Cellular uptake of metal oxide-based nanocomposites and targeting of chikungunya virus replication protein nsP3.

Emergence of new pathogenic viruses along with adaptive potential of RNA viruses has become a major public health concern. Therefore, it is increasingly crucial to investigate and assess the antiviral potential of nanocomposites, which is constantly advancing area of medical biology. In this study, two types of nanocomposites: Ag/NiO and Ag2O/NiO/ZnO with varying molar ratios of silver and silver oxide, respectively have been synthesised and characterised. Three metal/metal oxide (Ag/NiO) composites having different amounts of Ag nanoparticles (NPs) anchored on NiO octahedrons are AN-5 % (5 % Ag), AN-10 % (10 % Ag) and AN-15 % (15 % Ag)) and three ternary metal oxide nanocomposites (Ag2O/NiO/ZnO) i.e., A/N/Z-1, A/N/Z-2, and A/N/Z-3 with different molar ratios of silver oxide (10 %, 20 % and 30 %, respectively) were evaluated for their antiviral potential. Cellular uptake of nanocomposites was confirmed by ICP-MS. Intriguingly, molecular docking of metal oxides in the active site of nsP3 validated the binding of nanocomposites to chikungunya virus replication protein nsP3. In vitro antiviral potential of nanocomposites was tested by performing plaque reduction assay, cytopathic effect (CPE) analysis and qRT-PCR. The nanocomposites showed significant reduction in virus titre. Half-maximal inhibitory concentration (IC50) for A/N/Z-3 and AN-5 % were determined to be 2.828 and 3.277 µg/mL, respectively. CPE observation and qRT-PCR results were consistent with the data obtained from plaque reduction assay for A/N/Z-3 and AN-5 %. These results have opened new avenues for development of nanocomposites based antiviral therapies.

Surface decorated reporter-tagged chikungunya virus-like particles for clinical diagnostics and identification of virus entry inhibitors.

The ever-evolving and versatile VLP technology is becoming an increasingly popular area of science. This study presents surface decorated reporter-tagged VLPs of CHIKV, an enveloped RNA virus of the genus alphavirus and its applications. Western blot, IFA and live-cell imaging confirm the expression of reporter-tagged CHIK-VLPs from transfected HEK293Ts. CryoEM micrographs reveal particle diameter as ∼67nm and 56-70 nm, respectively, for NLuc CHIK-VLPs and mCherry CHIK-VLPs. Our study demonstrates that by exploiting NLuc CHIK-VLPs as a detector probe, robust ratiometric luminescence signal in CHIKV-positive sera compared to healthy controls can be achieved swiftly. Moreover, the potential activity of the Suramin drug as a CHIKV entry inhibitor has been validated through the reporter-tagged CHIK-VLPs. The results reported in this study open new avenues in the eVLPs domain and offer potential for large-scale screening of clinical samples and antiviral agents targeting entry of CHIKV and other alphaviruses.

Chikungunya virus titration, detection and diagnosis using N-Acetylglucosamine (GlcNAc) specific lectin based virus capture assay.

Re-emergence and global expansion of Chikungunya virus (CHIKV) from Africa to Indian Subcontinent in 2013, has significantly resulted in chronic morbidities in infected individuals. The burden of CHIKV on human population is still uncertain, owing to lack of vaccine and underdiagnosis. Due to the absence of vaccine or antiviral therapeutics, timely diagnosis and detection of CHIKV is vital for minimizing virus transmission. Commercially available diagnostic and titration kits relies on the traditional methods such as real-time PCR (RT-PCR), serodiagnostic assays, and plaque assay, which are expensive, time-consuming and technically challenging. To overcome these limitations and to increase the diagnostic coverage of CHIKV infections, a rapid and economical antigen capture assay has been developed in this study for serological diagnosis of CHIKV, using tamarind chitinase (chi)-like lectin (TCLL). TCLL extracted and purified from tamarind seeds (Tamarindus indica), has been reported recently to bind to N-acetylglucosamine (GlcNAc) containing glycan on the envelope protein of virus. Evaluation of antigen capture assay for serological diagnosis of CHIKV signified that the developed assay is able to detect CHIKV in both laboratory and clinical samples efficiently. Furthermore, a standard graph using different concentrations of CHIKV has been established using samples with known virus titer, to assist in quantification of viral load in a given sample. The feasibility of antigen capture assay for broad-spectrum diagnosis of alphaviral infections was evaluated using Sindbis virus (SINV) belonging to the same alphavirus genus, and the results obtained were in agreement with those of CHIKV. In summary, the developed glycan-based virus capture assay can be potentially applied as point-of-care routine diagnostic and titration assay for CHIKV as well for other re-emerging alphaviral infections.

Alphavirus capsid protease inhibitors as potential antiviral agents for Chikungunya infection.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus and currently, no antiviral drug is available to combat it. Capsid protein (CP) of alphaviruses present at the N-terminus of the structural polyprotein possesses auto-proteolytic activity which is essential for initiating the structural polyprotein processing. We are reporting for the first time antiviral molecules targeting capsid proteolytic activity. Structure-assisted drug-repositioning identified three molecules: P1,P4-Di(adenosine-5') tetraphosphate (AP4), Eptifibatide acetate (EAC) and Paromomycin sulphate (PSU) as potential capsid protease inhibitors. A FRET-based proteolytic assay confirmed anti-proteolytic activity of these molecules. Additionally, in vitro cell-based antiviral studies showed that EAC, AP4, and PSU drastically stifled CHIKV at the post-entry step with a half-maximal effective concentration (EC50) of 4.01 µM, 10.66 µM and 22.91 µM; respectively. Interestingly, the inhibitors had no adverse effect on viral RNA synthesis and treatment of cells with inhibitors diminished levels of CP in virus-infected cells, which confirmed inhibition of capsid auto-proteolytic activity. In conclusion, the discovery of antiviral molecules targeting capsid protease demystifies the alphavirus capsid protease as a potential target for antiviral drug discovery.

Evaluation of the Antiviral Potential of Halogenated Dihydrorugosaflavonoids and Molecular Modeling with nsP3 Protein of Chikungunya Virus (CHIKV).

Antiviral therapy is crucial for the circumvention of viral epidemics. The unavailability of a specific antiviral drug against the chikungunya virus (CHIKV) disease has created an alarming situation to identify or develop potent chemical molecules for remedial management of CHIKV. In the present investigation, in silico studies of dihydrorugosaflavonoid derivatives (5a-f) with non-structural protein-3 (nsP3) were carried out. nsP3 replication protein has recently been considered as a possible antiviral target in which crucial inhibitors fit into the adenosine-binding pocket of the macrodomain. The 4'-halogenated dihydrorugosaflavonoids displayed intrinsic binding with the nsp3 macrodomain (PDB ID: 3GPO) of CHIKV. Compounds 5c and 5d showed docking scores of -7.54 and -6.86 kcal mol-1, respectively. Various in vitro assays were performed to confirm their (5a-f) antiviral potential against CHIKV. The non-cytotoxic dose was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was found to be <100 µM. The compounds 5c and 5d showed their inhibitory potential for CHIKV, which was determined through cytopathic effect assay and plaque reduction assay, which show inhibition up to 95 and 92% for 70 µM concentration of the compounds, respectively. The quantitative real-time polymerase chain reaction assay result confirmed the ability of 5c and 5d to reduce the viral RNA level at 70 µM concentration of compounds to nearly 95 and 93% concentration, respectively, in cells with CHIKV infection. Further, the CHIKV-inhibitory capacity of these compounds was corroborated by execution of immunofluorescence assay. The executed work will be meaningful for the future research of studied dihydrorugosaflavonoids against prime antiviral entrants, leading to remedial management to preclude CHIKV infection.

Chikungunya virus inhibition by peptidomimetic inhibitors targeting virus-specific cysteine protease.

Chikungunya virus (CHIKV), a mosquito-borne pathogenic virus that reemerged and caused epidemic in the Indian Ocean island of La Réunion, is a potential public health threat. Currently there is no antiviral drug or vaccine commercially available for the treatment of chikungunya fever, which necessitates the urge for an effective antiviral therapy for chikungunya treatment. In the present study, a FRET based protease assay was used to analyze the proteolytic activity of chikungunya nsP2 protease (CHIKV nsP2pro) - an essential viral enzyme, with fluorogenic substrate peptide. This protease assay was used to assess the inhibitory activity of Pep-I (MMsINC® database ID MMs03131094) and Pep-II (MMsINC® database ID MMs03927237), peptidomimetic compounds identified in a previous study by our group. Both compounds inhibited CHIKV nsP2pro with half maximal inhibition concentration (IC50) values of ∼34 µM and ∼42 µM, respectively. Kinetic studies showed that the inhibition constant (Ki) value is 33.34 ±â€¯2.53 µM for Pep-I and 45.89 ±â€¯4.38 µM for Pep-II. Additionally, these two compounds significantly inhibited CHIKV replication in BHK-21 cells at concentrations much lower than their cytotoxic concentrations. Intriguingly, these compounds did not show inhibitory effect on Sindbis virus. This suggests that Pep-I and Pep-II compounds identified as CHIKV nsP2 substrate peptidomimetics, specifically inhibit CHIKV replication.

Evaluation of autoantibody signatures in meningioma patients using human proteome arrays.

Meningiomas are one of the most common tumors of the Central nervous system (CNS). This study aims to identify the autoantibody biomarkers in meningiomas using high-density human proteome arrays (~17,000 full-length recombinant human proteins). Screening of sera from 15 unaffected healthy individuals, 10 individuals with meningioma grade I and 5 with meningioma grade II was performed. This comprehensive proteomics based investigation revealed the dysregulation of 489 and 104 proteins in grades I and II of meningioma, respectively, along with the enrichment of several signalling pathways, which might play a crucial role in the manifestation of the disease. Autoantibody targets like IGHG4, CRYM, EFCAB2, STAT6, HDAC7A and CCNB1 were significantly dysregulated across both the grades. Further, we compared this to the tissue proteome and gene expression profile from GEO database. Previously reported upregulated proteins from meningioma tissue-based proteomics obtained from high-resolution mass spectrometry demonstrated an aggravated autoimmune response, emphasizing the clinical relevance of these targets. Some of these targets like SELENBP1 were tested for their presence in tumor tissue using immunoblotting. In the light of highly invasive diagnostic modalities employed to diagnose CNS tumors like meningioma, these autoantibody markers offer a minimally invasive diagnostic platform which could be pursued further for clinical translation.

Protein microarray applications: Autoantibody detection and posttranslational modification.

The discovery of DNA microarrays was a major milestone in genomics; however, it could not adequately predict the structure or dynamics of underlying protein entities, which are the ultimate effector molecules in a cell. Protein microarrays allow simultaneous study of thousands of proteins/peptides, and various advancements in array technologies have made this platform suitable for several diagnostic and functional studies. Antibody arrays enable researchers to quantify the abundance of target proteins in biological fluids and assess PTMs by using the antibodies. Protein microarrays have been used to assess protein-protein interactions, protein-ligand interactions, and autoantibody profiling in various disease conditions. Here, we summarize different microarray platforms with focus on its biological and clinical applications in autoantibody profiling and PTM studies. We also enumerate the potential of tissue microarrays to validate findings from protein arrays as well as other approaches, highlighting their significance in proteomics.