ABSTRACT
Aim: People with type 1 or type 2 diabetes have a higher hospital admission rate following Covid-19 infection. This study aims to determine the degree to which the results of a previous study in Greater Manchester (GM) could be replicated in national-level data for England. Method(s): We focussed on the univariable regression analysis, which shows the association between admission and Covid-19 infection in people with diabetes. Modelling was conducted using logistic regression on data from the Covid-IMPACT database. Odds ratios were compared descriptively with the previous study. Result(s): In people with type 2 diabetes, factors associated with an increased risk of hospitalisation similar to the previous study were: older age, male sex, higher social deprivation, higher body mass index (BMI), higher cholesterol, lower eGFR, taking an ACE-inhibitor/ ARB, not taking metformin, and having asthma or hypertension. Patients with COPD, and those taking aspirin or clopidogrel also had increased risk, but the national data showed a greater risk (GM COPD odds ratio 1.89 [1.63-2.19] vs national 2.34 [2.28-2.40] / aspirin 1.49 [1.34-1.66] vs 1.66 [1.63-1.70] / clopidogrel 1.71 [1.47-1.98] vs 1.99 [1.94-2.04]). Similar results were observed in patients with type 1 diabetes. However, due to the increase in sample size, many factors which were previously not statistically significant have become significant, such as in type 2 diabetes BMI, low HDL-cholesterol. Conclusion(s): We have successfully replicated the methods, results and conclusions of our previous study in relation to factors associated with increased risk of hospital admission in diabetes individuals. Regional databases are suitable for large cohort studies, and in this instance produced similar results to a national database, validating our previous findings.
ABSTRACT
The TG6002.03 trial is a dose-escalation phase 1 clinical trial of TG6002 infusion via the hepatic artery in patients with liver-dominant colorectal cancer metastases. TG6002 is an engineered Copenhagen strain oncolytic Vaccinia virus, deleted of thymidine kinase and ribonucleotide reductase to enhance tumor selective viral replication and expressing FCU1, an enzyme converting the non-cytotoxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). In this trial, patients with advanced unresectable liver-dominant metastatic colorectal cancer who had failed previous oxaliplatin and irinotecan-based chemotherapy were treated with up to 2 cycles of TG6002 infusion 6 weeks apart via the hepatic artery on day 1 combined with oral 5-FC on days 5 to 14 (where day 1 = TG6002 infusion). TG6002 infusion was performed over 30 minutes via selective catheterization of the hepatic artery proper. 5-FC oral dosing was 50mg/kg x4 daily. Blood was sampled for TG6002 pharmacokinetics and 5-FC and 5-FU measurements. Sampling of liver metastases was performed at screening and on day 4 or day 8 for virus detection and 5-FC and 5-FU quantification. In total, 15 patients (median age 61 years, range 37-78) were treated in 1 UK centre and 2 centres in France and received a dose of TG6002 of 1 x 106 (n=3), 1 x 107 (n=3), 1 x 108 (n=3), or 1 x 109 pfu (n=6). Fourteen of the 15 patients received a single cycle of treatment, including one patient who did not received 5-FC, and one patient received two cycles. TG6002 was transiently detected in plasma following administration, suggesting a strong tissue selectivity for viral replication. In the highest dose cohort, a virus rebound was observed on day 8, concordant with replication time of the virus. In serum samples, 5-FU was present on day 8 in all patients with a high variability ranging from 0.8 to 1072 ng/mL and was measurable over several days after initiation of therapy. Seven of the 9 patients evaluable showed the biodistribution of the virus in liver lesions by PCR testing on day 4 or day 8. Translational blood samples showed evidence for T-cell activation and immune checkpoint receptor-ligand expression. At 1 x 109 pfu, there was evidence for T-cell proliferation and activation against tumour-associated antigens by ELISpot and for immunogenic cell death. In terms of safety, a total of 34 TG6002-related adverse events were reported, of which 32 were grade 1-2 and 2 were grade 3. The maximum tolerated dose was not reached, and a single dose-limiting toxicity was observed consisting of a myocardial infarction in a context of recent Covid-19 infection in a 78-year-old patient. These results indicate that TG6002 infused via the hepatic artery in combination with oral 5-FC was well tolerated, effectively localized and replicated in the tumor tissues, expressed its therapeutic payload and showed anti-tumoral immunological activity.
ABSTRACT
It is well known that vitamin D's general immunomodulatory actions are helpful in viral infections and that a shortage is linked to a more serious prognosis for Covid-19. In this sistematic review, we examined the existing literature on evidence as to whether there is also link between vitamin D range levels in pediatric population and the outcome of the Covid-19 infection. We looked for studies that measured vitamin D blood concentrations and examined the effects of vitamin D supplementation in young infected patients. Vitamin D may decrease the risk of respiratory infections in a number of ways through its interactions with numerous cells, including by decreasing viral survival and replication, reducing the cytokine storm, raising angiotensin-converting enzyme 2 concentrations (ACE2) while not damaging the endothelial integrity. The incidence or severity of Covid-19 is linked with blood 25-hydroxyvitamin D concentrations, according to many observational studies. However experimental verification is still needed. Given their safety and broad therapeutic window, vitamin D supplements seem to be an effective way for individuals and doctors to prevent or treat Covid-19. Nonetheless, the outcomes of significant vitamin D randomized controlled trials are further needed.Copyright © 2022 Maria Nicolae et al., published by Sciendo.
ABSTRACT
Background: The emergence of Coronavirus disease (COVID-19) has been declared a pandemic and made a medical emergency worldwide. Various attempts have been made, including optimizing effective treatments against the disease or developing a vaccine. Since the SARS-CoV-2 protease crystal structure has been discovered, searching for its inhibitors by in silico technique becomes possible. Objective(s): This study aims to virtually screen the potential of phytoconstituents from the Begonia genus as 3Cl pro-SARS-CoV- 2 inhibitors, based on its crucial role in viral replication, hence making these proteases "promising" for the anti-SARS-CoV-2 target. Method(s): In silico screening was carried out by molecular docking on the web-based program DockThor and validated by a retrospective method. Predictive binding affinity (Dock Score) was used for scoring the compounds. Further molecular dynamics on Desmond was performed to assess the complex stability. Result(s): Virtual screening protocol was valid with the area under curve value 0.913. Molecular docking revealed only beta-sitosterol-3-O-beta-D-glucopyranoside with a lower docking score of -9.712 kcal/mol than positive control of indinavir. The molecular dynamic study showed that the compound was stable for the first 30 ns simulations time with Root Mean Square Deviation <3 A, despite minor fluctuations observed at the end of simulation times. Root Mean Square Fluctuation of catalytic sites HIS41 and CYS145 was 0.756 A and 0.773 A, respectively. Conclusion(s): This result suggests that beta-sitosterol-3-O-beta-Dglucopyranoside might be a prospective metabolite compound that can be developed as anti-SARS-CoV-2.Copyright © 2023, Page Press Publications. All rights reserved.
ABSTRACT
Background/Objectives: Rosmarinus Officinalis L.(Rosemary) extract Carnosic acid(CA) has been investigated for its antimicrobial and antioxidative properties(1). Only limited number of publications reported the utilization of this extract in SARSCoV-2 infection. Also, the mechanistic understanding of CA remains to be determined. Our goal was to elucidate the potential role of CA in COVID19. To obtain mechanistic insight of pharmacogenomic action of CA, comprehensive in silico analyses were performed. Further in vitro experiments were done to illustrate the cytotoxicity of CA and confirm in silico findings. Method(s): CA was extracted from Rosmarinus Officinalis L. by HPLC. Stimulation assays were performed using the COVID19 samples. In silico pharmacogenomic properties of CA were performed by using SwissADME. SwissTargetPrediction tool was utilized to define the possible targets. SARS-CoV-2-interacting proteins were evaluated using STRING(2). To verify in silico findings, gene expression levels were analyzed using qPCR. Result(s): Among the top 15 SwissTargetPrediction target molecules(out of 100), Prostaglandin E synthase(PTGES) had the highest probability for CA. Among 332 proteins identified using the STRING, PGES2 was found to be interacting with the nsp7, important molecule for viral replication. The stimulation assays and gene expression analyses confirmed the viral inhibitory role of CA through PTGES pathway. Conclusion(s): To our knowledge, our work is the first to reveal the inhibitory role of CA in COVID19 through PTGES pathway. Given the crucial role of PTGES in inflammation, it is noteworthy to examine CA as potential anti-SARS-CoV2 therapeutics.
ABSTRACT
Cumulative data regardingCOVID-19 infection during pregnancy have demonstrated the ability of SARS-CoV-2 to infect the placenta. However, the mechanisms of SARS-CoV-2 placental viral entry are yet to be defined. SARS-CoV-2 infects cells by binding to the ACE2 receptor. However, SARS-CoV-2 cell entry also requires co-localization of spike protein cleavage by the serine protease TMPRSS2. However, the co-expression of ACE2 and TMPRSS2 in placental cells is debated, raising the question of whether potential non-canonical molecular mechanismsmay be involved in SARS-CoV-2 placental cells' viral entry. Although published data regarding the ability of the SARS-CoV- 2 to infect the fetus are contradicting, the placenta appears to be an immunological barrier to active SARS-CoV-2 infection and vertical transmission;however, the mechanism is unclear. Our experiments demonstrated the ability of the SARS-CoV-2 virus to directly infect the placenta and induce transcriptomic responses in COVID-positive mothers. These transcriptomic responses were characterized by differential expression of specific mRNAs and miRNAs associated with SARS-CoV-2 infection, with induction of specific placental miRNAs that can inhibit viral replication. Failure in such mechanisms may be associated with vertical transmission. Since the start of the COVID-19 pandemic, the COVID-19 mRNA vaccines have been widely used to reduce the morbidity and mortality of SARS-CoV-2 infection. Historically, non-live vaccines have not caused any harm to pregnant mothers;however, it is unclear whether our current understanding of the effects of non-live vaccines serves as a reliable precedent owing to the novel technology used to create these mRNA vaccines. Since there are no definitive data on the possible biodistribution of mRNA vaccines to the placenta, the likelihood of vaccine mRNA reaching the fetus remains uncertain. Little has been reported on the tissue localization of the lipid nanoparticles (LNPs) after intramuscular (IM) administration of the mRNA vaccine. The biodistribution of LNPs containing the mRNA vaccine has been investigated in animal models but not humans. In the murine model, the vaccine LNPs were rapidly disseminated to several organs, including the heart, liver, kidney, lung, and spleen, following IM administration. However, no traditional pharmacokinetic or biodistribution studies have been performed with the mRNA vaccines, including possible biodistribution to breast milk or the placenta.
ABSTRACT
Within the conditions of the ongoing COVID-19 pandemic, when many questions regarding prevention and treatment strategies remain unsolved and the search for the best antiviral agents is underway, attention should be paid to the role of trace elements zinc and selenium in increasing the body's resistance to viral infections and their direct antiviral activity against SARS-CoV-2. Experimental data show that trace elements zinc and selenium not only actthrough regulating the immune response at all levels of humoral and cellular immunity, but also can play a significant role in adjuvant therapy for viral diseases. This is especially relevant in the case of COVID-19. Studies of the direct antiviral effect of these micro-elements testify to its 3 main ways to SARS-Cov-2: I - counteraction to virus replication and its transcription through: (i) their covalent binding to the SH-group of the cysteine of the main protease M(Pro) of the virus;(ii) inhibition of its RNA polymerase activity by zinc;II - preventing the penetration of the virus into cells due to blocking SH-groups of protein disulfide isomerase (RDI) of the protein of its spikes (peplomers);III - decreasing the adsorption capacity of the virus due to the blocking of the electrostatic interaction of SARS-CoV-2 peplomers and angiotensin-converting enzyme (ACE-2) in ultra-low, uncharacteristic oxidation states (Zn+1and Se-2). The intensity of the antiviral action of these trace elements may depend on their chemical form. It was found that zinc citrate (a five-membered complex of zinc with citric acid) and monoselenium citric acid obtained with the help of nanotechnology have a greater intensity of action and higher chemical purity. Taking into account the immunostimulating and direct antiviral effect of zinc and selenium, their use in the form of pharmaceuticals and dietary supplements should be considered as adjunctive therapy for SARS-CoV-2 in patients, or as a preventive strategy for uninfected people from risk groups during the spread of COVID-19.Copyright © Publisher PH <<Akademperiodyka>> of the NAS of Ukraine, 2023.
ABSTRACT
Plain language summaryMERS-CoV is a virus that causes a severe illness in the nose, mouth and throat of humans. It is a zoonotic virus, which means that it can spread from animals to humans. MERS-CoV was first found in Saudi Arabia in 2012 and continues to pose a threat to public health. Interactions between the virus and human cells and proteins are important to establishing infection. Understanding these interactions is important for the development of drugs to treat viral infections. Here, we have identified some proteins that interact with MERS-CoV. Tweetable A proteomic approach for the identification of cellular proteins that interact with the 5 '-terminal region of MERS-CoV RNA genome. #MERS-CoV #RNA_viruses. Aim: The aim of this study was to identify host factors that interact with the 5 ' end of the MERS-CoV RNA genome. Materials & methods: RNA affinity chromatography followed by mass spectrometry analysis was used to identify the binding of host factors in Vero E6 cells. Results: A total of 59 host factors that bound the MERS-CoV RNA genome in non-infected Vero E6 cells were identified. Most of the identified cellular proteins were previously reported to interact with the genome of other RNA viruses. We validated our mass spectrometry results using western blotting. Conclusion: These data enhance our knowledge about the RNA-host interactions of coronaviruses, which could serve as targets for developing antiviral therapeutics against MERS-CoV.
ABSTRACT
OBJECTIVE: This study aims to review the role of the oral cavity in SARS-CoV-2- and other viral upper respiratory tract infections. MATERIAL AND METHODS: Data reviewed in the text have been researched online and also reflect personal expertise. RESULTS: Numerous respiratory and other viruses replicate in the oral cavity and are transmitted via aerosols (< 5 µm) and droplets (> 5 µm). SARS-CoV-2 replication has been documented in the upper airways as well as in oral mucosa and salivary glands. These sites are also virus reservoirs that can infect other organs, e.g., the lungs and gastrointestinal tract, as well as other individuals. Laboratory diagnosis of viruses in the oral cavity and upper airways focuses on real-time PCR; antigen tests are less sensitive. For screening and monitoring infections, nasopharyngeal and oral swabs are tested; saliva is a good and more comfortable alternative. Physical means like social distancing or masks have been proven successful to reduce the risk of infection. Both wet-lab and clinical studies confirm that mouth rinses are effective against SARS-CoV-2 and other viruses. Antiviral mouth rinses can inactivate all viruses that replicate in the oral cavity. CONCLUSIONS: The oral cavity plays an important role in viral infections of the upper respiratory tract: it serves as a portal of entry, a site of replication, and a source of infection by droplets and aerosols. Physical means but also antiviral mouth rinses can help reduce the spread of viruses and contribute to infection control.
Subject(s)
COVID-19 , Virus Diseases , Humans , SARS-CoV-2 , Mouthwashes , Respiratory Aerosols and Droplets , Mouth , Antiviral AgentsABSTRACT
Prior to the coronavirus disease-19 (COVID-19) pandemic, the germicidal effects of visible light (λ = 400 - 700 nm) were well known. This review provides an overview of new findings that suggest there are direct inactivating effects of visible light - particularly blue wavelengths (λ = 400 - 500 nm) - on exposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions, and inhibitory effects on viral replication in infected cells. These findings complement emerging evidence that there may be clinical benefits of orally administered blue light for limiting the severity of COVID-19. Possible mechanisms of action of blue light (e.g., regulation of reactive oxygen species) and important mediators (e.g., melatonin) are discussed.
ABSTRACT
Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. In this study, Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin (GLY) for 2 hours, and then infected with different concentrations of SADSCoV, aiming to investigate the inhibitory effect of GLY on SADS-CoV. Western blot and TCID50 results revealed a significantly decreased N protein expression and viral titer, indicating that GLY can inhibit the infection of SADS-CoV. Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV. Western blot results showed that when the concentration of GLY was 0.8 mmol/L, the expression of N protein decreased significantly, indicating that GLY inhibited the invasion of the virus. At first, cells were treated with 0.4 mmol/L GLY, and cell samples were collected at 2 hours, 6 hours and 12 hours after being infected with SADS-CoV for analysis, and the expression of N protein were found to be significantly reduced at all points, indicating that GLY had a significant inhibitory effect on the replication of the virus. GLY is a competitive inhibitor of high mobility group box 1 (HMGB1), and the receptors of HMGB1 mainly include TLR4 and RAGE. Based on this fact, the mutant plasmid at the key sites of HMGB1 (C45S, C106S, C45/106S) and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV, and the cell supernatant and samples were harvested. The western blot and TCID50 results showed that the expression of N protein and the virus titer were decreased, suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection. To further explore the signaling pathway through which GLY functions, Vero E6 and IPI-2I cells were inoculated with SADS-CoV, and cell samples were harvested, western blot was used to detect the changes of MAPK proteins. The results showed that the protein expression levels of p-p38, p-JNK and p-ERK were up-regulated in the early and late stages, indicating that the MAPK pathway was activated by SADS-CoV infection. Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV. Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins, while other proteins showed no significant changes. These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK. Also, Vero E6 cells were treated with HMGB1 antibody, the siRNA of HMGB1 and HMGB1 mutants plasmid, and infected with SADS-CoV. Protein samples were harvested, western blot results showed that phosphorylation of JNK decreased, indicating that HMGB1 affected JNK phosphorylation. Finally, Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV, western blot, TCID50 and IFA results showed that the expression of N protein and virus titer, as well as virus replication were reduced, indicating that SP600125 inhibited virus replication. In conclusion, our results revealed that GLY can inhibit in vitro replication of SADS- CoV, mainly through the HMGB1/TLR4/JNK signaling pathway. The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.
ABSTRACT
Introduction: The world is currently facing a pandemic initiated by the new coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus. Viral transcription and replication are the fundamental processes of any virus. They allow the synthesis of genetic material and the consequent multiplication of the virus to infect other cells or organisms.Methods: The most important protein in SARS-CoV-2 is the RNA polymerase (RdRp or nsp12), responsible for both processes. The structure of this protein (PDB ID: 6M71) was used as a target in the application of computational strategies for a drug search, like virtual screening and molecular docking. Here, the Pathogen Box database of chemical compounds was used together with Remdesivir, Beclabuvir, and Sofosbuvir drugs as potential inhibitors of nsp12.Results: The results showed Top10 potential target inhibitors with binding energy (ΔG) higher than those of the positive controls, of which TCMDC-134153 and TCMDC-135052, both with ΔG = −7.53 kcal/mol, present interactions with three important residues of the nsp12 catalytic site.Conclusion: These proposed ligands would be used for subsequent validation by molecular dynamics, where they can be considered as drugs for the development of effective treatments against this new pandemic.
ABSTRACT
SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CLpro, and Mpro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.
Subject(s)
COVID-19 , Humans , SARS-CoV-2 , COVID-19 Vaccines , Pandemics/prevention & control , Caco-2 Cells , Drug Discovery , Antiviral Agents/therapeutic use , Nucleocapsid ProteinsABSTRACT
Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , RNA-Dependent RNA Polymerase/metabolism , Nucleotides/metabolism , RNA, Viral/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
Background: Implementation of vaccination programmes has had a transformational impact on control of the SARS-CoV-2 pandemic, but the need for effective antiviral drugs remains. Molnupiravir (MPV) targets viral RNA polymerase inhibiting replication via lethal mutagenesis and nirmatrelvir (NTV) is a protease inhibitor boosted with ritonavir when given clinically. This study aimed to assess the virological efficacy of NTV and MPV individually and in combination against the SARS-CoV-2 BA.1 Omicron variant in a K18-hACE2 mouse model. Method(s): K18-hACE2 mice were inoculated intranasally with 103 PFU of SARSCoV-2 BA.1 Omicron (B.1.1.529). After 24 hours, mice were orally dosed q12H, as outlined in Figure 1. At 2, 3, and 4-days post infection mice were sacrificed, and lung samples harvested. Animals were weighed and monitored daily throughout. Subsequently, viral replication in the lung was quantified using qRT-PCR to measure total (N-gene) and sub-genomic (E-gene) viral RNA. Data were normalized to 18S for quantitation. Viral exposures expressed as Areas Under viral load Curves (AUCs) were calculated by the trapezoidal method using mean values at each timepoint. Separate studies in Syrian golden hamsters using individual drugs were also conducted, and total serum IgG was measured by ELISA at 4-days post infection. Result(s): Mice gained weight in all groups post-treatment, with no significant difference between groups. A reduction in lung viral exposure was evident in all treatment groups compared to the vehicle control dosed mice (Figure 1). Coadministration of NTV with MPV displayed a trend towards lower lung viral exposure compared to the vehicle control with ~40-and ~45-fold reduction in AUC for N-and SgE-gene assays, respectively. Dosed individually, NTV and MPV reduced viral exposure 5.7-and 7.7-fold for the N-gene assay, respectively. Differences in total serum IgG concentrations were evident between vehicle and NTV-(34-fold reduction, P=0.018), and MPV-(4.2-fold reduction, P=0.053) treated hamsters. Conclusion(s): These data show virological efficacy of NTV and MPV against the SARS-CoV-2 BA.1 Omicron variant. The combination of NTV and MPV demonstrated a lower viral RNA exposure in the lung than either drug alone, albeit not statistically significant. Initial data indicate potential immune alterations in NTV and MPV dosed hamsters. Studies to clarify the utility of NTV/ MPV combinations and further characterize the impact of antiviral therapy on IgG are warranted.
ABSTRACT
Background: The SARS-CoV-2 Omicron variant is highly immune evasive but is attenuated in cell and animal models of infection, which many reports attribute to spike mutations. However, the phenotype and contribution to viral fitness of Omicron non-spike mutations remain unknown. Method(s): To study mutations across the entire genome independent of spike, we developed a novel cloning and replicon system capable of generating mutants within 6 hours and obtaining phenotypic results within 3-4 days. Result(s): Using a series of Omicron replicons, we found that ORF1ab harbors critical mutations, especially in the nonstructural protein 6 (NSP6), which lower viral fitness and are currently evolving in Omicron subvariants. In addition, Omicron mutations in several NSPs epistatically interact and are critical for viral replication and polyprotein processing. Conclusion(s): Collectively, we describe a robust replicon technology to study mutations across the genome and our data highlight the need to vigilantly study and monitor non-spike mutations in emerging Omicron subvariants.
ABSTRACT
Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.
ABSTRACT
The re-emergence of SARS-CoV, known as SARS-CoV-2, has proven extremely infectious that has infected a huge population worldwide. SARS-CoV-2 genome is translated into polyproteins that is processed by virus-specific protease enzymes. 3CLprotease is named as the main protease (Mpro) enzyme that cleaves nsp4 to nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. Till date, there is no effective commercially available drug against COVID-19 and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. We have used a high-throughput virtual screening approach to examine FDA approved library, natural compound library, and LOPAC 1280 (Library of Pharmacologically Active Compounds, Sigma-Aldrich, St. Louis, MO) library against Mpro. Primary screening identified potential drug molecules for the target, among which ten molecules were studied further using biophysical and biochemical techniques. SPR was used to validate the binding of inhibitors to purified Mpro and using FRET-based biochemical protease assay these inhibitors were confirmed to have Mpro inhibitory activity. Based on the kinetic studies, the antiviral efficacy of these compounds was further analysed by cell-culture based antiviral assays. Four out of ten molecules inhibited SARS-CoV-2 replication in Vero cells at a concentration range of 12.5 to 50 muM. The antiviral activity was evaluated by RT-PCR assay and TCID50 experiments. The co-crystallization of Mpro in complex with inhibitor for determining their structures is being carried out. Collectively, this study will provide valuable mechanistic and structural insights for development of effective antiviral therapeutics against SARS-CoV-2.