Vaccine development: Current trends and technologies.

Despite the effectiveness of vaccination in reducing or eradicating diseases caused by pathogens, there remain certain diseases and emerging infections for which developing effective vaccines is inherently challenging. Additionally, developing vaccines for individuals with compromised immune systems or underlying medical conditions presents significant difficulties. As well as traditional vaccine different methods such as inactivated or live attenuated vaccines, viral vector vaccines, and subunit vaccines, emerging non-viral vaccine technologies, including viral-like particle and nanoparticle vaccines, DNA/RNA vaccines, and rational vaccine design, offer new strategies to address the existing challenges in vaccine development. These advancements have also greatly enhanced our understanding of vaccine immunology, which will guide future vaccine development for a broad range of diseases, including rapidly emerging infectious diseases like COVID-19 and diseases that have historically proven resistant to vaccination. This review provides a comprehensive assessment of emerging non-viral vaccine production methods and their application in addressing the fundamental and current challenges in vaccine development.

CTAB Protocol for Obtaining High-Quality Total RNA from Hibiscus rosa-sinensis and Other Related Plants of the Family Malvaceae.

Malvaceae family, also known as the Mallow family, is a family of flowering plants containing Hibiscus rosa-sinensis and other plants of high medicinal value. This study focuses on the challenges associated with high-quality RNA extraction from Hibiscus rosa-sinensis and its related plants characterized by high levels of mucilage and phenolic compounds in their tissues. High mucilage and secondary metabolite content pose obstacles in obtaining high-quality RNA, negatively impacting downstream applications, such as gene expression analysis. Our research aimed to develop an efficient RNA extraction method tailored to the unique characteristics of Malvaceae family plants especially Hibiscus rosa-sinensis. Through the substitution of NaCl with KCl, a crucial component of the CTAB buffer, our methodology successfully addressed the challenges posed by high mucilage and phenolic compound levels. This modification led to a significant reduction in sample viscosity, which is because of the high mucilage in these plants. Our modified CTAB extraction method yielded significantly more RNA with higher purity than the conventional CTAB methods alone. The extracted RNA was largely intact, as indicated by 28S/18S ratios and RIN values, yielding high-quality RNA with improved purity suggested by the 260/280 and 260/230 ratios. The proposed approach not only serves as a solution to the specific challenges encountered in Hibiscus rosa-sinensis but also holds promise for broader applications across different plants within the family.

Whole genome characterization and diagnostics of prunus necrotic ringspot virus (PNRSV) infecting apricot in India.

Prunus necrotic ringspot virus (PNRSV) is a pathogen that infects Prunus species worldwide, causing major economic losses. Using one and two-step RT-PCR and multiplex RT-PCR, the whole genome of the PNRSV-infecting apricot was obtained and described in this study. Computational approaches were used to investigate the participation of several regulatory motifs and domains of the Replicase1, Replicase2, MP, and CP. A single degenerated reverse and three forward oligo primers were used to amplify PNRSV's tripartite genome. The size of RNA1 was 3.332 kb, RNA2 was 2.591 kb, and RNA3 was 1.952 kb, according to the sequencing analysis. The Sequence Demarcation Tool analysis determined a percentage pair-wise identity ranging between 91 and 99% for RNA1 and 2, and 87-98% for RNA3. Interestingly, the phylogenetic analysis revealed that closely related RNA1, RNA2, and RNA3 sequences of PNRSV strains from various geographical regions of the world are classified into distinct clades or groups. This is the first report on the characterization of the whole genome of PNRSV from India, which provides the cornerstone for further studies on the molecular evolution of this virus. This study will assist in molecular diagnostics and management of the diseases caused by PNRSV.

Microbial Biofilms: Applications, Clinical Consequences, and Alternative Therapies.

Biofilms are complex communities of microorganisms that grow on surfaces and are embedded in a matrix of extracellular polymeric substances. These are prevalent in various natural and man-made environments, ranging from industrial settings to medical devices, where they can have both positive and negative impacts. This review explores the diverse applications of microbial biofilms, their clinical consequences, and alternative therapies targeting these resilient structures. We have discussed beneficial applications of microbial biofilms, including their role in wastewater treatment, bioremediation, food industries, agriculture, and biotechnology. Additionally, we have highlighted the mechanisms of biofilm formation and clinical consequences of biofilms in the context of human health. We have also focused on the association of biofilms with antibiotic resistance, chronic infections, and medical device-related infections. To overcome these challenges, alternative therapeutic strategies are explored. The review examines the potential of various antimicrobial agents, such as antimicrobial peptides, quorum-sensing inhibitors, phytoextracts, and nanoparticles, in targeting biofilms. Furthermore, we highlight the future directions for research in this area and the potential of phytotherapy for the prevention and treatment of biofilm-related infections in clinical settings.

A Novel Frameshift Mutation, Deletion of HBB:c.199_202delAAAG [Codon 66/67 (-AAAG)] in ß-Thalassemia Major Patients from the Western Region of Uttar Pradesh, India.

PURPOSE: Beta thalassemia is one of the most common inherited disorders in India with heterogenous clinical phenotypes from silent carrier to clinically severe ones. Our study aimed to characterize the mutation spectrum in thalassemia patients who are coming to the hospital for follow-up from the western region of Uttar Pradesh India. PATIENTS AND METHODS: For the study, a case series of the retrospective bi-centre study was conducted. The patients from two thalassemia centers in the major hospitals (LLRMC Meerut, and JNMC, Aligarh administered by the Ministry of Health and Family Welfare (MoHFW)) in the Western Uttar Pradesh, India were considered for the study. A total of 77 blood samples were obtained from individuals (both related and unrelated) diagnosed with ß-thalassemia after their consent. After DNA extraction, HBB gene amplification, mutation-specific polymerase chain reaction and gene sequencing were carried out to analyze the mutations. RESULTS: In this study, seven different types of mutations were reported for the first time in Western Uttar Pradesh, India. A novel frameshift mutation, deletion of 4 nucleotides Codon 66/67 (-AAAG) in exon 2 region, is reported for the first time. IVS 1-5 (G>C) and Codon 41/42 (-CTTT) are the most frequently reported mutations. The molecular spectrum for these two cases consists of 44 and 42 alleles out of 108 alleles, respectively. CONCLUSION: A total of 108 ß-thalassemia alleles were studied from 46 homozygous and 31 compound heterozygous patients. All the individuals were from 20 districts of the Western Uttar Pradesh, India.

Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits.

A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees. ACLSV was detected up to a dilution of 5-5, whereas ApMV, ASGV, CVA, PPV and PNRSV up to 5-4. The developed assay was validated following testing a total of 45 symptomatic leaf samples collected from different geographical regions of Jammu and Kashmir (India), and the presence of the viruses was further confirmed by RT-PCR and sequencing. The polyprobe, designed for performing molecular hybridization assay can be developed quickly and avoid the tedious transformation and cloning procedures. Apart from simultaneously detecting viruses in stone and pome fruit trees, it holds great potential for virus indexing programmes to ascertain the supply of virus-free plant materials to the growers.

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.