Effect of insilico predicted and designed potential siRNAs on inhibition of SARS-CoV-2 in HEK-293 cells.

Objectives: The COVID-19 was identified for the first time from the sea food market, Wuhan city, China in 2019 and the pathogenic organism was identified as SARS-CoV-2. Currently, this virus has spread to 223 countries and territories and known as a serious issue for the global human community. Many vaccines have been developed and used for immunization. Methods: We have reported the insilico prediction, designing, secondary structure prediction, molecular docking analysis, and in vitro assessment of siRNAs against SARS-CoV-2. The online bioinformatic approach was used for siRNAs selection and designing. The selected siRNAs were evaluated for antiviral efficacy by using Lipofectamine 2000 as delivery agent to HEK-293 cells. The MTT assay was used for cytotoxicity determination. The antiviral efficacy of potential siRNAs was determined based on the Ct value of q-RT-PCR and the data analysis was done by Prism-GraphPad software. Results: The analyzed data resulted in the selection of only three siRNAs out of twenty-six siRNAs generated by online software. The secondary structure prediction and molecular docking analysis of siRNAs revealed the efficient binding to the target. There was no cellular toxicity observed in the HEK-293 cells at any tested concentrations of siRNAs. The purification of RNA was completed from inoculated cells and subjected to q-RT-PCR. The highest Ct value was observed in siRNA 3 than the others. The results offered valuable evidence and invigorated us to assess the potency of siRNAs by using alone or in combination in other human cells. Conclusion: The data generated from this study indicates the significance of in silico prediction and narrow down the potential siRNA' against SARS-CoV-2, and molecular docking investigation offered the effective siRNAs binding with the target. Finally, it is concluded that the online bioinformatics approach provided the prediction and selection of siRNAs with better antiviral efficacy. The siRNA-3 was observed to be the best for reduction of viral RNA in cells.

In Vitro Inhibitory Analysis of Rationally Designed siRNAs against MERS-CoV Replication in Huh7 Cells.

MERS-CoV was identified for the first time in Jeddah, Saudi Arabia in 2012 in a hospitalized patient. This virus subsequently spread to 27 countries with a total of 939 deaths and 2586 confirmed cases and now has become a serious concern globally. Camels are well known for the transmission of the virus to the human population. In this report, we have discussed the prediction, designing, and evaluation of potential siRNA targeting the ORF1ab gene for the inhibition of MERS-CoV replication. The online software, siDirect 2.0 was used to predict and design the siRNAs, their secondary structure and their target accessibility. ORF1ab gene folding was performed by RNAxs and RNAfold software. A total of twenty-one siRNAs were selected from 462 siRNAs according to their scoring and specificity. siRNAs were evaluated in vitro for their cytotoxicity and antiviral efficacy in Huh7 cell line. No significant cytotoxicity was observed for all siRNAs in Huh7 cells. The in vitro study showed the inhibition of viral replication by three siRNAs. The data generated in this study provide preliminary and encouraging information to evaluate the siRNAs separately as well as in combination against MERS-CoV replication in other cell lines. The prediction of siRNAs using online software resulted in the filtration and selection of potential siRNAs with high accuracy and strength. This computational approach resulted in three effective siRNAs that can be taken further to in vivo animal studies and can be used to develop safe and effective antiviral therapies for other prevalent disease-causing viruses.

Structure-Based Identification of Natural Products as SARS-CoV-2 Mpro Antagonist from Echinacea angustifolia Using Computational Approaches.

Coronavirus disease-19 (COVID-19) pandemic, caused by the novel SARS-CoV-2 virus, continues to be a global threat. The number of cases and deaths will remain escalating due to the lack of effective therapeutic agents. Several studies have established the importance of the viral main protease (Mpro) in the replication of SARS-CoV-2 which makes it an attractive target for antiviral drug development, including pharmaceutical repurposing and other medicinal chemistry approaches. Identification of natural products with considerable inhibitory potential against SARS-CoV-2 could be beneficial as a rapid and potent alternative with drug-likeness by comparison to de novo antiviral drug discovery approaches. Thereof, we carried out the structure-based screening of natural products from Echinacea-angustifolia, commonly used to prevent cold and other microbial respiratory infections, targeting SARS-CoV-2 Mpro. Four natural products namely, Echinacoside, Quercetagetin 7-glucoside, Levan N, Inulin from chicory, and 1,3-Dicaffeoylquinic acid, revealed significant docking energy (>-10 kcal/mol) in the SARS-CoV-2 Mpro catalytic pocket via substantial intermolecular contacts formation against co-crystallized ligand (<-4 kcal/mol). Furthermore, the docked poses of SARS-CoV-2 Mpro with selected natural products showed conformational stability through molecular dynamics. Exploring the end-point net binding energy exhibited substantial contribution of Coulomb and van der Waals interactions to the stability of respective docked conformations. These results advocated the natural products from Echinacea angustifolia for further experimental studies with an elevated probability to discover the potent SARS-CoV-2 Mpro antagonist with higher affinity and drug-likeness.

Designing and evaluation of MERS-CoV siRNAs in HEK-293 cell line.

BACKGROUND: The MERS-CoV was identified for the first time from Jeddah, Saudi Arabia in 2012 from a hospitalized patient. This virus has now been spread to 27 countries with a total of 858 deaths and 2494 confirmed cases and has become a serious concern for the human population. Camels are well known for the transmission of the virus to the human population. METHODS: In this report, we have discussed the designing, prediction, and evaluation of potential siRNAs against the orf1ab gene of MERS-CoV. The online software was used to predict and design the siRNAs and finally, total twenty-one siRNA were filtered out from four hundred and sixty-two sIRNAs as per their scoring and specificity criteria. We have used only ten siRNAs to evaluate their cytotoxicity and efficacy by reverse transfection approach in HEK-293-T cell lines. RESULTS: Based on the results and data generated; no cytotoxicity was observed for any siRNAs at various concentrations in HEK-293-T cells. The ct value of real-time PCR showed the inhibition of viral replication in siRNA-1, 2, 4, 6, and 9. The data generated provided the preliminary information and encouraged us to evaluate the remaining siRNAs separately as well as in combination to analyses the replication of MERS-CoV inhibition in other cell lines. CONCLUSION: Based on the results obtained; it is concluded that the prediction of siRNAs using online software resulted in the filtration of potential siRNAs with high accuracy and strength. This technology can be used to design and develop antiviral therapy not only for MERS-CoV but also against other viruses.

Seroprevalence of Dromedary Camel HEV in Domestic and Imported Camels from Saudi Arabia.

Hepatitis E Virus (HEV) imposes a major health concern in areas with very poor sanitation in Africa and Asia. The pathogen is transmitted mainly through ingesting contaminated water or food, coming into contact with affected people, and blood transfusions. Very few reports including old reports are available on the prevalence of HEV in Saudi Arabia in humans and no reports exist on HEV prevalence in camels. Dromedary camel trade and farming are increasing in Saudi Arabia with importation occurring unidirectionally from Africa to Saudi Arabia. DcHEV transmission to humans has been reported in one case from the United Arab Emeritus (UAE). This instigated us to perform this investigation of the seroprevalence of HEV in imported and domestic camels in Saudi Arabia. Serum samples were collected from imported and domestic camels. DcHEV-Abs were detected in collected sera using ELISA. The prevalence of DcHEV in the collected samples was 23.1% with slightly lower prevalence in imported camels than domestic camels (22.4% vs. 25.4%, p value = 0.3). Gender was significantly associated with the prevalence of HEV in the collected camels (p value = 0.015) where males (31.6%) were more infected than females (13.4%). This study is the first study to investigate the prevalence of HEV in dromedary camels from Saudi Arabia. The high seroprevalence of DcHEV in dromedaries might indicate their role as a zoonotic reservoir for viral infection to humans. Future HEV seroprevalence studies in humans are needed to investigate the role of DcHEV in the Saudi human population.

IgY antibodies for the immunoprophylaxis and therapy of respiratory infections.

Emergence of drug resistance among the causative organisms for respiratory tract infections represents a critical challenge to the global health care community. Further, although vaccination can prevent disease, vaccine development is impeded by several factors. Therefore, novel approaches to treat and manage respiratory infections are urgently needed. Passive immunization represents a possible alternative to meet this need. Immunoglobulin Y antibodies (IgYs) from the yolk of chicken eggs have previously been used against bacterial and viral infections in human and animals. Their advantages include lack of reaction with mammalian Fc receptors, low production cost, and ease of extraction. Compared to mammalian IgGs, they have higher target specificity and greater binding avidity. They also possess remarkable pathogen-neutralizing activity in the respiratory tract and lungs. In this review, we provide an overview of avian IgYs and describe their potential therapeutic applications for the prevention and treatment of respiratory infections.

Analysis of Hepatitis B virus (HBV) mutations in patients from Western Saudi Arabia with chronic disease.

INTRODUCTION: Extensive research has provided a link between HBV variants and the clinical complications of liver diseases. This study was performed to further investigate the relationship between HBV variants in preS, S and BCP/PC regions and disease progression in chronic hepatitis B (CHB) cases in Jeddah, Saudi Arabia. METHODOLOGY: 182 CHB patients were recruited for this study. HBV DNA was amplified by PCR in the PreS, S, and BCP/PC regions. Sequences were generated from 31 and 26 treated cases in PreS and S regions respectively and from 72 cases in the BCP/PC region. RESULTS: The majority of cases (86.7%) were genotype D. Mutations at preS1-A2922C, X-A1624C and PC-G1887A were detected only in cases with either a high fibrosis score or hepatocellular carcinoma (HCC), while mutations at positions PC-C1982A, PC-G1951T, X-C1628T and X-A1630G were detected more frequently in HCC cases, without reaching statistical significance. Seven deletions were detected in the PreS-region. No deletions were detected in the CCAAT box. The accumulation of mutations per sample in the preS1-2 and S regions were associated with elevated ALT (p < 0.001, 0.001 and 0.001; respectively) and increased fibrosis (p = 0.018, 0.02 and 0.013; respectively). The accumulation of mutations per sample in the BCP/PC region is associated with high viral load. Occult hepatitis B infection (OBI) was identified in 5 samples. CONCLUSION: Our results add to the knowledge about HBV genotype-D variants. The accumulation of mutations per sample and OBI seem to play a role in the progression of HBV infection. G1896A was associated with the HBeAg negativity. The preS deletions did not play a role in liver disease progression.

Design and Delivery of Therapeutic siRNAs: Application to MERS-Coronavirus.

BACKGROUND: The MERS-CoV is a novel human coronavirus causing respiratory syndrome since April 2012. The replication of MERS-CoV is mediated by ORF 1ab and viral gene activity can be modulated by RNAi approach. The inhibition of virus replication has been documented in cell culture against multiple viruses by RNAi approach. Currently, very few siRNA against MERS-CoV have been computationally designed and published. METHODS: In this review, we have discussed the computational designing and delivery of potential siRNAs. Potential siRNA can be designed to silence a desired gene by considering many factors like target site, specificity, length and nucleotide content of siRNA, removal of potential off-target sites, toxicity and immunogenic responses. The efficient delivery of siRNAs into targeted cells faces many challenges like enzymatic degradation and quick clearance through renal system. The siRNA can be delivered using transfection, electroporation and viral gene transfer. Currently, siRNAs delivery has been improved by using advanced nanotechnology like lipid nanoparticles, inorganic nanoparticles and polymeric nanoparticles. CONCLUSION: The efficacy of siRNA-based therapeutics has been used not only against many viral diseases but also against non-viral diseases, cancer, dominant genetic disorders, and autoimmune disease. This innovative technology has attracted researchers, academia and pharmaceuticals industries towards designing and development of highly effective and targeted disease therapy. By using this technology, effective and potential siRNAs can be designed, delivered and their efficacy with toxic effects and immunogenic responses can be tested against MERS-CoV.