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Objectives: The World Health Organization priority zoonotic pathogen Middle East respiratory syndrome (MERS) coronavirus (CoV) has a high case fatality rate in humans and circulates in camels worldwide. Methods: We performed a global analysis of human and camel MERS-CoV infections, epidemiology, genomic sequences, clades, lineages, and geographical origins for the period January 1, 2012 to August 3, 2022. MERS-CoV Surface gene sequences (4061 bp) were extracted from GenBank, and a phylogenetic maximum likelihood tree was constructed. Results: As of August 2022, 2591 human MERS cases from 26 countries were reported to the World Health Organization (Saudi Arabia, 2184 cases, including 813 deaths [case fatality rate: 37.2%]) Although declining in numbers, MERS cases continue to be reported from the Middle East. A total of 728 MERS-CoV genomes were identified (the largest numbers were from Saudi Arabia [222: human = 146, camels = 76] and the United Arab Emirates [176: human = 21, camels = 155]). A total of 501 ‘S’-gene sequences were used for phylogenetic tree construction (camels [n = 264], humans [n = 226], bats [n = 8], other [n=3]). Three MERS-CoV clades were identified: clade B, which is the largest, followed by clade A and clade C. Of the 462 clade B lineages, lineage 5 was predominant (n = 177). Conclusion: MERS-CoV remains a threat to global health security. MERS-CoV variants continue circulating in humans and camels. The recombination rates indicate co-infections with different MERS-CoV lineages. Proactive surveillance of MERS-CoV infections and variants of concern in camels and humans worldwide, and development of a MERS vaccine, are essential for epidemic preparedness.

Background: Infection with SARS-CoV-2 may perturb normal microbiota, leading to secondary infections that can complicate the viral disease. The aim of this study was to probe the alteration of nasopharyngeal (NP) microbiota in the context of SARS-CoV-2 infection and obesity and to identify other respiratory pathogens among COVID-19 cases that may affect patients’ health. Methods: A total of 107 NP swabs, including 22 from control subjects and 85 from COVID-19 patients, were processed for 6S amplicon sequencing. The respiratory pathogens causing secondary infections were identified by RT-PCR assay, using a kit that contained specific primers and probes combinations to amplify 33 known respiratory pathogens. Results: No significant (p > 0.05) difference was observed in the alpha and beta diversity analysis, but specific taxa differed significantly between the control and COVID-19 patient groups. Genera of Sphingomonas, Kurthia, Microbacterium, Methylobacterium, Brevibacillus, Bacillus, Acinetobacter, Lactococcus, and Haemophilus was significantly abundant (p

The risk of transmission of respiratory tract infections is considerably enhanced at mass gathering (MG) religious events. Hajj is an annual Islamic MG event with approximately 3 million Muslim pilgrims from over 180 countries concentrated in Makkah, Saudi Arabia. This study aimed to investigate the genetic diversity of influenza viruses circulating among pilgrims during the Hajj pilgrimage. We performed a cross-sectional analytical study where nasopharyngeal swabs (NPs) from pilgrims with respiratory tract illnesses presenting to healthcare facilities during the 2019 Hajj were screened for influenza viruses. Influenza A subtypes and influenza B lineages were determined by multiplex RT–PCR for positive influenza samples. The phylogenetic analysis was carried out for the hemagglutination (HA) gene. Out of 185 nasopharyngeal samples, 54 were positive for the human influenza virus. Of these, 27 were influenza A H1N1 and 19 H3N2, 4 were untypable influenza A, and 4 were influenza B. Phylogenetic analysis revealed that the H1N1 and H3N2 strains differentiated into different and independent genetic groups and formed close clusters with selected strains of influenza viruses from various locations. To conclude, this study demonstrates a high genetic diversity of circulating influenza A subtypes among pilgrims during the Hajj Season. There is a need for further larger studies to investigate in-depth the genetic characteristics of influenza viruses and other respiratory viruses during Hajj seasons.

High seroprevalence rates of several phleboviruses have been reported in domestic animals and humans in sandfly-infested regions. Sandfly Fever Sicilian virus (SFSV) and Toscana virus (TOSV) are two of these viruses commonly transmitted by Phlebotomus sandflies. While SFSV can cause rapidly resolving mild febrile illness, TOSV could involve the central nervous system (CNS), causing diseases ranging from aseptic meningitis to meningoencephalitis. Sandfly-associated phleboviruses have not been investigated before in Saudi Arabia and are potential causes of infection given the prevalence of sandflies in the country. Here, we investigated the seroprevalence of SFSV and TOSV in the western region of Saudi Arabia in samples collected from blood donors, livestock animals, and animal handlers. An overall seroprevalence of 9.4% and 0.8% was found in humans for SFSV and TOSV, respectively. Seropositivity was significantly higher in non-Saudis compared to Saudis and increased significantly with age especially for SFSV. The highest seropositivity rate was among samples collected from animal handlers. Specifically, in blood donors, 6.4% and 0.7% tested positive for SFSV and TOSV nAbs, respectively. Animal handlers showed higher seroprevalence rates of 16% and 1% for anti-SFSV and anti-TOSV nAbs, respectively, suggesting that contact with livestock animals could be a risk factor. Indeed, sera from livestock animals showed seropositivity of 53.3% and 4.4% in cows, 27.5% and 7.8% in sheep, 2.2% and 0.0% in goats, and 10.0% and 2.3% in camels for SFSV and TOSV, respectively. Together, these results suggest that both SFSV and TOSV are circulating in the western region of Saudi Arabia in humans and livestock animals, albeit at different rates, and that age and contact with livestock animals could represent risk factors for infection with these viruses.

Objectives: Acute respiratory tract infections (ARIs) due to human rhinoviruses (HRVs) are common in pilgrims during the annual Hajj pilgrimage. The objective of this study was to investigate the genetic diversity of HRV among pilgrims with respiratory symptoms during Hajj 2019. Methods: HRV infection was detected using multiplex real-time reverse transcription polymerase chain reaction. Cycle sequencing was performed on positive samples and the sequences were subjected to phylogenetic analysis. Results: A total of 19 HRV-positive respiratory samples were sequenced. All three serotypes of HRV were identified: HRV-A (13; 68.42%) was more common than HRV-B (2; 10.53%) and HRV-C (4; 21.05%). HRV-A species were found to be of genotypes A101, A21, A30, A57, A23, A60, and A11. HRV-B species belonged to genotypes B4 and B84, and HRV-C species were of genotypes C15, C3, and C56. Conclusion: Sequencing studies of respiratory tract viruses in pilgrims are important. We provide preliminary evidence of high diversity of HRV genotypes circulating in pilgrims in a restricted area during Hajj. This requires further clinical and sequencing studies of viral pathogens in larger cohorts of overseas and local pilgrims.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a more severe strain of coronavirus (CoV) that was first emerged in China in 2019. Available antiviral drugs could be repurposed and natural compounds with antiviral activity could be safer and cheaper source of medicine for SARS-CoV-2. 78 natural antiviral compounds database was identified from literature and virtual screening technique was applied to identify potential 3-chymotrypsin-like protease (3CLpro) inhibitors. Molecular docking studies were conducted to analyze the main protease (3CLpro) and inhibitors interactions with key residues of active site of target protein (PDB ID: 6LU7), active site constitute the part of active domain I and II of 3CLpro. 10 compounds with highest dock score were subjected to calculate ADMET parameters to figure out drug-likeness. Molecular dynamic (MD) simulation of the selected lead was performed by Amber simulation package to understand the conformational changes in docked complex. MD simulations analysis (RMSD, RMSF, Rg, BF, HBs, and SASA plots) of lead bounded with 3CLpro, hence revealed the important structural turns and twists during MD simulations from 0 to 100 ns. MM-PBSA/GBSA methods has also been applied for the estimation binding free energy (BFE) of the selected lead-complex. The present study has identified lead compound “Forsythoside A” an active extract of Forsythia suspense as SARS-CoV-2 3CLpro inhibitor that can block the viral replication and translation. Structural analysis of target protein and lead compound performed in this study could contribute to the development of potential drug against SARS-CoV-2 infection.

Background: Rheumatic diseases patients receiving Rituximab had severe COVID-19 disease. Although they had impaired humoral immune responses following COVID-19 vaccine, they had preserved cellular immune responses. Waning of COVID-19 antibody responses was observed within six months post vaccination among immunocompromised patients. Recent reports showed fatal outcome of breakthrough SARS-CoV-2 infections among vaccinated high-risk rheumatic diseases patients receiving Rituximab. SAR-CoV-2 serological tests were not performed. Objective: Evaluation of COVID-19 vaccine humoral responses and breakthrough infections among low risk fully vaccinated rheumatic patients during the Delta Variant Era. Methods: A case series of 19 fully vaccinated patients with rheumatic diseases were followed to determine post vaccine SARS-CoV-2 neutralizing antibody titers and to monitor the development of breakthrough infections up to eight months post vaccine at our tertiary care center in Jeddah, Saudi Arabia from 1st April until 30th November 2021. Results: The mean age of patients was 49 years old. 10% of patients were receiving Rituximab. 73% of patients had positive SARS-CoV-2 serological testing post second vaccine. Two mild breakthrough COVID-19 infections were diagnosed six months post second dose of vaccine. Patients were less than 65 years, did not receive Rituximab, did not have interstitial lung diseases and had positive post vaccine serological testing. Conclusions: We demonstrated high SARS-CoV-2 neutralizing antibodies seroprevalence and self-limiting breakthrough infections in low risk rheumatic diseases patients during the Delta Era. Future studies are needed to study the outcome of rheumatic diseases patients in the Era of Omicron in view of viral immune escape responses.

Abstract Camels gained attention since the discovery of MERS-CoV as intermediary hosts for potentially epidemic zoonotic viruses. DcHEV is a novel zoonotic pathogen associated with camel contact. This study aimed to genetically characterize DcHEV in domestic and imported camels in Saudi Arabia. DcHEV was detected by RT-PCR in serum samples, PCR-positive samples were subjected to sequencing and phylogenetic analyses. DcHEV was detected in 1.77% of samples with higher positivity in domestic DCs. All positive imported dromedaries were from Sudan with age declining prevalence. Domestic DcHEV sequences clustered with sequences from Kenya, Somalia, and UAE while imported sequences clustered with one DcHEV isolate from UAE and both sequences clustered away from isolates reported from Pakistan. Full-genome sequences showed 24 amino acid difference with reference sequences. Our results confirm the detection of DcHEV in domestic and imported DCs. Further investigations are needed in human and camel populations to identify DcHEV potential zoonosis threat.

Antibiotic resistant bacteria are a growing threat to global health security. Whilst the emergence of antimicrobial resistance (AMR) is a natural phenomenon, it is also driven by antibiotic exposure in health care, agriculture, and the environment. Antibiotic pressure and inappropriate use of antibiotics are important factors which drive resistance. Apart from their use to treat bacterial infections in humans, antibiotics also play an important role in animal husbandry. With limited antibiotic options, alternate strategies are required to overcome AMR. Passive immunization through oral, nasal and topical administration of egg yolk-derived IgY antibodies from immunized chickens were recently shown to be effective for treating bacterial infections in animals and humans. Immunization of chickens with specific antigens offers the possibility of creating specific antibodies targeting a wide range of antibiotic-resistant bacteria. In this review, we describe the growing global problem of antimicrobial resistance and highlight the promising potential of the use of egg yolk IgY antibodies for the treatment of bacterial infections, particularly those listed in the World Health Organization priority list.

Data integration with phenotypes such as gene expression, pathways or function, and protein-protein interactions data has proven to be a highly promising technique for improving human complex diseases, particularly cancer patient outcome prediction. Hepatocellular carcinoma is one of the most prevalent cancers, and the most common cause is chronic HBV and HCV infection, which is linked to the majority of cases, and HBV and HCV play a role in multistep carcinogenesis progression. We examined the list of known hepatocellular carcinoma biomarkers with the publicly available expression profile dataset of hepatocellular carcinoma infected with HCV from day 1 to day 10 in this study. The study covers an overexpression pattern for the selected biomarkers in clinical hepatocellular carcinoma patients, a combined investigation of these biomarkers with the gathered temporal dataset, temporal expression profiling changes, and temporal pathway enrichment following HCV infection. Following a temporal analysis, it was discovered that the early stages of HCV infection tend to be more harmful in terms of expression shifting patterns, and that there is no significant change after that, followed by a set of genes that are consistently altered. PI3K, cAMP, TGF, TNF, Rap1, NF-kB, Apoptosis, Longevity regulating pathway, signaling pathways regulating pluripotency of stem cells, Cytokine-cytokine receptor interaction, p53 signaling, Wnt signaling, Toll-like receptor signaling, and Hippo signaling pathways are just a few of the most commonly enriched pathways. The majority of these pathways are well-known for their roles in the immune system, infection and inflammation, and human illnesses like cancer. We also find that ADCY8, MYC, PTK2, CTNNB1, TP53, RB1, PRKCA, TCF7L2, PAK1, ITPR2, CYP3A4, UGT1A6, GCK, and FGFR2/3 appear to be among the prominent genes based on the networks of genes and pathways based on the copy number alterations, mutations, and structural variants study.

In the era of SARS-CoV-2 variants and COVID-19 vaccination, the duration of infectious viral shedding and isolation in post vaccine breakthrough infections is challenging and depends on disease severity. The current study described a case of SARS-CoV-2 Delta variant pneumonia requiring hospitalization. The patient received two doses of BNT162b2 COVID-19 vaccines, and he had positive SARS-CoV-2 viral cultures 12 days post symptom onset. The time between the second dose of vaccine and the breakthrough infection was 6 months. While immunosuppression is a known risk factor for prolonged infectious viral shedding, age and time between vaccination and breakthrough infection are important risk factors that warrant further studies.

Safe, passive immunization methods are required against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants. Immunization of chickens with antigen is known to induce specific IgY antibodies concentrated in the egg yolk and has a good safety profile, high yield of IgY per egg, can be topically applied, not requiring parenteral delivery. Our data provide the first evidence of the prophylactic efficacy of Immunoglobulin Y antibodies against SARS-CoV-2 in mice. Lohmann hens were injected with recombinant SARS-CoV-2 RBD protein; IgY-Abs were extracted from the eggs and characterized using SDS-PAGE. Antiviral activity was evaluated using plaque reduction neutralization tests. In additional experiments, IgY-RBD efficacy was examined in mice sensitized to SARS-CoV-2 infection by transduction with Ad5-hACE2 (mild disease) or by using mouse-adapted virus (severe disease). In both cases, prophylactic intranasal administration of IgY-Abs reduced SARS-CoV-2 replication, and reduced morbidity, inflammatory cell infiltration, hemorrhage, and edema in the lungs and increased survival compared to control groups that received non-specific IgY-Abs. These results indicate that further evaluation of IgY-RBD antibodies in humans is warranted. Author summary We report the generation, characterization and antiviral activity of egg yolk derived IgY-Abs against the SARS-CoV-2 Receptor Binding Domain. Using two different models of murine SARS-CoV-2 infection, we show that intranasal prophylactic administration of IgY-Abs reduced viral replication, and reduced inflammatory cell infiltration, hemorrhage, and edema in the lungs.

Iron is a crucial micronutrient for immunity induction in response to infections and vaccinations. This study aimed to investigate the effect of iron deficiency on COVID-19-vaccine-induced humoral immunity. We investigated the effectiveness of COVID-19 vaccines (BNT162b2, mRNA-1273, and ChAdOx nCov-2019) in iron-deficient individuals (n = 63) and provide a side-by-side comparison to healthy controls (n = 67). The presence of anti-SARS-CoV-2 spike (S) and anti-nucleocapsid (NP) IgG were assessed using in-house S- and NP-based ELISA followed by serum neutralization test (SNT). High concordance between S-based ELISA and SNT results was observed. The prevalence of neutralizing antibodies was 95.24% (60/63) in the study group and 95.52% (64/67) in the controls with no significant difference. The presence/absence of past infection, period since vaccination, vaccine type, and being iron-deficient or having iron-deficiency anemia did not exert any significant effect on the prevalence or titer of anti-SARS-CoV-2 neutralizing antibodies. NP-based ELISA identified individuals unaware of exposure to SARS-CoV-2. Moreover, absence of anti-NP IgG was noted in participants who were previously diagnosed with COVID-19 suggesting the unpredictability of after-infection immunity. To sum up, this study demonstrated an initial lack of evidence on the association between iron deficiency and the effectiveness of COVID-19-vaccine-induced neutralizing humoral immunity. Similar studies with larger sample size remain necessary to obtain comprehensive conclusions about the effect or lack of effect of iron on COVID-19-vaccine effectiveness.

Background: The Hepatitis E virus (HEV) is a common cause of viral hepatitis worldwide. Little is known about the seroprevalence of HEV in the general population of Saudi Arabia. Methods: A community-based cross-sectional HEV seroprevalence study was conducted in Makkah, Saudi Arabia. Anti-HEV IgG antibodies were detected in sera using an in-house ELISA. The frequency of HEV sageerology and its correlation with demographic, and environmental factors were evaluated. Results: Enrollment consisted of 1329 individuals, ages ranged from 8 to 88 years, the mean age was 30.17 years, the median age was 28yrs, and the male: female ratio was 1.15. The overall seroprevalence was 23.8% (316/1329). Males had significantly higher seroprevalence than females (66.1 vs. 33.9%; p < 0.001). Seroprevalence had significant correlations with age, occupation, and lack of regular water supply and housing conditions. Conclusions: This is the first HEV community-based seroprevalence study from Saudi Arabia. Results show that the HEV is endemic in Makkah and affects all age groups and occupations. HEV affects more males than females and those living in crowded accommodations without a regular supply of water. Further studies are required across all regions of Saudi Arabia to determine the country’s seroprevalence of active or past infection using tests for HEV IgG, HEV IgM antibodies and/or HEV RNA and underlying determinants of transmission.

Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the orf1ab region of the virus genome because of its high sequence conservation and vital role in virus replication. The designed siRNAs were used for experimental evaluation in selected cell lines: Vero cells, HEK-293-T, and Huh-7. Virus inhibition was assessed according to the cycle threshold value during a quantitative real-time polymerase chain reaction. Out of 462 potential siRNAs, we filtered out 21 based on specific selection criteria without off-target effect. The selected siRNAs did not show any cellular toxicity in the tested cell lines at various concentrations. Based on our results, it was obvious that the combined use of siRNAs exhibited a reduction in MERS-CoV replication in the Vero, HEK-293-T, and Huh-7 cell lines, with the highest efficacy displayed in the Vero cells.

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes viral encephalitis leading to neural damage, is a major threat in most Asian countries. The RNA-dependent RNA polymerase (RdRp) present in the viral genome is the key component for genome replication, making it an attractive target for antiviral drug development. In this study, the natural products from Echinacea angustifolia were retrieved for structure-based virtual screening against JEV–RdRp. The top six compounds (Echinacoside, Echinacin, Rutin, Cynaroside, Quercetagetin 7-glucoside, and Kaempferol-3-glucoside) were obtained based on the highest negative docking score, ADMET (absorption, distribution, metabolism, excretion, and toxicity), and molecular interaction. The computational analysis of these selected compounds against the co-crystallized ligands, i.e., ATP and GTP, were performed. Further, 100 ns molecular dynamic simulation and post-free binding energy calculation of all the selected compounds complexed with JEV–RdRP were performed to check the stability of the complexes. The obtained results showed considerable stability and intermolecular interaction with native ligand-binding site residues of JEV–RdRp. Hence, selected natural compounds are admissible inhibitors of JEV–RdRp protein and can be considered for future antiviral drug development studies.

Zika virus (ZIKV) has been characterized as one of many potential pathogens and placed under future epidemic outbreaks by the WHO. However, a lack of potential therapeutics can result in an uncontrolled pandemic as with other human pandemic viruses. Therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted a strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the NS2B-NS3 protease (ZIKVpro) and NS5 RNA dependent RNA polymerase (ZIKVRdRp) proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of the top four common bioflavonoids, viz. Rutin, Nicotiflorin, Isoquercitrin, and Hyperoside. These compounds showed substantial docking energy (−7.9 to −11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (B:His51, B:Asp75, and B:Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Besides, long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals stability of the respective docked poses contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp against ZIKV for further experimental assessment.

Background: The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. Methods: We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), and incubated at 65 °C using real-time PCR 7500. Results: Our direct detection with the RT-LAMP protocol showed 100% concordance (sensitivity and specificity) with the standard protocol used for the detection of SARS-CoV-2 nucleic acid. Conclusions: In this study, we set up a rapid, simple, and sensitive RT-LAMP assay for the detection of SARS-CoV-2 in clinical samples. The assay is suitable for point of care detection in public hospitals, medical centers in rural areas, and in transportation hubs.

Without effective antivirals, the COVID-19 pandemic will likely continue to substantially affect public health. Medicinal plants and phytochemicals are attractive therapeutic options, particularly those targeting viral proteins essential for replication cycle. Herein, a total 179 phytochemicals of licorice (Glycyrrhiza glabra) were screened and scrutinized against the SARS-CoV-2 main protease (Mpro) with considerable binding affinities in the range of −9.831 to −2.710 kcal/mol. The top 10 compounds with the best docking scores, licuraside, glucoliquiritin apioside, 7,3′-Dihydroxy-5′-methoxyisoflavone, licuroside, kanzonol R, neoisoliquiritin, licochalcone-A, formononetin, isomucronulatol, and licoricone, were redocked using AutoDock Vina, yielding −8.7 to −7.3 kcal/mol binding energy against Glycyrrhizin (−8.0 kcal/mol) as a reference ligand. Four compounds, licuraside, glucoliquiritin apioside, 7,3′-Dihydroxy-5′-methoxyisoflavone, and licuroside, with glycyrrhizin (reference ligand) were considered for the 100 ns MD simulation and post-simulation analysis which support the stability of docked bioactive compounds with viral protein. In vitro studies demonstrated robust anti-SARS-CoV-2 activity of licorice and glycyrrhizin under different treatment protocols (simulations treatment with viral infection, post-infection treatment, and pre-treatment), suggesting multiple mechanisms for action. Although both compounds inhibited SARS-CoV-2 replication, the half-maximal inhibitory concentration (IC50) of glycyrrhizin was substantially lower than licorice. This study supports proceeding with in vivo experimentation and clinical trials and highlights licorice and glycyrrhizin as potential therapeutics for COVID-19.

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.

Objective: The acute cases of pneumonia (COVID-19) were first reported from China in December 2019, and the pathogen was identified as SARS-CoV-2. Currently, many vaccines have been developed against this virus by using multiple genes, applying different platforms, and used for the vaccinations of the human population. Spike protein genes play an important role in host cell attachment and viral entry and have been extensively used for the development of vaccine and antiviral therapeutics. Short interfering RNA is also known as silencing RNA and contribute a significant role to regulate the expression of a specific gene. By using this technology, virus inhibition has been demonstrated against many viral diseases. Methods: In this work, we have reported the Insilico prediction, designing, and experimental validation of siRNAs antiviral potency against SARS-CoV-2-S-RBD. The siDirect 2.0 was selected for siRNAs prediction, and secondary structure was predicted by RNAfold while the HNADOCK was used for molecular docking analysis and specific binding of siRNAs to the selected target. We have used and evaluated four siRNAs for cellular toxicity and determination of antiviral efficiency based on the Ct value of q-real-time PCR in Vero E6 cells. Results: Based on the experimental evaluation and analysis of results from generated data, we observed that there is no cytotoxicity for any tested siRNAs in Vero E6 cells. Total four siRNA were filtered out from twenty-one siRNAs following the strict selection and scoring criteria. The better antiviral efficiency was observed in 3rd siRNAs based on the Ct value of q-real-time PCR. The results that emerged from this study encouraged us to validate the efficiency of these siRNAs in multiple cells by using alone and in a combination of two or more siRNAs to inhibit the SARS-CoV-2 proliferation. Conclusion: The Insilico prediction, molecular docking analysis provided the selection of better siRNAs. Based on the experimental evaluation only 3rd siRNA was found to be more effective than others and showed better antiviral efficiency. These siRNAs should also be evaluated in other cell lines either separately or in combination against SARS-CoV-2 to determine their antiviral efficiency.