Systematic identification and repurposing of FDA-approved drugs as antibacterial agents against Streptococcus pyogenes: In silico and in vitro studies.

Streptococcus pyogenes (Group A Streptococcus - GAS) is a human pathogen causing wide range of infections and toxin-mediated diseases in human beings of all age groups with fatality of 10-30 %. The limited success of antibiotics and the non-availability of vaccines makes GAS a global burden. The multi-subunit RNA polymerase (RNAP) is a validated bacterial therapeutic target as it is involved in transcription and can arrest growth. Of the five subunits of this enzyme complex, the ß-subunit (RpoC) has attracted specific attention as a drug target, particularly in the switch region. Here we attempt to repurpose non-antimicrobial drugs to act as RpoC inhibitors against S. pyogenes. In this study, 1826 FDA approved drugs have been identified through high-throughput virtual screening. Free Energy Perturbation (FEP) based binding free energy calculations have been performed at the final step of the virtual screening funnel to ensure high accuracy in silico results. Three compounds identified have been tested for susceptibility of S. pyogenes MTCC 442 strain and two antibiotic-resistant clinical isolates of S. pyogenes using microdilution assay. Among the three, two drugs Amlodipine Besylate (Amd) and Ranitidine hydrochloride (Rnt) have shown inhibition against all the tested strains and its mechanism of interaction with RpoC has been studied. The docked complexes were analyzed to understand the binding mode of the drugs to the target. Classical Molecular Dynamics studies for RpoC-Rnt complex and the two stable conformations of RpoC-Amd complex was carried out. Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (RoG) and Solvent Accessible Surface Area (SASA) of the complexes were plotted and studied. The thermodynamic parameters of protein-drug were experimentally determined using Isothermal Titration Calorimetry (ITC). Infrared spectroscopic studies and Fluorescence quenching studies provided insights into the secondary structural changes in RpoC on binding to the drugs.

Distribution and Functional Analyses of Mutations in Spike Protein and Phylogenic Diversity of SARS-CoV-2 Variants Emerged during the Year 2021 in India.

Introduction: Prolonged COVID-19 pandemic accelerates the emergence and transmissibility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants through the accumulation of adaptive mutations. Particularly, adaptive mutations in spike (S) protein of SARS-CoV-2 leads to increased viral infectivity, severe morbidity and mortality, and immune evasion. This study focuses on the phylodynamic distribution of SARS-CoV-2 variants during the year 2021 in India besides analyzing the functional significance of mutations in S-protein of SARS-CoV-2 variants. Methods: Whole genome of SARS-CoV-2 sequences (n = 87957) from the various parts of India over the period of January to December 2021 was retrieved from Global Initiative on Sharing All Influenza Data. All the S-protein sequences were subjected to clade analysis, variant calling, protein stability, immune escape potential, structural divergence, Furin cleavage efficiency, and phylogenetic analysis using various in silico tools. Results: Delta variant belonging to 21A, 21I, and 21J clades was found to be predominant throughout the year 2021 though many variants were also present. A total of 4639 amino acid mutations were found in S-protein. D614G was the most predominant mutation in the S-protein followed by P681R, L452R, T19R, T478K, and D950N. The highest number of mutations was found in the N-terminal domain of S-protein. Mutations in the crucial sites of S-protein impacting pathogenicity, immunogenicity, and fusogenicity were identified. Intralineage diversity analysis showed that certain variants of SARS-CoV-2 possess high diversification. Conclusions: The study has disclosed the distribution of various variants including the Delta, the predominant variant, in India throughout the year 2021. The study has identified mutations in S-protein of each SARS-CoV-2 variant that can significantly impact the virulence, immune evasion, increased transmissibility, high morbidity, and mortality. In addition, it is found that mutations acquired during each viral replication cycle introduce new sub-lineages as studied by intralineage diversity analysis.

Molecular investigation of human metapneumovirus in children with acute respiratory infections in Chennai, South India, from 2016-2018.

Human metapneumovirus (hMPV) has emerged as a frequent cause of acute respiratory infections (ARI) among young children. The prevalence and genetic diversity of hMPV circulating in Chennai, Southern India, has not been studied yet. Hence, this study was aimed to investigate the prevalence, co-infection with other respiratory viruses like HRSV A and B, influenza A and B, hRV and HPIV 1-4 viruses, socio-demographic associations, and genotypes of hMPV among children in Chennai. A total of 350 nasal swab specimens were collected from children with ARI during April 2016 to August 2018 and tested for hMPV by real time PCR method. In this study, hMPV was detected in 4% (14/350) of the samples. One hMPV positive sample was found to be co-infected with influenza B virus. The mean and median ages of the children with hMPV infection were 61.5 months (5.1 years) and 83 months (6.9 years), respectively. Phylogenetic analysis of the partial F gene revealed the presence of A2c subcluster among the study strains as well as with B1 and B2 lineages. The prevalence data obtained in this study is important in evaluating the role of hMPV in childhood ARI and emphasizes the importance of routine viral diagnosis in hospitals. To the best of our knowledge, this is the first study to report the prevalence, seasonality, and genetic diversity of hMPV in Chennai as well as the first study to report A2c subcluster of hMPV among children in India.

Epitope Identification and Designing a Potent Multi-epitope Vaccine Construct against SARS-CoV-2 Including the Emerging Variants.

Introduction: The emergence of a novel coronavirus in China has turned into a SARS-CoV-2 pandemic with high fatality. As vaccines are developed through various strategies, their immunogenic potential may drastically vary and thus pose several challenges in offering immune responses against the virus. Methods: In this study, we adopted an immunoinformatics-aided approach for developing a new multi-epitope vaccine construct (MEVC). In silico approach was taken for the identification of B-cell and T-cell epitopes in the Spike protein, for MEVC various cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes with the highest affinity for the respective HLA alleles were assembled and joined by linkers. Results: The computational data suggest that the MEVC is nontoxic, nonallergenic and thermostable and elicit both humoral and cell-mediated immune responses. Subsequently, the biological activity of MEVC was assessed by bioinformatic tools using the interaction between the vaccine candidate and the innate immune system receptors TLR3 and TLR4. The epitopes of the construct were analyzed with that of the strains belonging to various clades including the emerging variants having multiple unique mutations in S protein. Conclusions: Due to the advantageous features, the MEVC can be tested in vitro for more practical validation and the study offers immense scope for developing a potential vaccine candidate against SARS-CoV-2 in view of the public health emergency associated with COVID-19 disease caused by SARS-CoV-2.

Phylodynamic Pattern of Genetic Clusters, Paradigm Shift on Spatio-Temporal Distribution of Clades, and Impact of Spike Glycoprotein Mutations of SARS-CoV-2 Isolates from India.

INTRODUCTION: The COVID-19 pandemic is associated with high morbidity and mortality, with the emergence of numerous variants. The dynamics of SARS-CoV-2 with respect to clade distribution is uneven, unpredictable and fast changing. METHODS: Retrieving the complete genomes of SARS-CoV-2 from India and subjecting them to analysis on phylogenetic clade diversity, Spike (S) protein mutations and their functional consequences such as immune escape features and impact on infectivity. Whole genome of SARS-CoV-2 isolates (n = 4,326) deposited from India during the period from January 2020 to December 2020 is retrieved from Global Initiative on Sharing All Influenza Data (GISAID) and various analyses performed using in silico tools. RESULTS: Notable clade dynamicity is observed indicating the emergence of diverse SARS-CoV-2 variants across the country. GR clade is predominant over the other clades and the distribution pattern of clades is uneven. D614G is the commonest and predominant mutation found among the S-protein followed by L54F. Mutation score prediction analyses reveal that there are several mutations in S-protein including the RBD and NTD regions that can influence the virulence of virus. Besides, mutations having immune escape features as well as impacting the immunogenicity and virulence through changes in the glycosylation patterns are identified. CONCLUSIONS: The study has revealed emergence of variants with shifting of clade dynamics within a year in India. It is shown uneven distribution of clades across the nation requiring timely deposition of SARS-CoV-2 sequences. Functional evaluation of mutations in S-protein reveals their significance in virulence, immune escape features and disease severity besides impacting therapeutics and prophylaxis.

Genetic diversity of human respiratory syncytial virus in children with acute respiratory infections in Chennai, South India.

Introduction: Human respiratory syncytial virus (HRSV) an RNA virus belonging to Pneumoviridae family, is an important cause of acute respiratory infections (ARIs) in young children. HRSV circulates as two subgroups A and B, which are further categorised into several genotypes. New genotypes may replace existing ones over successive epidemic seasons and multiple genotypes may cocirculate in the same community rendering it important to monitor them at the molecular level. The present study assessed the circulating genotypes of HRSV in Chennai. Materials and Methods: Two hundred and sixty-seven children with ARI were recruited during the study from April 2016 to March 2018 for detecting HRSV A and B by real-time reverse transcription-polymerase chain reaction. Phylogeny and selection pressure analysis were done. Results: Fifty-seven of the 267 samples (21.3%) were positive for HRSV, of which 7.1% and 14.2% were HRSV A and B, respectively, indicating that HRSV B was the major subgroup circulating in Chennai. Peak activity of HRSV was observed during the monsoon and winter months. Phylogenetic analysis of 2nd hypervariable region (HVR) of attachment glycoprotein gene (G gene) revealed that the HRSV A strains belonged to ON1 and HRSV B strains belonged to BA9 genotypes. Several unique amino acid substitutions were observed among the study strains. The Shannon entropy plot revealed that the HRSV A strains from our study have a high potential for amino acid substitutions in the 2nd HVR of G gene. Conclusion: This study underlines the genetic diversity of HRSV and emphasises the need for continued molecular surveillance for infection management and prevention strategies.

Influenza Virus Among Children with Acute Respiratory Infections in Chennai, India.

Influenza is a major public health concern. Information on the prevalence of influenza virus in respiratory tract infections in Indian children is very sparse. In the present study, 267 nasal swabs were collected from children with acute respiratory infections in Chennai, India, out of which 22 (8.2%) and 6 (2.3%) samples were positive for influenza A and B virus respectively.

Native Valve Endocarditis Caused by Escherichia Coli.

Escherichia coli is a rare cause of infective endocarditis. This report describes a case of native valve endocarditis caused by Escherichia coli in a 58-year-old male renal transplant patient who had a concurrent urinary tract infection caused by the same organism. The patient was successfully treated with antibiotics and recovered without surgical intervention.

Influenza Virus Among Children with Acute Respiratory Infections in Chennai, India

nfluenza is a major public health concern. Information on theprevalence of influenza virus in respiratory tract infections inIndian children is very sparse. In the present study, 267 nasalswabs were collected from children with acute respiratoryinfections in Chennai, India, out of which 22 (8.2%) and 6 (2.3%)samples were positive for influenza A and B virus respectively.