in the CICP were randomized into intervention and control groups. OHEMA provided information on customized oral health care management, oral exercises, and intraoral and extraoral massage methods for 50 min/session, once a week, for 6 weeks. Pre- and post-intervention surveys assessed the unstimulated salivary flow rate, subjective oral dryness, tongue pressure, and SWAL-QoL, which were analyzed using ANCOVA and repeated measures ANOVA. In the intervention group, tongue pressure increased significantly from pre- (17.75) to post-intervention (27.24) (p < 0.001), and subjective oral dryness decreased from pre- (30.75) to post-intervention (18.50). The unstimulated salivary flow rate had a higher mean score in the intervention group (7.19) than in the control group (5.04) (p < 0.001). The SWAL-QoL significantly improved from pre- (152.10) to post-intervention (171.50) in the intervention group (p < 0.001) but did not change significantly in the control group (p > 0.05). OHEMA appears to be a useful tool for oral health education for the elderly as it improved the SWAL-QoL, with increased tongue pressure and reduced oral dryness.

Assay System for Simultaneous Detection of SARS-CoV-2 and Other Respiratory Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers disease with nonspecific symptoms that overlap those of infections caused by other seasonal respiratory viruses (RVs), such as the influenza virus (Flu) or respiratory syncytial virus (RSV). A molecular assay for accurate and rapid detection of RV and SARS-CoV-2 is crucial to manage these infections. Here, we compared the analytical performance and clinical reliability of Allplex™ SARS-CoV-2/FluA/FluB/RSV (SC2FabR; Seegene Inc., Seoul, South Korea) kit with those of four commercially available RV detection kits. Upon testing five target viral strains (SARS-CoV-2, FluA, FluB, RSV A, and RSV B), the analytical performance of SC2FabR was similar to that of the other kits, with no significant difference (p ≥ 0.78) in z-scores. The efficiency of SC2FabR (E-value, 81-104%) enabled reliable SARS-CoV-2 and seasonal RV detection in 888 nasopharyngeal swab specimens processed using a fully automated nucleic acid extraction platform. Bland-Altman analyses revealed an agreement value of 95.4% (SD ± 1.96) for the kits, indicating statistically similar results for all five. In conclusion, SC2FabR is a rapid and accurate diagnostic tool for both SARS-CoV-2 and seasonal RV detection, allowing for high-throughput RV analysis with efficiency comparable to that of commercially available kits. This can be used to help manage respiratory infections in patients during and after the coronavirus disease 2019 pandemic.

Immunization with porcine epidemic diarrhea virus harbouring Fc domain of IgG enhances antibody production in pigs.

Background: Outbreaks of porcine epidemic diarrhea virus (PEDV) infection have re-emerged and spread rapidly worldwide, resulting in significant economic losses. Vaccination is the best way to prevent PEDV infection in young piglets.Objective: To enhance the efficacy of an inactivated vaccine against PEDV, we evaluated the adjuvant properties of Fc domain of IgG.Methods: Fifteen crossbred gilts (180 ∼ 210 days old) were used. Five pigs in group 1 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV. Five pigs in group 2 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV-sFc. Five pigs in group 3 were not vaccinated and served as negative controls. Serum samples were collected at farrowing and subjected to ELISA, a serum neutralizing (SN) test, and a cytokine assay. Statistical analysis was performed by a two-tailed unpaired t-test.Results: Vero cells expressing swine IgG Fc on its surface was established. When PEDV was propagated in the cells expressing the swine Fc, PEDV virion incorporated the Fc. Immunization of pigs with inactivated PEDV harbouring Fc induced significantly higher antibody production against PEDV, comparing to the immunization with normal inactivated PEDV. In addition, we observed significantly increased IFN-γ levels in sera.Conclusion: Our results indicate that Fc molecule facilitate immune responses and PEDV harbouring Fc molecule could be a possible vaccine candidate. However, a challenge experiment would be needed to investigate the protective efficacy of PEDV harbouring Fc.