A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

UNASSIGNED: Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines.
UNASSIGNED: In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning.
UNASSIGNED: We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system.
UNASSIGNED: These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

This book chapter presents a concise overview of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. It explores viral classification based on morphology and nucleic acid composition with a focus on DNA and RNA viruses, the SARS-CoV-2 structure including the structural as well as nonstructural proteins in detail, and the viral replication mechanisms. The chapter then delves into the characteristics and diversity of coronaviruses, particularly SARS-CoV-2, highlighting its similarities with other beta-coronaviruses. The replication and transcription complex, RNA elongation, and capping, as well as the role of accessory proteins in viral replication and modulation of the host immune response is discussed extensively.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-stranded RNA virus that sits at the centre of the recent global pandemic. As a member of the coronaviridae family of viruses, it shares features such as a very large genome (>30 kb) that is replicated in a purpose-built replication organelle. Biogenesis of the replication organelle requires significant and concerted rearrangement of the endoplasmic reticulum membrane, a job that is carried out by a group of integral membrane non-structural proteins (NSP3, 4 and 6) expressed by the virus along with a host of viral replication enzymes and other factors that support transcription and replication. The primary sites for RNA replication within the replication organelle are double membrane vesicles (DMVs). The small size of DMVs requires generation of high membrane curvature, as well as stabilization of a double-membrane arrangement, but the mechanisms that underlie DMV formation remain elusive. In this review, we discuss recent breakthroughs in our understanding of the molecular basis for membrane rearrangements by coronaviruses. We incorporate established models of NSP3-4 protein-protein interactions to drive double membrane formation, and recent data highlighting the roles of lipid composition and host factor proteins (e.g. reticulons) that influence membrane curvature, to propose a revised model for DMV formation in SARS-CoV-2.

Dengue, a mosquito-borne viral disease spread by the dengue virus (DENV), has become one of the most alarming health issues in the global scenario in recent days. The risk of infection by DENV is mostly high in tropical and subtropical areas of the world. The mortality rate of patients affected with DENV is ever-increasing, mainly due to a lack of anti-dengue viral-specific synthetic drug components.
Repurposing synthetic drugs has been an effective tool in combating several pathogens, including DENV. However, only the Dengvaxia vaccine has been developed so far to fight against the deadly disease despite the grave situation, mainly because of the limitations of understanding the actual pathogenicity of the disease.
To address this particular issue and explore the actual disease pathobiology, several potential targets, like three structural proteins and seven non-structural (NS) proteins, along with their inhibitors of synthetic and natural origin, have been screened using docking simulation.
Exploration of these targets, along with their inhibitors, has been extensively studied in culmination with molecular docking-based screening to potentiate the treatment.
These screened inhibitors could possibly be helpful for the designing of new congeneric potential compounds to combat dengue fever and its complications.

Promyelocytic leukemia (PML) protein constitutes an indispensable element within PML-nuclear bodies (PML-NBs), playing a pivotal role in the regulation of multiple cellular functions while coordinating the innate immune response against viral invasions. Simultaneously, numerous viruses elude immune detection by targeting PML-NBs. Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis, a severe neurological disease that affects humans and animals. However, the mechanism through which JEV evades immunity via PML-NBs has been scarcely investigated. In the present study, PK15 cells were infected with JEV, and the quantity of intracellular PML-NBs was enumerated. The immunofluorescence results indicated that the number of PML-NBs was significantly reduced in JEV antigen-positive cells compared to viral antigen-negative cells. Subsequently, ten JEV proteins were cloned and transfected into PK15 cells. The results revealed that JEV non-structural proteins, NS2B, NS3, NS4A, NS4B, and NS5, significantly diminished the quantity of PML-NBs. Co-transfection was performed with the five JEV proteins and various porcine PML isoforms. The results demonstrated that NS2B colocalized with PML4 and PML5, NS4A colocalized with PML1 and PML4, NS4B colocalized with PML1, PML3, PML4, and PML5, while NS3 and NS5 interacted with all five PML isoforms. Furthermore, ectopic expression of PML isoforms confirmed that PML1, PML3, PML4, and PML5 inhibited JEV replication. These findings suggest that JEV disrupts the structure of PML-NBs through interaction with PML isoforms, potentially leading to the attenuation of the host\'s antiviral immune response.

SARS-CoV-2 was the cause of the global pandemic that caused a total of 14.9 million deaths during the years 2020 and 2021, according to the WHO. The virus presents a mutation rate between 10-5 and 10-3 substitutions per nucleotide site per cell infection (s/n/c). Due to this, studies aimed at knowing the evolution of this virus could help us to foresee (through the future development of new detection strategies and vaccines that prevent the infection of this virus in human hosts) that a pandemic caused by this virus will be generated again. In this research, we performed a functional annotation and identification of changes in Nsp (non-structural proteins) domains in the coronavirus genome. The comparison of the 13 selected coronavirus pangenomes demonstrated a total of 69 protein families and 57 functions associated with the structural domain\'s differentials between genomes. A marked evolutionary conservation of non-structural proteins was observed. This allowed us to identify and classify highly pathogenic human coronaviruses into alpha, beta, gamma, and delta groups. The designed Nsp cluster provides insight into the trajectory of SARS-CoV-2, demonstrating that it continues to evolve rapidly. An evolutionary marker allows us to discriminate between phylogenetically divergent groups, viral genotypes, and variants between the alpha and betacoronavirus genera. These types of evolutionary studies provide a window of opportunity to use these Nsp as targets of viral therapies.

The β-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution.

Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, we develop a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. We demonstrate the utility of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. We show that NSP8 engages in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.