CoV Infection Research Articles

Designing a Candidate Multi-Epitope Vaccine against Transmissible Gastroenteritis Virus Based on Immunoinformatic and Molecular Dynamics.

Transmissible gastroenteritis virus (TGEV) is an etiological agent of enteric disease that results in high mortality rates in piglets. The economic impact of the virus is considerable, causing significant losses to the pig industry. The development of an efficacious subunit vaccine to provide promising protection against TGEV is of the utmost importance. The viral antigen, spike glycoprotein (S), is widely regarded as one of the most effective antigenic components for vaccine research. In this study, we employed immunoinformatics and molecular dynamics approaches to develop an 'ideal' multi-epitope vaccine. Firstly, the dominant, non-toxic, highly antigenic T (Th, CTL) and B cell epitopes predicted from the TGEV S protein were artificially engineered in tandem to design candidate subunit vaccines. Molecular docking and dynamic simulation results demonstrate that it exhibits robust interactions with toll-like receptor 4 (TLR4). Of particular significance was the finding that the vaccine was capable of triggering an immune response in mammals, as evidenced by the immune simulation results. The humoral aspect is typified by elevated levels of IgG and IgM, whereas the cellular immune aspect is capable of eliciting the robust production of interleukins and cytokines (IFN-γ and IL-2). Furthermore, the adoption of E. coli expression systems for the preparation of vaccines will also result in cost savings. This study offers logical guidelines for the development of a secure and efficacious subunit vaccine against TGEV, in addition to providing a novel theoretical foundation and strategy to prevent associated CoV infections.

Neurotoxic effects of coronavirus: Potential implications in Alzheimer's onset and progression

The COVID-19, caused by SARS-CoV-2, first affects the respiratory tract but evidence is emerging that the virus, reaching the central nervous system (CNS), can lead to severe neurological disorders. In particular, CoV infection could cause an acceleration of the neurodegenerative process. On the other hand, patients diagnosed with Alzheimer's disease (AD) develop more serious forms of COVID-19 with worse relapses. Therefore, understanding the connection between the two pathologies, AD and infection by coronavirus, could help in the development of new therapeutic approaches to counter them.We used the SH-SY5Y cell line differentiated into neurons, as widely used in studies of AD if supplemented with exogenous fibrillary β-amyloid (Aβ). As a glial counterpart, human microglia (HMC3) and astrocytic (D54MG) cell lines were used to create co-cultures with neurons via transwell systems. In these experimental models, we generated infection with the Human Coronavirus OC43 (HCoV-OC43), a low-risk model of SARS-CoV-2.Our results suggest that the infection by HCoV-OC43 leads to a neurotoxic effect not depending on an already present event of Aβ deposition. Indeed, unlike microglia, neurons and even more astrocytes are susceptible to CoV infection and, although the infection does not show a cytotoxic effect in the neurons in the first few days, significant alterations at a biochemical and morphological level have been observed, suggesting that the neurons are reacting to a stressful condition, including the prodromal and neurodegenerative features of AD. Interestingly, the interaction of infected astrocytes with the neurons resulted in the manifestation of signs of neurodegeneration, such as amyloid-beta deposition.By using exogenous fibrillary Aβ, as an AD in vitro model, our data suggest that there is an aggravating effect both on the infection itself and on the neurological disease progression.In conclusion, the results of this study suggest a causal interplay between HCoV-OC43 and neurological diseases and demonstrate that the co-presence of different CNS cell populations is the necessary condition to study the pathogenic effects in vitro as a whole.

Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells

Coronaviruses (CoVs) are enveloped single-stranded RNA viruses that predominantly attack the human respiratory system. In recent decades, several deadly human CoVs, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have brought great impact on public health and economics. However, their high infectivity and the demand for high biosafety level facilities restrict the pathogenesis research of CoV infection. Exacerbated inflammatory cell infiltration is associated with poor prognosis in CoV-associated diseases. In this study, we used human CoV 229E (HCoV-229E), a CoV associated with relatively fewer biohazards, to investigate the pathogenesis of CoV infection and the regulation of neutrophil functions by CoV-infected lung cells. Induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells (iAECIIs) exhibiting specific biomarkers and phenotypes were employed as an experimental model for CoV infection. After infection, the detection of dsRNA, S, and N proteins validated the infection of iAECIIs with HCoV-229E. The culture medium conditioned by the infected iAECIIs promoted the migration of neutrophils as well as their adhesion to the infected iAECIIs. Cytokine array revealed the elevated secretion of cytokines associated with chemotaxis and adhesion into the conditioned media from the infected iAECIIs. The importance of IL-8 secretion and ICAM-1 expression for neutrophil migration and adhesion, respectively, was demonstrated by using neutralizing antibodies. Moreover, next-generation sequencing analysis of the transcriptome revealed the upregulation of genes associated with cytokine signaling. To summarize, we established an in vitro model of CoV infection that can be applied for the study of the immune system perturbations during severe coronaviral disease.

Porcine lung tissue slices: a culture model for PRCV infection and innate immune response investigations

Respiratory coronaviruses (RCoVs) significantly threaten human health, necessitating the development of an ex vivo respiratory culture system for investigating RCoVs infection. Here, we successfully generated a porcine precision-cut lung slices (PCLSs) culture system, containing all resident lung cell types in their natural arrangement. Next, this culture system was inoculated with a porcine respiratory coronavirus (PRCV), exhibiting clinical features akin to humans who were infected by SARS-CoV-2. The results demonstrated that PRCV efficiently infected and replicated within PCLSs, targeting ciliated cells in the bronchioles, terminal bronchioles, respiratory bronchioles, and pulmonary alveoli. Additionally, through RNA-Seq analysis of the innate immune response in PCLSs following PRCV infection, expression levels of interferons, inflammatory cytokines and IFN stimulated genes were significantly upregulated. This ex vivo model may not only offer new insights into PRCV infection in the porcine respiratory tract but also serve as a valuable tool for studying human respiratory CoVs infection.

Bat species assemblage predicts coronavirus prevalence

Anthropogenic disturbances and the subsequent loss of biodiversity are altering species abundances and communities. Since species vary in their pathogen competence, spatio-temporal changes in host assemblages may lead to changes in disease dynamics. We explore how longitudinal changes in bat species assemblages affect the disease dynamics of coronaviruses (CoVs) in more than 2300 cave-dwelling bats captured over two years from five caves in Ghana. This reveals uneven CoV infection patterns between closely related species, with the alpha-CoV 229E-like and SARS-related beta-CoV 2b emerging as multi-host pathogens. Prevalence and infection likelihood for both phylogenetically distinct CoVs is influenced by the abundance of competent species and naïve subadults. Broadly, bat species vary in CoV competence, and highly competent species are more common in less diverse communities, leading to increased CoV prevalence in less diverse bat assemblages. In line with the One Health framework, our work supports the notion that biodiversity conservation may be the most proactive measure to prevent the spread of pathogens with zoonotic potential.

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.

An ascidian Polycarpa aurata-derived pan-inhibitor against coronaviruses targeting Mpro

Coronaviruses (CoVs) are responsible for a wide range of illnesses in both animals and human. The main protease (Mpro) of CoVs is an attractive drug target, owing its critical and highly conserved role in viral replication. Here, we developed and refined an enzymatic technique to identify putative Mpro inhibitors from 189 marine chemicals and 46 terrestrial natural products. The IC50 values of Polycarpine (1a), a marine natural substance we studied and synthesized, are 30.0 ± 2.5 nM for SARS-CoV-2 Mpro and 0.12 ± 0.05 μM for PEDV Mpro. Our research further demonstrated that pretreatment with Polycarpine (1a) inhibited the betacoronavirus SARS-CoV-2 and alphacoronavirus PEDV multiplication in Vero-E6 cells. As a result, Polycarpine (1a), a pan-inhibitor of Mpro, will function as an effective and promising antiviral option to combat CoVs infection and as a foundation for further therapeutic research.

Fungal metabolite 6-pentyl-α-pyrone reduces canine coronavirus infection

Canine coronavirus (CCoV) can produce a self-limited enteric disease in dogs but, because of notable biological plasticity of coronaviruses (CoVs), numerous mutations as well as recombination events happen leading to the emergence of variants often more dangerous for both animals and humans.Indeed, the emergence of new canine-feline recombinant alphacoronaviruses, recently isolated from humans, highlight the cross-species transmission potential of CoVs.Consequently, new effective antiviral agents are required to treat CoV infections. Among the candidates for the development of drugs against CoVs infection, fungal secondary metabolites (SMs) represent an important source to investigate. Herein, antiviral ability of 6-pentyl-α-pyrone (6 PP), a SM obtained by Trichoderma atroviride, was assessed against CCoV. During in vitro infection, nontoxic concentration of 6 PP significantly increased cell viability, reduced morphological signs of cell death, and inhibited viral replication of CCoV. In addition, we found a noticeable lessening in the expression of aryl hydrocarbon receptor (AhR), a strategic modulator of CoVs infection.Overall, due to the variety of their chemical and biological properties, fungal SMs can decrease the replication of CoVs, thus identifying a suitable in vitro model to screen for potential drugs against CoVs, using a reference strain of CCoV (S/378), non-pathogenic for humans.

1103. Endemic Coronavirus Infections in a US Cohort of Children from Birth to 4 Years

Abstract Background Endemic coronaviruses (“CoVs”) OC43, HKU1, NL63 and 229E are “common cold viruses” related to SARS-CoV-2, but their natural histories are poorly understood. We documented endemic CoV infections in a prospective cohort of US infants and children to determine the extent to which natural infection protects against subsequent homotypic and heterotypic endemic CoV infections and SARS-CoV-2 infections. Methods Cincinnati mother-child pairs were enrolled in the third trimester of pregnancy in 2017-18 and children were followed from birth to 4 years with weekly collection of mid-turbinate nasal swabs. Blood was collected at 6 weeks; 6, 12, 18, 24 months; and annually thereafter. Mothers reported on socio demographics, risk factors, and the child’s weekly symptoms. Medical visits were documented from pediatric care providers. CoV infections were followed for the first 4 years of life (focusing on the most compliant subset of 116 children having >70% weekly sample collection). Infections were identified through nasal swabs tested using a RT-PCR multiplex pathogen panel, and by serum IgG responses using a validated kit at CDC and interpreted using classification and regression tree (CART) analysis. Results We detected 398 endemic CoV infections over 317.5 child-years of follow-up (1.1 infections/child-year). Endemic beta-coronaviruses, OC43 and HKU1, were associated with statistically significant homotypic protection (77% and 84%, respectively) after a single infection. Similarly protective homotypic associations (73%) were elicited by NL63, the dominant alpha-coronavirus, after two infections. 229E infections were uncommon. No heterotypic protective association was found for any of the endemic CoVs or for SARS-CoV-2 infections from June 2020 to Nov 2021. The majority of endemic CoVs and SARS-CoV-2 infections were asymptomatic, but this proportion varied by CoV strain. Symptomatic infections were mild for all CoV strains with no hospitalizations. Conclusion Natural infection resulted in homotypic immunity but not heterotypic immunity against other CoVs to the 4th birthday. Children were not protected against SARS-CoV-2 by prior endemic CoV infections. CoV infections in these young children were largely asymptomatic or mild. Disclosures Elizabeth P. Schlaudecker, MD, MPH, Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Mary A. Staat, MD, MPH, CDC: Grant/Research Support|Cepheid: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria

Broad antagonism of coronaviruses nsp5 to evade the host antiviral responses by cleaving POLDIP3.

Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.

Induction of antiviral gene expression by cyclosporine A, but not inhibition of cyclophilin A or B, contributes to its restriction of human coronavirus 229E infection in a lung epithelial cell line

The development of antivirals with an extended spectrum of activity is an attractive possibility to protect against future emerging coronaviruses (CoVs). Cyclosporine A (CsA), a clinically approved immunosuppressive drug, has established antiviral activity against diverse unrelated viruses, including several CoVs. However, its antiviral mechanisms of action against CoV infection have remained elusive, precluding the rational design of non-immunosuppressive derivatives with improved antiviral activities. In this study, we evaluated the mechanisms of CsA against HCoV-229E infection in a human lung epithelial cell line. We demonstrate that the antiviral activity of CsA against HCoV-229E is independent of classical CsA target proteins, cyclophilin A or B, which are not required host factors for HCoV-229E in A549 cells. Instead, CsA treatment induces expression of antiviral genes in a manner dependent on interferon regulatory factor 1, but independent of classical interferon responses, which contributes to its inhibitory effect against HCoV-229E infection. Our results also point to a role for the HCoV-229E nucleoprotein in antagonizing activation of type I interferon, but we show that CsA treatment does not affect evasion of innate immune signalling pathways by HCoV-229E. Overall, our findings further the understanding of the antiviral mechanisms of CsA against CoV infection and highlight a novel immunomodulatory strategy to inhibit CoV infection that may inform future drug development efforts.

Reverse Genetics Systems for Emerging and Re-Emerging Swine Coronaviruses and Applications.

Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination.

Identification of TMEM53 as a novel SADS-CoV restriction factor that targets viral RNA-dependent RNA polymerase

ABSTRACT Zoonotic transmission of coronaviruses (CoVs) poses a serious public health threat. Swine acute diarrhea syndrome coronavirus (SADS-CoV), originating from a bat HKU2-related CoV, causes devastating swine diseases and poses a high risk of spillover to humans. Currently, licensed therapeutics that can prevent potential human outbreaks are unavailable. Identifying the cellular proteins that restrict viral infection is imperative for developing effective interventions and therapeutics. We utilized a large-scale human cDNA screening and identified transmembrane protein 53 (TMEM53) as a novel cell-intrinsic SADS-CoV restriction factor. The inhibitory effect of TMEM53 on SADS-CoV infection was found to be independent of canonical type I interferon responses. Instead, TMEM53 interacts with non-structural protein 12 (NSP12) and disrupts viral RNA-dependent RNA polymerase (RdRp) complex assembly by interrupting NSP8-NSP12 interaction, thus suppressing viral RdRp activity and RNA synthesis. Deleting the transmembrane domain of TMEM53 resulted in the abrogation of TMEM53-NSP12 interaction and TMEM53 antiviral activity. Importantly, TMEM53 exhibited broad antiviral activity against multiple HKU2-related CoVs. Our findings reveal a novel role of TMEM53 in SADS-CoV restriction and pave the way to host-directed therapeutics against HKU2-related CoV infection.

Identification, Isolation, and Molecular Characterization of Betacoronavirus in Oryx leucoryx.

Coronaviruses (CoVs) are enveloped viruses with a large RNA genome (26 to 32 kb) and are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. CoV infections cause respiratory, enteric, and neurologic disorders in mammalian and avian species. In 2019, Oryx leucoryx animals suffered from severe hemorrhagic diarrhea and high morbidity rates. Upon initial diagnosis, we found that the infected animals were positive for coronavirus by pancoronavirus reverse transcriptase RT-PCR. Next, we detected the presence of CoV particles in these samples by electron microscopy and immunohistochemistry. CoV was isolated and propagated on the HRT-18G cell line, and its full genome was sequenced. Full-genome characterization and amino acid comparisons of this viral agent demonstrated that this virus is an evolutionarily distinct Betacoronavirus belonging to the subgenus Embecovirus and the Betacoronavirus 1 species. Furthermore, we found that it is most similar to the subspecies dromedary camel coronavirus HKU23 by phylogenetic analysis. Here, we present the first report of isolation and characterization of Betacoronavirus associated with enteric disease in Oryx leucoryx. IMPORTANCE CoVs cause enteric and respiratory infections in humans and animal hosts. The ability of CoVs to cross interspecies barriers is well recognized, as emphasized by the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of novel CoV strains and surveillance of CoVs in both humans and animals are relevant and important to global health. In this study, we isolated and characterized a newly identified Betacoronavirus that causes enteric disease in a wild animal, Oryx leucoryx (the Arabian oryx). This work is the first report describing CoV infection in Oryx leucoryx and provides insights into its origin.

MLST8 is essential for coronavirus replication and regulates its replication through the mTORC1 pathway.

Coronaviruses (CoVs), which pose a serious threat to human and animal health worldwide, need to hijack host factors to complete their replicative cycles. However, the current study of host factors involved in CoV replication remains unknown. Here, we identified a novel host factor, mammalian lethal with sec-13 protein 8 (mLST8), which is a common subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), and is critical for CoV replication. Inhibitor and knockout (KO) experiments revealed that mTORC1, but not mTORC2, is essential for transmissible gastroenteritis virus replication. Furthermore, mLST8 KO reduced the phosphorylation of unc-51-like kinase 1 (ULK1), a factor downstream of the mTORC1 signaling pathway, and mechanistic studies revealed that decreased phosphorylation of the mTORC1 downstream factor ULK1 promoted the activation of autophagy, which is responsible for antiviral replication in mLST8 KO cells. Then, transmission electron microscopy indicated that both mLST8 KO and autophagy activator inhibited the formation of double-membrane vesicles in early viral replication. Finally, mLST8 KO and autophagy activator treatment could also inhibit the replication of other CoVs, indicating a conserved relationship between autophagy activation and CoV replication. In summary, our work reveals that mLST8 is a novel host regulator of CoV replication, which provides new insights into the mechanism of CoV replication and can facilitate the development of broad-spectrum antiviral drugs. IMPORTANCE CoVs are highly variable, and existing CoV vaccines are still limited in their ability to address mutations in CoVs. Therefore, the need to improve our understanding of the interaction of CoVs with the host during viral replication and to find targets for drugs against CoVs is urgent. Here, we found that a novel host factor, mLST8, is critical for CoV infection. Further studies showed that mLST8 KO inhibited the mTORC1 signaling pathway, and we found that autophagy activation downstream of mTORC1 was the main cause of antiviral replication in mLST8 KO cells. Autophagy activation impaired the formation of DMVs and inhibited early viral replication. These findings deepen our understanding of the CoV replication process and provide insights into potential therapeutic applications.

Human interactions with bats and bat coronaviruses in rural Côte d'Ivoire

Bats are presumed reservoirs of diverse α- and β- coronaviruses (CoVs) and understanding the diversity of bat-CoVs and the role bats play in CoV transmission is highly relevant in the context of the current COVID pandemic. We sampled bats in Côte d'Ivoire (2016–2018) living at ecotones between anthropogenic and wild habitats in the Marahoué National Park, a recently encroached protected area, to detect and characterize the CoVs circulating in bats and humans. A total of 314 bats were captured, mostly during the rainy season (78%), and CoV RNA was detected in three of the bats (0.96%). A CoV RNA sequence similar to Chaerephon bat coronavirus/Kenya/KY22/2006 (BtKY22) was found in a Chaerephon cf. pumilus and a Mops sp. fecal swab, while a CoV RNA sequence similar to the two almost identical Kenya bat coronaviruses BtKY55 and BtKY56 (BtKY55/56) was detected in an Epomops buettikoferi oral swab. Phylogenetic analyses indicated differences in the degree of evolutionary host-virus co-speciation for BtKY22 and BtKY55/56. To assess potential for human exposure to these viruses, we conducted human syndromic and community-based surveillance in clinics and high-risk communities. We collected data on participant characteristics, livelihoods, animal contact, and high-risk behaviors that may be associated with exposure to zoonotic diseases. We then collected biological samples for viral testing from 401 people. PCR testing of these biological samples revealed no evidence of CoV infection among the enrolled individuals. We identified higher levels of exposure to bats in people working in crop production and in hunting, trapping and fishing. Finally, we used the ‘Spillover’ risk-ranking tool to assess the potential for viral spillover and concluded that, while there is no evidence to suggest imminent risk of spillover for these CoVs, their host range and other traits suggest caution and vigilance are warranted in people with high exposure risk.

Antiviral Activity of Svarnvir-IV Tablet Assayed for Activity Against SARS-CoV-2 In Vitro.

The coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, has become a global pandemic in a very short time span. While several vaccines have been developed in the last year, specific treatments for CoV infection are still being explored. Thus, the situation highlights the need to develop safe and efficacious antiviral therapeutics. AyurvedicRasayanatherapy has been traditionally used in India for its holistic healing systems and proven history of empirical use. There is emerging evidence that Ayurvedic treatment methodologies and herbal medicines may be effective strategies in combating COVID-19. The present study is aimed at evaluating the antiviral and therapeutic activity of an Ayurvedic herbomineral formulation (Svarnvir-IV tablet, 450 mg) against the SARS-CoV-2 virusin vitro. A cell-based assay was conducted to evaluate the cytotoxicity of the Svarnvir-IV tablets (Aimil Pharmaceuticals, Delhi, India) for the determination of virucidal activity assessment (at 2 hours) and therapeutic activity assessment (at 1 hour, 2 hours, and 4 hours). When incubated with SARS-CoV-2 virus at 0.1 multiplicity of infection (MoI) for two hours, Svarnvir-IV tablet exhibited virucidal activity against SARS-CoV-2 with an EC50 value of 0.0058 mg/ml. It also exhibited therapeutic activity when treated with cells infected with the SARS-CoV-2 virus (0.1 MoI) for 1 hour, 2 hours and 4 hours post-infection, with an EC50 value of 0.094 mg/ml, 0.023 mg/ml, and 0.05 mg/ml, respectively. The original supporting data obtained from this study, along with existing Ayurvedic traditional information, has shown encouraging results.

Abstract 419: Liberation Of Soluble Urokinase Plasminogen Receptor (supar) Occurs In Response To Coronavirus Infection But Not To Influenza Virus Infection

Objectives: Urokinase (uPA), and its receptor, uPAR, mediate fibrinolysis and matrix remodeling. Liberation of monocyte/macrophage (MO/Mϕ) bound uPAR to yield soluble uPAR (suPAR) occurs after infection with coronaviruses (CoVs) such as SARS-CoV-2, and suPAR levels correlate with systemic inflammation and degree of end organ injury. How suPAR liberation from MO/Mϕs occurs after CoV infection, and whether suPAR induction occurs after pneumotropic viral infections such as influenza A (IAV) is unclear. Methods: Murine bone marrow derived Mϕs (BMDMs) and immortalized Mϕs (RAW264.7) were infected with CoV [MHVA59 (murine model for SARS-CoV-2 infection) or MHV1] or with IAV (A/PR/8/34) and levels of mRNA and protein were measured using qRT-PCR and ELISA, respectively. uPA activity of cell supernatants was quantitated using a commercially available assay. uPA knockdown of Mϕs was performed using siRNA transfection. C57BL6/J mice underwent intranasal inoculation of either 2x10 5 pfu of MHVA59, 25 pfu of IAV, or with PBS (sham). Plasma levels of suPAR and splenic Mϕ (surrogate for circulating MOs) levels of mRNA and protein were analyzed at post-infection day 3. Results: Though both CoV or IAV infection resulted in induction of mRNA and protein levels of cellular uPAR and uPA, and uPA activity, only CoV infection yielded liberation of suPAR from Mϕs (Fig. 1A-C). Indicating a functional role in suPAR liberation, infection of uPA-silenced Mϕs yielded a blunted induction of suPAR release despite not affecting mRNA levels of uPAR (Fig 1D). Finally, inoculation of mice with MHVA59, but not with IAV, resulted in elevated plasma levels of suPAR, despite inducing a robust induction of uPAR in splenic Mϕs (Fig. 1E). Conclusions: suPAR induction results after CoV infection, but not IAV infection, and occurs through a uPA-dependent pathway. Antagonism of co-regulators of uPAR and uPA may prove to be a therapeutic strategy to combat post-CoV tissue injury mediated by Mϕs.

Complete Recovery of 94 Vaccinated and Non-Vaccinated Cases from COVID-19 Treated at Home with Favipiravir, Dexamethasone, Antibiotics, and Other Supportive Medicines and Care

Objective: In 2002-2003 an extraordinary outbreak of coronavirus infection known as Severe Acute Respiratory Syndrome Corona virus or SARS CoV infection occurred in south China and eventually spread in 28 countries around the world with 8096 cases. In December 2019, Corona virus infected disease 19 or COVID-19 or SARS CoV-2 first occurred in Wuhan, South China and rapidly spread all over the world. All the leading viral disease research group and clinical Institutions have been trying hard to find an effective antiviral regime to curve the COVID-19 mortality and morbidity. The preventive vaccinations for COVID-19 are in vogue with mixed response. The objective of this study was to provide treatment for COVID-19 cases at home at the earliest with proper selection and combination of medicines to stop the mortality, morbidity and heavy hospital expenditure. Methods: All the study cases were from Kolkata, India. Considering infectivity and mortality of SARSCoV-2, primarily all the clinical history including clinical data of the cases were collected through mobile media (smart phone). The accumulated data had been recorded in history sheets which were finally completed after the recovery of the cases by personal interview or interview of the family members in case of death. The majority cases were diagnosed by RT-PCR and rest by Rapid antigen test or by symptoms. The study could be labelled as an open label study considering all the facts and figures and outcome involved in the study design of this clinical research. This study included 123 COVID-19 cases, at an age range, 10-85 Years (mean age 48·66±17·69; median age 51 years). Out of these cases, 94 cases between 22-85 years (mean age 50·44±16·83; median age 53years) were selected for home treatment up to 2 weeks with our formulated protocol consisting of Favipiravir, Dexamethasone Antibiotics (Azithromycin/ Clarithromycin or Doxycycline), Pantoprazole and Doxophylline. Out of 94 cases only two cases had experienced little fall of oxygen saturation and treated with oxygen at home and Doxophylline. All the 94 cases treated with the protocol completely recovered after two weeks without any residual symptoms. The rest 29 cases, treated outside in hospitals or clinics and designated as external cases were used for comparison. Results: Out of total of 29 COVID-19 external cases, 25 cases had taken ivermectin for COVID-19 treatment of which 19 cases recovered with various post COVID symptoms. Six cases were unresponsive to ivermectin and were admitted to hospital and out of them one patient expired. All the 94 cases, treated at home by our protocol, had recovered completely from the disease without any residual symptoms and the recovery period was shortened. we found that, our formulated protocol for COVID-19 was an absolute success. Conclusion: From the study it is concluded that COVID-19 cases can be treated effectively at home without any casualty provided that the cases are diagnosed and treated with proper medicines at the earliest.

Peptide-based inhibitors hold great promise as the broad-spectrum agents against coronavirus.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), and the recent SARS-CoV-2 are lethal coronaviruses (CoVs) that have caused dreadful epidemic or pandemic in a large region or globally. Infections of human respiratory systems and other important organs by these pathogenic viruses often results in high rates of morbidity and mortality. Efficient anti-viral drugs are needed. Herein, we firstly take SARS-CoV-2 as an example to present the molecular mechanism of CoV infection cycle, including the receptor binding, viral entry, intracellular replication, virion assembly, and release. Then according to their mode of action, we provide a summary of anti-viral peptides that have been reported in peer-reviewed publications. Even though CoVs can rapidly evolve to gain resistance to the conventional small molecule drugs, peptide-based inhibitors targeting various steps of CoV lifecycle remain a promising approach. Peptides can be continuously modified to improve their antiviral efficacy and spectrum along with the emergence of new viral variants.