Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of the FBXW7 Protein via the Ubiquitin-Proteasome Pathway.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Genome Analysis of the G6P6 Genotype of Porcine Group C Rotavirus in China.

Swine enteric disease is the predominant cause of morbidity and mortality, and viral species involved in swine enteric disease include rotaviruses and coronaviruses, among others. Awareness of the circulating porcine rotavirus group C (PoRVC) in pig herds is critical to evaluate the potential impact of infection. At present, due to the lack of disease awareness and molecular diagnostic means, the research on RVC infection in China is not well-studied. In this study, diarrhea samples collected from pig farms were detected positive for RVC by PCR, and the full-length RVC was not previously reported for Chinese pig farms. This rotavirus strain was designated as RVC/Pig/CHN/JS02/2018/G6P6. A natural recombination event was observed with breakpoints at nucleotides (nt) 2509 to 2748 of the VP2 gene. Phylogenetic analysis based on nsp1 revealed that a new branch A10 formed. Collectively, our data suggest a potentially novel gene recombination event of RVC in the VP2 gene. These findings provide a new insight into the evolution of the rotavirus.

Bioinformatics and systems biology approach to identify the pathogenetic link of Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Background The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global crisis. Although many people recover from COVID-19 infection, they are likely to develop persistent symptoms similar to those of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) after discharge. Those constellations of symptoms persist for months after infection, called Long COVID, which may lead to considerable financial burden and healthcare challenges. However, the mechanisms underlying Long COVID and ME/CFS remain unclear. Methods We collected the genes associated with Long COVID and ME/CFS in databases by restricted screening conditions and clinical sample datasets with limited filters. The common genes for Long COVID and ME/CFS were finally obtained by taking the intersection. We performed several advanced bioinformatics analyses based on common genes, including gene ontology and pathway enrichment analyses, protein–protein interaction (PPI) analysis, transcription factor (TF)–gene interaction network analysis, transcription factor–miRNA co-regulatory network analysis, and candidate drug analysis prediction. Results We found nine common genes between Long COVID and ME/CFS and gained a piece of detailed information on their biological functions and signaling pathways through enrichment analysis. Five hub proteins (IL-6, IL-1B, CD8A, TP53, and CXCL8) were collected by the PPI network. The TF–gene and TF–miRNA coregulatory networks were demonstrated by NetworkAnalyst. In the end, 10 potential chemical compounds were predicted. Conclusion This study revealed common gene interaction networks of Long COVID and ME/CFS and predicted potential therapeutic drugs for clinical practice. Our findings help to identify the potential biological mechanism between Long COVID and ME/CFS. However, more laboratory and multicenter evidence is required to explore greater mechanistic insight before clinical application in the future.

SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.

A New Neutralization Epitope in the Spike Protein of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infects piglets and causes serious diarrhea as well as vomiting, dehydration, and death. The trimeric S protein plays a crucial role in the induction of neutralizing antibodies, and many neutralizing monoclonal antibodies (mAbs) against PEDV S protein have been developed. However, these mAbs exclusively target the S1 protein. In this study, we obtained a neutralizing mAb, 5F7, against the S2 protein of PEDV, and this mAb could neutralize new variant genotype 2 PEDV strains (LNCT2), as well as a genotype 1 PEDV strain (CV777), in vitro. The core sequence of the epitope was found in amino acid sequence 1261 aa~1337 aa. These findings confirm that the S2 protein possessed neutralizing epitopes and provided knowledge to aid further research on this virus.

Design and Evaluation of a Novel Peptide-Drug Conjugate Covalently Targeting SARS-CoV-2 Papain-like Protease.

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide-drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 µM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

Porcine deltacoronavirus infection is inhibited by Griffithsin in cell culture.

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus that causes severe diarrhea in piglets and results in serious economic losses. There are no effective vaccines and antiviral drugs to prevent and treat PDCoV infection currently. Griffithsin (GRFT) is a lectin with potent antiviral activity against enveloped viruses because of its ability to specifically bind N-linked high-mannose oligosaccharides. GRFT has been reported to possess antiviral activity against severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and porcine epidemic diarrhea virus (PEDV). Here, we first confirmed the antiviral activity of GRFT against PDCoV in vitro. The infected cells (%) and virus titers were significantly decreased at concentration 1 µg/mL or above of GRFT. Time-course experiments revealed that GRFT inhibits PDCoV infection at the adsorption and penetration step. GRFT binding to PDCoV spike (S) protein on the surface wraps the virus and blocks its entry. The outstanding antiviral potency indicates that GRFT has the potential value as a candidate drug for the prevention and treatment of PDCoV infection.

Exploring the interaction of quercetin-3-O-sophoroside with SARS-CoV-2 main proteins by theoretical studies: A probable prelude to control some variants of coronavirus including Delta.

The aim of this study was to investigate the mechanism of interaction between quercetin-3-O-sophoroside and different SARS-CoV-2's proteins which can bring some useful details about the control of different variants of coronavirus including the recent case, Delta. The chemical structure of the quercetin-3-O-sophoroside was first optimized. Docking studies were performed by CoV disease-2019 (COVID-19) Docking Server. Afterwards, the molecular dynamic study was done using High Throughput Molecular Dynamics (HTMD) tool. The results showed a remarkable stability of the quercetin-3-O-sophoroside based on the calculated parameters. Docking outcomes revealed that the highest affinity of quercetin-3-O-sophoroside was related to the RdRp with RNA. Molecular dynamic studies showed that the target E protein tends to be destabilized in the presence of quercetin-3-O-sophoroside. Based on these results, quercetin-3-O-sophoroside can show promising inhibitory effects on the binding site of the different receptors and may be considered as effective inhibitor of the entry and proliferation of the SARS-CoV-2 and its different variants. Finally, it should be noted, although this paper does not directly deal with the exploring the interaction of main proteins of SARS-CoV-2 Delta variant with quercetin-3-O-sophoroside, at the time of writing, no direct theoretical investigation was reported on the interaction of ligands with the main proteins of Delta variant. Therefore, the present data may provide useful information for designing some theoretical studies in the future for studying the control of SARS-CoV-2 variants due to possible structural similarity between proteins of different variants.

Epidemiology of Sleep Disturbances and Their Effect on Psychological Distress During the COVID-19 Outbreak: A Large National Study in China.

Background: The purpose of the current study was to assess the prevalence of sleep disturbances among Chinese people during the COVID-19 pandemic in a large national survey, analyze the relationship between sleep disturbances and mental health status, and explore the influencing factors of the relationship between sleep disturbances and mental health status. Methods: An online survey was accessed by 19,740 people throughout China from February 14 to 21, 2020. The survey included the Depression Anxiety Stress Scale-21 (DASS-21) to measure psychological distress and two questions about sleep disturbances. Logistic regression analyses and moderation analysis were performed. Results: (1) Among the 14,505 respondents included in analyses, 3,783 (26.08%) reported sleep disturbances at least 3 days during the past week. (2) Sleep disturbances increased the risk of depression, anxiety, and stress (p < 0.05). (3) Gender, age, education, occupation, frequency of attending to epidemic information, nervousness about supplies, receiving provisions of living necessities from the service department during the outbreak, number of correct responses to questions about the epidemic, and isolation/quarantine affected the risk of mental health problems among participants experiencing sleep disturbances (p < 0.05). (4) A moderation analysis found that sleep problems were more likely to affect depression, anxiety, and stress scores in men than women during the COVID-19 outbreak. Conclusion: During the COVID-19 outbreak, 26.08% people surveyed experienced sleep disturbances, and the presence of sleep disturbances was positively related to depression, anxiety, and stress, especially among front-line anti-epidemic workers, younger people, people living in isolation/quarantine, people with a college or greater education, and males.

Risk and clinical outcomes of COVID-19 in patients with rheumatic diseases compared with the general population: a systematic review and meta-analysis.

Patients with rheumatic diseases are often more susceptible to different bacteria and viruses because of immune impairment, but it is not clear whether there is a higher risk of infection and a more serious course of disease for novel coronavirus (SARS-CoV-2). We performed this systematic review and meta analysis to assess the risk and clinical outcomes of COVID-19 in patients with rheumatic diseases compared with the general population. We searched PubMed, EMBASE, Scopus and Web of Science databases from January 1, 2020 to October 20, 2020 to determine epidemiological information related to patients with rheumatic diseases and COVID-19, including clear risk estimate or data that could be converted and extracted. We included 26 observational studies, totaling about 2000 patients with rheumatic diseases of whom were infected with COVID-19. Meta-analysis showed that the risk of COVID-19 infection in rheumatic patients was significantly higher than that in the general population (OR = 1.53, 95% CI 1.24-1.88, P = 0.000). In terms of hospitalization and severe clinical outcomes associated with COVID-19, we found that rheumatic patients showed similar results to the reference population (hospitalization OR = 1.36, 95% CI 0.81-2.29, P = 0.247; admitted to ICU OR = 1.94, 95% CI 0.88-4.27, P = 0.098; death OR = 1.29, 95% CI 0.84-1.97, P = 0.248). The presence of comorbidities, hypertension, lung diseases were significantly associated with the increased risk of COVID-19-related hospitalization in rheumatic patients and anti-TNF drugs were associated with lower hospitalization risk. Older age was related to severe COVID-19. Our meta-analysis indicated that rheumatic patients were at a higher risk of COVID-19 infection but might not lead to a more serious disease process.

Dynamics of binding ability prediction between spike protein and human ACE2 reveals the adaptive strategy of SARS-CoV-2 in humans.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing the COVID-19 pandemic in 2020. High adaptive plasticity on the spike protein of SASR-CoV-2 enables it to transmit across different host species. In the present study, we collected 2092 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny. We also analyzed the polymorphic interaction between spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 suggests that SARS-CoV-2 is probably originated from a recombination event on the spike protein between a bat coronavirus and a pangolin coronavirus that endows it humans infectivity. Compared with other regions in the S gene of SARS-CoV-2, the direct-binding sites of the receptor-binding domain (RBD) is more conserved. We focused on 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 and simulated their differential stability and binding affinity to hACE2 (S19-D615). The results indicate no preference for SARS-CoV-2 infectivity on people of different ethnic groups. The variants in the spike protein of SARS-CoV-2 may also be a good indicator demonstrating the transmission route of SARS-CoV-2 from its natural reservoir to human hosts.

Coronaviruses disease 2019 (COVID-19): Causative agent, mental health concerns, and potential management options.

Coronavirus disease-2019 (COVID-19) pandemic started from Wuhan, China has infected more than 6.7 million individuals and killed more than 390,000 individuals globally. Due to the higher transmissibility and infectiousness, asymptomatic infection, and lack of effective treatment options and vaccine, fatalities and morbidities are increasing day by day globally. Despite physical health consequences, COVID-19 pandemic has created stress and anxiety, as result there is an increased risk of mental illnesses both in the infected and normal individuals. To eradicate these risks, it is necessary to determine the COVID-19 zoonotic source of transmission to humans and clinical manifestations in infected individuals. Although, identification or development of the highly effective therapeutic agents is necessary, however, development of protective strategies against the COVID-19 by enhancing immune responses will be an asset in the current scenarios of the COVID-19 pandemic. In this paper, we discuss the transmission, health consequences, and potential management (therapeutic and preventive) options for COVID-19 disease.

Mental health consequences of infections by coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

BACKGROUND: Anxiety and stress like mental illnesses are the common outcomes of viral epidemics and pandemics. Novel coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) was first reported in Wuhan, China, and then spread all over the world in a short time. OBJECTIVES: To highlight and discuss the impact of COVID-19 pandemic on mental or psychological health. METHOD: Literature search and collection of the information were performed using PubMed, the reports from the World health organization, and the Center for disease control and prevention. RESULTS: COVID-19 infection has already been declared as a global pandemic, which in association with infodemic has increased the risk of psychiatric/psychological disorders. A large population of the world is prone to develop anxiety, depressive disorders, and other mental abnormalities. Therefore, timely psychological interventions and preventive strategies are required. Moreover, the infection has been reported to be linked with cerebrovascular conditions; therefore, patients with underlying cerebrovascular diseases should be given attention. CONCLUSION: COVID-19-mediated mental health complications and cerebrovascular conditions may cause a huge burden on healthcare communities in the future. Therefore, timely intervention and the development or application of preventive strategies are required to decrease the risk of neurological consequences.

Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum.

Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-SIgA secretory component (SC) mAb probe for the detection of anti-PEDV-specific SIgA in swine. On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Results showed that the immunochromatographic test strip had high sensitivity and specificity. When compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect PEDV specific SIgA in colostrum samples. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. We found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of PEDV specific SIgA, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying PEDV.