Genomic Diversity and Recombination Analysis of the Spike Protein Gene from Selected Human Coronaviruses.

Human coronaviruses (HCoVs) are seriously associated with respiratory diseases in humans and animals. The first human pathogenic SARS-CoV emerged in 2002-2003. The second was MERS-CoV, reported from Jeddah, the Kingdom of Saudi Arabia, in 2012, and the third one was SARS-CoV-2, identified from Wuhan City, China, in late December 2019. The HCoV-Spike (S) gene has the highest mutation/insertion/deletion rate and has been the most utilized target for vaccine/antiviral development. In this manuscript, we discuss the genetic diversity, phylogenetic relationships, and recombination patterns of selected HCoVs with emphasis on the S protein gene of MERS-CoV and SARS-CoV-2 to elucidate the possible emergence of new variants/strains of coronavirus in the near future. The findings showed that MERS-CoV and SARS-CoV-2 have significant sequence identity with the selected HCoVs. The phylogenetic tree analysis formed a separate cluster for each HCoV. The recombination pattern analysis showed that the HCoV-NL63-Japan was a probable recombinant. The HCoV-NL63-USA was identified as a major parent while the HCoV-NL63-Netherland was identified as a minor parent. The recombination breakpoints start in the viral genome at the 142 nucleotide position and end at the 1082 nucleotide position with a 99% CI and Bonferroni-corrected p-value of 0.05. The findings of this study provide insightful information about HCoV-S gene diversity, recombination, and evolutionary patterns. Based on these data, it can be concluded that the possible emergence of new strains/variants of HCoV is imminent.

Exploring plant-derived small molecules as inhibitors of Marburg virus RNA binding protein activity.

The search for antiviral medications is greatly influenced by the hunt for potent inhibitors of viral proteins. To find possible inhibitors of the RNA binding activity of the Marburg virus VP35 protein, we used a thorough in silico drug discovery approach in this investigation. A comprehensive virtual screening process, followed by a detailed MMGBSA analysis, led to the discovery of four potential inhibitory compounds viz. Kudzuisoflavone A, Miquelianin, Rutin, and Protopseudohypericin. They were identified from an extensive library of phytomolecules derived from three medicinal plants: Adiantum capillus-veneris, Hypericum perforatum, and Pueraria montana. In molecular dynamics (MD) simulations, all these compounds showed steady binding to the target protein and favourable interactions. Notably, the free binding energies of all the selected compounds were better than the myricetin, a well-known blocker of the VP35 protein of the Ebola virus. Overall, this investigation offers insightful information about the molecular interactions and binding dynamics of the identified inhibitors' binding to the VP35 protein of the Marburg virus. The findings highlight the potential of three particular medicinal plants as sources of key chemicals for the creation of brand-new Marburg virus antiviral drugs. More experimental validation and optimization of the identified inhibitors are necessary in order to transform these findings into effective medicines for treating Marburg virus infections.Communicated by Ramaswamy H. Sarma.

In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea.

Hypericum perforatum and Echinacea are reported to have antiviral activities against several viral infections. In this study, H. perforatum (St. John's Wort) and Echinacea were tested in vitro using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) through its infectious cycle from 0 to 48 h post infection. The hypericin of H. perforatum and the different parts (roots, seeds, aerial) of two types of Echinacea species (Echinacea purpurea and Echinacea angustifolia) were tested for their anti-viral activities to measure the inhibition of viral load using quantitative real-time polymerase chain reaction (qRT-PCR) on cell culture assay. Interestingly, the H. perforatum-Echinacea mixture (1:1 ratio) of H. perforatum and Echinacea was tested as well on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti-viral treatment. Therefore, the results H. perforatum and Echinacea species, applied in this study showed significant anti-viral and virucidal effects in the following order of potency: H. perforatum, H. perforatum-Echinacea mixture, and Echinacea on SARS-CoV-2 infectious cycle. Additionally, molecular simulation analysis of the compounds with essential proteins (Mpro and RdRp) of the SARS-CoV-2 revealed the most potent bioactive compounds such as Echinacin, Echinacoside, Cyanin, Cyanidin 3-(6''-alonylglucoside, Quercetin-3-O-glucuronide, Proanthocyanidins, Rutin, Kaempferol-3-O-rutinoside, and Quercetin-3-O-xyloside. Thus, based on the outcome of this study, it is demanding the setup of clinical trial with specific therapeutic protocol.

Glycosylated Flavonoid Compounds as Potent CYP121 Inhibitors of Mycobacterium tuberculosis.

Due to the concerning rise in the number of multiple- and prolonged-drug-resistant (MDR and XDR) Mycobacterium tuberculosis (Mtb) strains, unprecedented demand has been created to design and develop novel therapeutic drugs with higher efficacy and safety. In this study, with a focused view on implementing an in silico drug design pipeline, a diverse set of glycosylated flavonoids were screened against the Mtb cytochrome-P450 enzyme 121 (CYP121), which is established as an approved drug target for the treatment of Mtb infection. A total of 148 glycosylated flavonoids were screened using structure-based virtual screening against the crystallized ligand, i.e., the L44 inhibitor, binding pocket in the Mtb CYP121 protein. Following this, only the top six compounds with the highest binding scores (kcal/mol) were considered for further intermolecular interaction and dynamic stability using 100 ns classical molecular dynamics simulation. These results suggested a considerable number of hydrogen and hydrophobic interactions and thermodynamic stability in comparison to the reference complex, i.e., the CYP121-L44 inhibitor. Furthermore, binding free energy via the MMGBSA method conducted on the last 10 ns interval of MD simulation trajectories revealed the substantial affinity of glycosylated compounds with Mtb CYP121 protein against reference complex. Notably, both the docked poses and residual energy decomposition via the MMGBSA method demonstrated the essential role of active residues in the interactions with glycosylated compounds by comparison with the reference complex. Collectively, this study demonstrates the viability of these screened glycosylated flavonoids as potential inhibitors of Mtb CYP121 for further experimental validation to develop a therapy for the treatment of drug-resistant Mtb strains.

Multi-staged gene expression profiling reveals potential genes and the critical pathways in kidney cancer.

Cancer is among the highly complex disease and renal cell carcinoma is the sixth-leading cause of cancer death. In order to understand complex diseases such as cancer, diabetes and kidney diseases, high-throughput data are generated at large scale and it has helped in the research and diagnostic advancement. However, to unravel the meaningful information from such large datasets for comprehensive and minute understanding of cell phenotypes and disease pathophysiology remains a trivial challenge and also the molecular events leading to disease onset and progression are not well understood. With this goal, we have collected gene expression datasets from publicly available dataset which are for two different stages (I and II) for renal cell carcinoma and furthermore, the TCGA and cBioPortal database have been utilized for clinical relevance understanding. In this work, we have applied computational approach to unravel the differentially expressed genes, their networks for the enriched pathways. Based on our results, we conclude that among the most dominantly altered pathways for renal cell carcinoma, are PI3K-Akt, Foxo, endocytosis, MAPK, Tight junction, cytokine-cytokine receptor interaction pathways and the major source of alteration for these pathways are MAP3K13, CHAF1A, FDX1, ARHGAP26, ITGBL1, C10orf118, MTO1, LAMP2, STAMBP, DLC1, NSMAF, YY1, TPGS2, SCARB2, PRSS23, SYNJ1, CNPPD1, PPP2R5E. In terms of clinical significance, there are large number of differentially expressed genes which appears to be playing critical roles in survival.

SARS-CoV-2 Mpro inhibitors: identification of anti-SARS-CoV-2 Mpro compounds from FDA approved drugs.

Recent outbreak of COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has raised serious global concern for public health. The viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication and essential for viral life cycle, has been established as an essential drug discovery target for SARS-CoV-2. Herein, we employed computationally screening of Druglib database containing FDA approved drugs against active pocket of SARS-CoV-2 Mpro using MTiopen screen web server, yields a total of 1051 FDA approved drugs with docking energy >-7 kcal/mol. The top 10 screened potential compounds against SARS-CoV-2 Mpro were then studied by re-docking, binding affinity, intermolecular interaction, and complex stability via 100 ns all atoms molecular dynamics (MD) simulation followed by post-simulation analysis, including end point binding free energy, essential dynamics, and residual correlation analysis against native crystal structure ligand N3 inhibitor. Based on comparative molecular simulation and interaction profiling of the screened drugs with SARS-CoV-2 Mpro revealed R428 (-10.5 kcal/mol), Teniposide (-9.8 kcal/mol), VS-5584 (-9.4 kcal/mol), and Setileuton (-8.5 kcal/mol) with stronger stability and affinity than other drugs and N3 inhibitor; and hence, these drugs are advocated for further validation using in vitro enzyme inhibition and in vivo studies against SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Understanding the role of potential pathways and its components including hypoxia and immune system in case of oral cancer.

There are a few biological functions or phenomenon which are universally associated with majority of the cancers and hypoxia and immune systems are among them. Hypoxia often occurs in most of the cancers which helps the cells in adapting different responses with respect to the normal cells which may be the activation of signaling pathways which regulate proliferation, angiogenesis, and cell death. Similar to it, immune signaling pathways are known to play critical roles in cancers. Moreover, there are a number of genes which are known to be associated with these hypoxia and immune system and appear to direct affect the tumor growth and propagations. Cancer is among the leading cause of death and oral cancer is the tenth-leading cause due to cancer death. In this study, we were mainly interested to understand the impact of alteration in the expression of hypoxia and immune system-related genes and their contribution to head and neck squamous cell carcinoma. Moreover, we have collected the genes associated with hypoxia and immune system from the literatures. In this work, we have performed meta-analysis of the gene and microRNA expression and mutational datasets obtained from public database for different grades of tumor in case of oral cancer. Based on our results, we conclude that the critical pathways which dominantly enriched are associated with metabolism, cell cycle, immune system and based on the survival analysis of the hypoxic genes, we observe that the potential genes associated with head and neck squamous cell carcinoma and its progression are STC2, PGK1, P4HA1, HK1, SPIB, ANXA5, SERPINE1, HGF, PFKM, TGFB1, L1CAM, ELK4, EHF, and CDK2.

Mechanistic insights into the Japanese encephalitis virus RNA dependent RNA polymerase protein inhibition by bioflavonoids from Azadirachta indica.

Japanese encephalitis (JE) virus is a flavivirus causing encephalitis causing neurological damage. RNA-dependent-RNA-polymerase (RdRp) is responsible for genome replication making it excellent anti-viral target. In this study, the crystal structure of JE RdRp (jRdRp) and bioflavonoids reported in Azadirachta indica were retrieved from specific databases. Structure-based virtual screening was employed using MTiOpenScreen server and top four compounds selected with the most negative docking scores. Conformations were redocked using AutoDock Vina; these complexes showed mechanistic interactions with Arg474, Gly605, Asp668, and Trp800 residues in the active site of jRdRp, i.e., guanosine-5'-triphosphate. Furthermore, 100 ns classical molecular dynamics simulation and binding free energy calculation showed stability of docked bioflavonoids in the active jRdRp pocket and significant contribution of van-der-Waals interactions for docked complex stability during simulation. Therefore, this study predicted the anti-viral activity of Gedunin, Nimbolide, Ohchinin acetate, and Kulactone against jRdRp and can be considered for further antiviral drug development.

Full-genome sequencing and analysis of DENV-3 serotype isolated from Yemen.

BACKGROUND: Dengue virus causes the dengue fever as well as hemorrhagic fever in tropical and sub-tropical countries. It is now endemic in most parts of the South East Asia. Full-genome information of dengue virus 3 is not available from Yemen. METHODS: In this study, the dengue virus 3 was detected by diagnostic tools like serology and RT-PCR in the samples isolated from a patient in Yemen. The full-genome was sequenced, and the identity, phylogenetic relationship and recombination analysis was performed by using BioEdit, MEGA X and RDP4 softwares. RESULTS: The full-genome of the Yemen isolate was found to be 10,643 nt long with 3390 amino acids. The Yemen dengue virus 3 isolate showed the sequence similarity (98.5-92.4%) with dengue virus 3 isolates from China, Pakistan, India and Bangladesh respectively. The significant non-synonymous substitutions of amino acid in Yemen isolate were observed with selected isolates. The phylogenetic tree of Yemen isolate formed a unique clade within genotype III and sub-clade into lineage III. The Dengue virus isolate from Jeddah formed separated cluster with lineage IV. CONCLUSIONS: This reveals the unique genetic variability among DENV-3 serotypes from Jeddah and earlier reported isolates from other regions.

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.

Isolation of Yasminevirus, the First Member of Klosneuvirinae Isolated in Coculture with Vermamoeba vermiformis, Demonstrates an Extended Arsenal of Translational Apparatus Components.

The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis This giant virus, called Yasminevirus, has a 2.1-Mb linear double-stranded DNA genome encoding 1,541 candidate proteins, with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery, with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids (aa), 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic, with ∼34% of genes having their closest homologues in other viruses, followed by ∼13.2% in Eukaryota, ∼7.2% in Bacteria, and less than 1% in Archaea Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses.IMPORTANCE Yasminevirus is an icosahedral double-stranded DNA virus isolated from sewage water by amoeba coculture. Here its structure and replicative cycle in the amoeba Vermamoeba vermiformis are described and genomic and evolutionary studies are reported. This virus belongs to the Klosneuvirinae group of giant viruses, representing the second isolated and cultivated giant virus in this group, and is the first isolated using a coculture procedure. Extended translational machinery pointed to Yasminevirus among the quasiautonomous giant viruses with the most complete translational apparatus of the known virosphere.

MERS-CoV, influenza and other respiratory viruses among symptomatic pilgrims during 2014 Hajj season.

More than two million Muslims visit Makkah, Saudi Arabia, annually to perform the religious rituals of Hajj where the risk of spreading respiratory infections is very common. The aim here was to screen symptomatic pilgrims for Middle East respiratory syndrome coronavirus (MERS-CoV) and other viral etiologies. Thus, 132 nasopharyngeal samples were collected from pilgrims presenting with acute respiratory symptoms at the healthcare facilities in the holy sites during the 5 days of the 2014 Hajj season. Samples were tested using real-time reverse transcription polymerase chain reactions and microarray. Demographic data including age, sex, and country of origin were obtained for all participants. While we did not detect MERS-CoV in any of the samples, several other viruses were detected in 50.8% of the cases. Among the detected viruses, 64.2% of the cases were due to a single-virus infection and 35.8% were due to the coinfections with up to four viruses. The most common respiratory virus was influenza A, followed by non-MERS human coronaviruses, rhinoviruses, and influenza B. Together, we found that it was not MERS-CoV but other respiratory viruses that caused acute respiratory symptoms among pilgrims. The observed high prevalence of influenza viruses underscores the need for more effective surveillance during the Hajj and adoption of stringent vaccination requirements from all pilgrims.

Legionella saoudiensis sp. nov., isolated from a sewage water sample.

A Gram-stain-negative, bacilli-shaped bacterial strain, LS-1T, was isolated from a sewage water sample collected in Jeddah, Saudi Arabia. The taxonomic position of strain LS-1T was investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences and those of four other genes indicated that strain LS-1T belongs to the genus Legionella in the family Legionellaceae. Regarding the 16S rRNA gene, the most closely related species are Legionella rowbothamii LLAP-6T (98.6 %) and Legionella lytica L2T (98.5 %). The mip gene sequence of strain LS-1T showed 94 % sequence similarity with that of L. lytica L2T and 93 % similarity with that of L. rowbothamii LLAP-6T. Strain LS-1T grew optimally at a temperature of 32 °C on a buffered charcoal yeast extract (BCYE) agar plate in a 5 % CO2 atmosphere and had a flagellum. The combined phylogenetic, phenotypic and genomic sequence data suggest that strain LS-1T represents a novel species of the genus Legionella, for which the name Legionella saoudiensis sp. nov. is proposed. The type strain is LS-1T (=DSM 101682T=CSUR P2101T).